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https://openalex.org/W2561258152	http://www.scielo.br/pdf/ambiagua/v12n1/1980-993X-ambiagua-12-01-00099.pdf	Portuguese	null	Vegetação arbustivo-arbórea em uma restinga de Jaguaruna, litoral sul do Estado de Santa Catarina, Brasil	Revista Ambiente & Água	2,017	cc-by	6,019	Robson dos Santos; Guilherme Alves Elias; Aline Votri Guislon; Iara Zaccaron Zanoni Universidade do Extremo Sul Catarinense (UNESC), Criciúma, SC, Brasil Programa de Pós-Graduação em Ciências Ambientais, Herbário Pe. Dr. Raulino Reitz (CRI) Autor correspondente: e-mail: guilherme@unesc.net, rsa@unesc.net, vg_aline@hotmail.com, iara_zanoni@hotmail.com Universidade do Extremo Sul Catarinense (UNESC), Criciúma, SC, Brasil Programa de Pós-Graduação em Ciências Ambientais, Herbário Pe. Dr. Raulino Reitz (CRI) Autor correspondente: e-mail: guilherme@unesc.net, rsa@unesc.net, vg_aline@hotmail.com, iara_zanoni@hotmail.com RESUMO Para ampliar o conhecimento sobre a planície costeira do Estado de Santa Catarina, foi realizado um estudo fitossociológico do componente arbustivo-arbóreo na restinga da Lagoa do Arroio Corrente, no município de Jaguaruna, sul do Estado. Para amostragem da vegetação, foi usado o método de parcelas, incluindo os indivíduos com diâmetro a altura do solo (DAS) mínimo de 2,5 cm. Constatou-se, diferença de altura na fitofisionomia do trecho superior (5 m) e do trecho inferior (10 m) das dunas, optando-se por realizar a amostragem separadamente. A vegetação da restinga da Lagoa do Arroio Corrente apresentou, no trecho superior, estratificação de baixo porte (1 a 5 m), destacando-se indivíduos arbustivos, conferindo aparência densa à vegetação, devido ao desenvolvimento aglomerado dessas plantas e, no trecho inferior, com indivíduos arbóreos emergentes, conferindo dois estratos, um mais baixo com predomínio de vegetação arbustiva (2 a 5 m) e um segundo com indivíduos arbóreos (até 10 m). A riqueza florística resultou em 17 famílias, 25 gêneros e 32 espécies. A área basal total foi de 4,3 m2.ha-1 (trecho superior) e 23,2 m2.ha-1 (trecho inferior). Guapira opposita (Vell.) Reitz apresentou maior valor de importância, destacando-se também nos demais parâmetros fitossociológicos analisados (frequência, densidade e dominância). As análises efetuadas contribuem com dados estruturais para as restingas de Santa Catarina, podendo auxiliar na caracterização da vegetação dos cordões arenosos do sul do Brasil. Palavras-chave: biodiversidade, fitossociologia, Floresta Atlântica, vegetação litorânea. Vegetação arbustivo-arbórea em uma restinga de Jaguaruna, litoral sul do Estado de Santa Catarina, Brasil doi:10.4136/ambi-agua.1952 Received: 06 Jun. 2016; Accepted: 29 Oct. 2016 Robson dos Santos; Guilherme Alves Elias; Aline Votri Guislon; Iara Zaccaron Zanoni Ambiente & Água - An Interdisciplinary Journal of Applied Science ISSN 1980-993X – doi:10.4136/1980-993X www.ambi-agua.net E-mail: ambi.agua@gmail.com Ambiente & Água - An Interdisciplinary Journal of Applied Science ISSN 1980-993X – doi:10.4136/1980-993X www.ambi-agua.net E-mail: ambi.agua@gmail.com Ambiente & Água - An Interdisciplinary Journal of Applied Science ISSN 1980-993X – doi:10.4136/1980-993X www.ambi-agua.net E-mail: ambi.agua@gmail.com ABSTRACT In order to develop a greater understanding of the coastal land of the state of Santa Catarina, we carried out a phytosociological study of shrub-tree component in the restinga of the Lagoa do Arroio Corrente, in the municipality of Jaguaruna, in the southern part of the state. The study used the “plots” method, including shrub-trees with a diameter at soil height Rev. Ambient. Água vol. 12 n. 1 Taubaté – Jan. / Feb. 2017 100 Robson dos Santos et al. (DSH) minimum of 2.5 cm. We found a difference in the height of shrub-trees individuals in the upper (10 m) and lower (5 m) sections, and opted to carry out sampling in these two vegetation types separately. The restinga’s vegetation of the Lagoa do Arroio Corrente had, in the upper section, low size stratification (1 to 5 m), due to shrubby individuals, with dense vegetation cover, due to the development of clusters of plants and, in the lower section, with emerging individual trees, resulting in two strata: a lower stratum, with predominance of shrubby (2 to 5) and an upper stratum, with tree individuals (up to 10 m). The floristic richness resulted in 17 botanical families, 25 genera and 32 species. The total basal area was 4.3 m2.ha-1 (upper section) and 23.2 m2.ha-1 (lower section). Guapira opposita (Vell.) Reitz exhibited the highest importance value, with other outstanding phytosociological parameters (frequency, density, and dominance). The analyses performed contribute to structural data for restingas of Santa Catarina, assisting in the characterization of vegetation of sand bars of Southern Brazil. Keywords: Atlantic Forest, biodiversity, coastal vegetation, phytosociology. Rev. Ambient. Água vol. 12 n. 1 Taubaté – Jan. / Feb. 2017 2. MATERIAL E MÉTODOS O estudo foi realizado no componente arbustivo-arbóreo da restinga da Lagoa do Arroio Corrente, município de Jaguaruna, litoral sul do estado de Santa Catarina, com distância de 2 km do mar. O solo foi classificado, como Espodossolo (Embrapa, 2013). O clima é Cfa (segundo a classificação de Köppen), ou seja, mesotérmico úmido, sem estação seca definida e com verão quente (Alvares et al., 2013), com índices pluviométricos médios de 1.400 mm ao ano e umidade relativa do ar de 82% (Back, 2009), influenciado pela umidade marítima. A vegetação se desenvolve sobre dunas fixas de aproximadamente 30 metros de altura, tendo sua base em contato com a água da lagoa e foi descrita por Reitz (1961) como “lagoa de barragem”. g Constatou-se, no reconhecimento da área de estudo, diferença de altura na fitofisionomia do trecho superior (5 m) e do trecho inferior (10 m) das dunas, optando-se por realizar a amostragem separadamente (Figura 1). A vegetação da restinga da Lagoa do Arroio Corrente apresentou, no trecho superior, estratificação de baixo porte (1 a 5 m), destacando-se indivíduos arbustivos, conferindo aparência densa à vegetação, devido ao desenvolvimento aglomerado dessas plantas e, no trecho inferior, com indivíduos arbóreos emergentes, conferindo dois estratos, um mais baixo com predomínio de vegetação arbustiva (2 a 5 m) e um segundo, com indivíduos arbóreos (até 10 m). A comparação entre os dois trechos foi feita por meio do índice de similaridade de Jaccard que expressa a semelhança entre ambientes, baseando-se no número de espécies comuns. A atualização taxonômica seguiu a Lista das Espécies da Flora do Brasil (Flora do Brasil 2020 em construção, 2016). O material coletado fértil foi depositado no Herbário Pe. Dr. Raulino Reitz (CRI) da Universidade do Extremo Sul Catarinense (UNESC). O estudo fitossociológico foi realizado empregando-se o método de parcelas, proposto por Mueller-Dombois e Ellenberg (2002). Foram traçadas em cada trecho da duna (superior e inferior) 30 parcelas de 5 m × 5 m (750 m2), distribuídas em três blocos de 10 parcelas contíguas, distantes 30 metros entre si e mensurados todos os indivíduos vivos com diâmetro a altura do solo (DAS) ≥ 2,5 cm. O diâmetro das plantas foi obtido com o auxílio de paquímetro digital e a altura foi estimada com bastão graduado. 1. INTRODUÇÃO 2017 101 Vegetação arbustivo-arbórea em uma restinga de Jaguaruna,… de restinga, sendo, portanto, de grande importância à produção científica sobre este tipo de ecossistema, uma vez que, constantemente vem sofrendo com as ações antrópicas (Cunha, 2005). Desta forma, diante da lacuna existente, este estudo teve como objetivo descrever as características estruturais do componente arbustivo-arbóreo de uma restinga no litoral sul do estado de Santa Catarina. 1. INTRODUÇÃO A vegetação da restinga no sul do Brasil possui características peculiares, e reúne um conjunto de ecossistemas com alta heterogeneidade ambiental, ocorrendo desde dunas até planícies costeiras. As condições ambientais, nas restingas, são extremas, com altas temperaturas, ventos constantes, elevada salinidade e solos com deficiência em nutrientes (Scarano et al., 2001). No entanto, a vegetação estabelecida nestes ambientes apresenta adaptabilidade e resistência, compondo um ecossistema com função de proteção da costa e da biodiversidade (Sevegnani e Comtois, 2013; Sevegnani et al., 2013). Mesmo apresentando tal importância, a restinga tem sofrido recorrentes impactos, principalmente, pela introdução de espécies animais e vegetais exóticas, pela pressão agrícola e pelo crescimento das cidades (Santos et al., 2012). Muitos são os papéis da vegetação de restinga sobre o ecossistema onde está inserida. Alguns autores associam essa vegetação à estabilização do substrato nesses ambientes, como a proteção da ação de ventos, que é considerada importante modificador da paisagem (Lindeman, 1906; Rambo, 1954; Assumpção e Nascimento, 2000; Santos et al., 2012), além de manter a drenagem natural, contribuindo também para a preservação da fauna endêmica e migratória (Falkenberg, 1999; Rocha et al., 2005). A restinga apresenta uma diversidade fisionômica que expressa uma composição, que geralmente, combina espécies próprias do litoral com outras provenientes de outros ecossistemas, como por exemplo, do bioma Mata Atlântica, e é representada por espécies aclimatadas à faixa litorânea (Araujo, 2000; Sacramento et al., 2007; IBGE, 2012), no entanto, podem ocorrer variações fenotípicas devido à diferença do habitat original (Freire, 1990; Leite e Klein, 1990; Scarano, 2002; Klein et al., 2007). Para melhor conhecimento das restingas são necessárias descrições sobre a vegetação que se pode dar, basicamente, por meio de métodos florísticos e estruturais (Kent e Coker, 1992; Santos et al., 2012). Dessa forma, a listagem da flora e a caracterização estrutural contribuem para designar e classificar as vegetações litorâneas, além de diferenciar suas fisionomias (Silva e Britez, 2005; Magnago et al., 2011; Melo Júnior et al., 2015), fornecendo mais características sobre as restingas de Santa Catarina. Em Santa Catarina, estudos florísticos e fitossociológicos que destacam a importância e diversidade da restinga, já foram tratados por Reitz (1954, 1961), Bresolin (1979), Falkenberg (1999), Korte et al. (2013) e Melo Júnior et al. (2015). Salienta-se que, no sul do estado de Santa Catarina, há poucos estudos sobre a ve Rev. Ambient. Água vol. 12 n. 1 Taubaté – Jan. / Feb. Rev. Ambient. Água vol. 12 n. 1 Taubaté – Jan. / Feb. 2017 2. MATERIAL E MÉTODOS Indivíduos ramificados no nível do solo foram considerados na amostragem quando, pelo menos, um dos ramos atendia ao critério de inclusão estabelecido (DAS ≥ 2,5 cm), conforme descrito por Moro e Martins (2011), que orientam que, se no nível do solo, dois eixos aparecem conspicuamente ligados (rameta), tendo uma base comum, ambos são considerados como um único indivíduo. Já, quando cada eixo emerge separadamente no nível do solo (geneta), eles são considerados como indivíduos distintos e devem ser medidos separadamente. p Para análise da organização do componente arbustivo-arbóreo foi calculado a densidade, a frequência e a dominância absoluta, o valor de importância (VI), o índice de diversidade de Shannon-Wiener (H’) e o índice de equabilidade de Pielou (J’). Foram elaborados histogramas do número de indivíduos por intervalos de altura (amplitude de um metro) e de diâmetro (amplitude de 2,5 cm). Foram consideradas como espécies raras aquelas representadas por apenas um indivíduo na amostragem (Martins, 1991). Rev. Ambient. Água vol. 12 n. 1 Taubaté – Jan. / Feb. 2017 102 Robson dos Santos et al. Figura 1. Perfil esquemático das fitofisionomias da restinga da Lagoa do Arroio Corrente, município de Jaguaruna, Santa Catarina. A = trecho superior e B = trecho inferior das dunas. A B A B Lagoa A A A B Lagoa A A A Figura 1. Perfil esquemático das fitofisionomias da restinga da Lagoa do Arroio Corrente, município de Jaguaruna, Santa Catarina. A = trecho superior e B = trecho inferior das dunas. B B Figura 1. Perfil esquemático das fitofisionomias da restinga da Lagoa do Arroio Corrente, município de Jaguaruna, Santa Catarina. A = trecho superior e B = trecho inferior das dunas. Aspectos das síndromes de dispersão das espécies arbustivo-arbóreas foram abordados e discutidos de acordo com van der Pijl (1972). Para esta análise, foram reunidas informações de cada espécie, principalmente as relativas ao tipo de fruto, com base em pesquisa bibliográfica e observações realizadas em campo. Aspectos das síndromes de dispersão das espécies arbustivo-arbóreas foram abordados e discutidos de acordo com van der Pijl (1972). Para esta análise, foram reunidas informações de cada espécie, principalmente as relativas ao tipo de fruto, com base em pesquisa bibliográfica e observações realizadas em campo. 2. MATERIAL E MÉTODOS Para o enquadramento das espécies nos grupos ecológicos, conforme proposto por Budowski (1965), consultou-se estudos realizados na região sul de Santa Catarina, bem como por observações feitas nos trabalhos de campo no momento da amostragem fitossociológica. Rev. Ambient. Água vol. 12 n. 1 Taubaté – Jan. / Feb. 2017 Rev. Ambient. Água vol. 12 n. 1 Taubaté – Jan. / Feb. 2017 3. RESULTADOS E DISCUSSÃO A composição florística foi representada por 32 espécies e 25 gêneros distribuídos em 17 famílias (Tabela 1), com maior número de espécies para Myrtaceae (cinco espécies), Aquifoliaceae e Euphorbiaceae com quatro espécies cada e Sapindaceae com três espécies. Rev. Ambient. Água vol. 12 n. 1 Taubaté – Jan. / Feb. 2017 103 Vegetação arbustivo-arbórea em uma restinga de Jaguaruna,… Essas quatro famílias representaram 50% do total de espécies amostradas. Entre as demais famílias, três foram representadas por duas espécies cada (19%) e dez por uma única espécie (31%). A família com maior número de indivíduos foi Nyctaginaceae (52%), seguida por Euphorbiaceae (13%), Myrtaceae (6%) e Lauraceae (4%). Tabela 1. Caracterização das fitofisionomias da restinga da Lagoa do Arroio Corrente, Jaguaruna, Santa Catarina. DA= densidade absoluta (indivíduos.ha-1 e DoA= dominância absoluta (m2.ha-1), índice de diversidade de Shannon-Wiener (H’), em nats.ind-1 e de equabilidade de Pielou (J’). Trecho Famílias Gêneros Espécies DA DoA Índice H’ J’ Superior 12 16 18 1.425 4,3 1,50 0,52 Inferior 15 23 27 2.732 23,2 2,30 0,70 Total 17 25 32 2.078 15,4 2,67 0,70 Tabela 1. Caracterização das fitofisionomias da restinga da Lagoa do Arroio Corrente, Jaguaruna, Santa Catarina. DA= densidade absoluta (indivíduos.ha-1 e DoA= dominância absoluta (m2.ha-1), índice de diversidade de Shannon-Wiener (H’), em nats.ind-1 e de equabilidade de Pielou (J’). ( ) Trecho Famílias Gêneros Espécies DA DoA Índice H’ J’ Superior 12 16 18 1.425 4,3 1,50 0,52 Inferior 15 23 27 2.732 23,2 2,30 0,70 Total 17 25 32 2.078 15,4 2,67 0,70 A família Myrtaceae, com riqueza superior às demais, geralmente permanece com representantes entre as cinco espécies de maior valor de importância, sobressaindo-se na composição florística e estrutural em diversos locais da costa brasileira (Silva et al., 2008; Scherer et al., 2005, 2009; Santos et al., 2012; Martins et al., 2013), além de suas espécies obterem, na restinga, um ambiente propício para seu desenvolvimento (Reitz, 1961). A similaridade florística (50%) evidenciou alta semelhança entre os dois trechos avaliados evidenciando existência de um padrão fitogeográfico baseado na distribuição das espécies. No trecho superior foram exclusivas as famílias Fabaceae e Lamiaceae, representadas por três espécies exclusivas, enquanto que no trecho inferior foram exclusivas as famílias Erythroxylaceae, Rosaceae, Sapindaceae e Sapotaceae, representadas por 13 espécies exclusivas (Tabela 2). Tabela 2. 3. RESULTADOS E DISCUSSÃO Parâmetros fitossociológicos, ordenados por valor de importância (VI), das espécies amostradas no trecho superior e inferior das dunas da restinga da Lagoa do Arroio Corrente, Jaguaruna, Santa Catarina. amostradas no trecho superior e inferior das dunas da restinga da Lagoa do Arroio Corrente, Jaguaruna, Santa Catarina. Trecho Superior Espécie Família FA DA DoA VI SD GE Guapira opposita (Vell.) Reitz Nyctaginaceae 76,7 960 2,82 59,0 Zoo Sin Campomanesia littoralis D.Legrand Myrtaceae 10,0 40 0,47 6,5 Zoo Cli Vitex megapotamica (Spreng.) Moldenke* Lamiaceae 13,3 80 0,12 5,4 Zoo Sin Handroanthus pulcherrimus (Sandwith) Mattos Bignoniaceae 10,0 53 0,18 4,6 Ane Sin Myrsine parvifolia A.DC.* Primulaceae 10,0 40 0,15 4,0 Zoo Sin Lithrea brasiliensis Marchand Anacardiaceae 6,7 27 0,10 2,7 Zoo Pio Myrsine umbellata Mart. Primulaceae 6,7 40 0,06 2,6 Zoo Sin Psidium cattleianum Sabine Myrtaceae 6,7 27 0,06 2,4 Zoo Sta Ilex paraguariensis A.St.-Hil. Aquifoliaceae 6,7 27 0,02 2,1 Zoo Pio Sebastiania serrata Müll.Arg. Euphorbiaceae 3,3 27 0,03 1,5 Aut Pio Casearia sylvestris Sw. Salicaceae 3,3 13 0,06 1,4 Zoo Sin Continua... 104 Robson dos Santos et al. Continuação… Sapium glandulosum (L.) Morong Euphorbiaceae 3,3 13 0,05 1,4 Aut Pio Enterolobium contortisiliquum (Vell.) Morong* Fabaceae 3,3 13 0,03 1,2 Aut Pio Myrcia splendens (Sw.) DC. Myrtaceae 3,3 13 0,03 1,2 Zoo Sin Ilex pseudobuxus Reissek* Aquifoliaceae 3,3 13 0,02 1,1 Zoo Pio Ocotea pulchella (Nees & Mart.) Mez Lauraceae 3,3 13 0,02 1,1 Zoo Pio Pera glabrata (Schott) Poepp. ex Baill. Peraceae 3,3 13 0,02 1,1 Zoo Sta Condalia buxifolia Reissek Rhamnaceae 3,3 13 0,01 1,0 Zoo Pio Total 177 1425 4,3 100 Trecho Inferior Espécie Família FA DA DoA VI SD GE Guapira opposita (Vell.) Reitz Nyctaginaceae 76,7 1200 12,41 41,1 Zoo Sin Sapium glandulosum (L.) Morong Euphorbiaceae 33,3 280 3,62 12,4 Aut Pio Allophylus edulis (A.St.-Hil. et al.) Hieron. ex Niederl.* Sapindaceae 23,3 120 0,63 5,0 Zoo Sin Ocotea indecora (Schott) Mez* Lauraceae 13,3 160 0,80 4,6 Zoo Cli Sebastiania serrata Müll.Arg. Euphorbiaceae 10,0 133 1,17 4,4 Zoo Sin Casearia sylvestris Sw. Salicaceae 10,0 93 0,94 3,6 Zoo Sin Condalia buxifolia Reissek Rhamnaceae 13,3 80 0,31 2,9 Zoo Pio Psidium cattleianum Sabine Myrtaceae 10,0 67 0,06 2,0 Zoo Sta Cupania vernalis Cambess.* Sapindaceae 10,0 53 0,17 2,0 Zoo Pio Ilex paraguariensis A.St.-Hil. Aquifoliaceae 10,0 67 0,04 2,0 Zoo Pio Chrysophyllum marginatum (Hook. 3. RESULTADOS E DISCUSSÃO A espécie com maior valor de importância, tanto no trecho superior quanto no trecho inferior, foi Guapira opposita (Vell.) Reitz, destacando-se das demais também nos demais parâmetros fitossociológicos (frequência, densidade e dominância). Já as espécies consideradas raras, aquelas representadas por um único indivíduo, perfizeram 44% no trecho superior e 22% no trecho inferior (Tabela 2). Dentre estas, destaca-se Prunus ulei Koehne, no trecho inferior da duna, que consta na lista das espécies ameaçadas de extinção no estado de Santa Catarina (CONSEMA, 2014), descrita como característica e exclusiva das restingas arbustivas, apresentando possivelmente restrita, descontínua e inexpressiva dispersão, estendendo-se em Santa Catarina, desde Laguna até Sombrio (Reitz, 1996). g Guapira opposita é característica da floresta ombrófila densa e da restinga litorânea no sul do Brasil, onde apresenta vasta e expressiva dispersão, além de elevada abundância (Reitz, 1970). A espécie figura entre as dominantes do componente arbustivo, formando densos agrupamentos nos terrenos arenosos e pouco ondulados nas proximidades das praias (Reitz, 1970). Na restinga da Lagoa do Arroio Corrente, G. opposita faz parte da vegetação típica de dunas fixas, situadas mais para o interior e atrás das dunas móveis ou semifixas. Ocorre nas associações juntamente com Sapium glandulosum (L.) Morong, Allophylus edulis (A.St.-Hil. et al.) Hieron. ex Niederl., Ocotea indecora (Schott) Mez, Sebastania serrata Müll.Arg., Casearia sylvestris Sw., Campomanesia littoralis D.Legrand e Vitex megapotamica (Spreng.) Moldenke. Os altos valores de densidade obtidos por espécies de ampla distribuição refletem a elevada plasticidade ecológica dessas espécies e a sua capacidade de adaptação a ambientes estressantes. A ocorrência de G. opposita, em diferentes ecossistemas, como na floresta ombrófila densa (Colonetti et al., 2009; Martins et al., 2013; Bosa et al., 2015) e na restinga (Santos et al., 2012), indica que a espécie apresenta plasticidade de caracteres morfológicos e fisiológicos, no sentido de que os filtros seletivos não restringiram seu estabelecimento e sobrevivência nos processos evolutivos. Uma vez que espécies com variação fenotípica têm maior habilidade para ocorrer ao longo de um gradiente de condições ambientais. A variação fenotípica de G. opposita possibilita seu estabelecimento em ambientes com diferentes intensidades luminosas, desde a restinga arbustiva até a restinga alta (Santos et al., 2010). Quando sombreados pelo dossel da floresta, a forma da copa mostra-se mais aberta, com muitas ramificações do caule, apresentando uma arquitetura de modo a constituir uma única camada coletora de luz. 3. RESULTADOS E DISCUSSÃO & Arn.) Radlk.* Sapotaceae 6,7 53 0,33 1,9 Zoo Pio Campomanesia littoralis D.Legrand Myrtaceae 6,7 53 0,29 1,8 Zoo Cli Lithrea brasiliensis Marchand Anacardiaceae 6,7 40 0,38 1,8 Zoo Pio Ilex theezans Mart. ex Reissek* Aquifoliaceae 6,7 40 0,17 1,5 Zoo Pio Ilex dumosa Reissek* Aquifoliaceae 6,7 27 0,27 1,5 Zoo Pio Myrsine umbellata Mart. Primulaceae 6,7 27 0,12 1,2 Zoo Sin Actinostemon concolor (Spreng.) Müll.Arg.* Euphorbiaceae 3,3 53 0,14 1,2 Aut Sta Pera glabrata (Schott) Poepp. ex Baill. Peraceae 6,7 27 0,10 1,2 Zoo Sta Matayba guianensis Aubl.* Sapindaceae 3,3 13 0,44 1,2 Zoo Sta Myrcia splendens (Sw.) DC. Myrtaceae 6,7 27 0,06 1,2 Zoo Sin Handroanthus pulcherrimus (Sandwith) Mattos Bignoniaceae 6,7 27 0,05 1,2 Ane Sin Erythroxylum deciduum A.St.-Hil.* Erythroxylaceae 3,3 27 0,16 0,9 Zoo Pio Myrcia palustris DC.* Myrtaceae 3,3 13 0,22 0,9 Zoo Pio Ocotea pulchella (Nees & Mart.) Mez Lauraceae 3,3 13 0,17 0,8 Zoo Pio Eugenia catharinae O.Berg* Myrtaceae 3,3 13 0,13 0,7 Zoo Sin Prunus ulei Koehne* Rosaceae 3,3 13 0,01 0,5 Zoo Cli Schinus polygamus (Cav.) Cabrera* Anacardiaceae 3,3 13 0,01 0,5 Zoo Sin total 297 2732 23,2 100 Nota: FA= frequência absoluta (%), DA= densidade absoluta (indivíduos.ha-1), DoA= dominância absoluta (m2.ha-1); síndrome de dispersão (SD), em que, Zoo= zoocoria, Ane= anemocoria e Aut= autocoria e grupo ecológico (GE), em que, Pio= pioneira, Sin= secundária inicial, Sta= secundária tardia e Cli= clímax. *espécie exclusiva do trecho amostrado (superior ou inferior). Rev. Ambient. Água vol. 12 n. 1 Taubaté – Jan. / Feb. 2017 105 Vegetação arbustivo-arbórea em uma restinga de Jaguaruna,… A diferença na composição das espécies e do porte dos indivíduos, relacionada aos trechos superior e inferior das dunas, pode estar relacionada à maior fertilidade decorrente do acúmulo de matéria orgânica no trecho inferior, o que pode favorecer o aparecimento e o desenvolvimento de espécies que atribuem um aspecto diferencial na fitofisionomia, fato também observado em outros estudos da costa brasileira (Sampaio, 2005; Almeida Junior et al., 2011). Adicionalmente, os ventos em excesso, vindos do mar, contribuem com a fitofisionomia presente no trecho superior das dunas. Apesar do tamanho reduzido do remanescente amostrado, a restinga da Lagoa do Arroio Corrente apresentou boa representatividade de espécies devido a maior riqueza quando comparada às demais áreas de mata arenosa amostradas no sul do Brasil (Scherer et al., 2009). 3. RESULTADOS E DISCUSSÃO Nos indivíduos da restinga, expostos diretamente à luz, a forma da copa mostra-se mais fechada, também com muitas ramificações do caule, porém mostrando as folhas dispostas em diversas camadas coletoras de luz (Santos et al., 2010). A altura média encontrada no componente arbustivo-arbóreo no trecho superior da duna foi 2,9 m e a máxima, 5,5 m; no trecho inferior à altura média foi 4,9 m e a máxima 10 m. A maioria dos indivíduos (92%) no trecho superior foi registrada entre a segunda e a quinta classe de altura, correspondendo aos intervalos de 2 a 5 m (Figura 2); no trecho inferior, a maioria dos indivíduos (74%) foi registrada entre a quarta e a sétima classe de altura, correspondendo aos intervalos de 3 a 7 m (Figura 2). Rev. Ambient. Água vol. 12 n. 1 Taubaté – Jan. / Feb. 2017 106 Robson dos Santos et al. Figura 2. Distribuição em classes de altura do componente arbustivo-arbóreo na restinga da Lagoa do Arroio Corrente, município de Jaguaruna, Santa Catarina, Brasil. 0 10 20 30 40 50 600-0,91-1,92-2,93-3,94-4,95-5,96-6,97-7,98-8,99-9,910-10,9 Número de indivíduos amostrados Classes de altura Trecho superior da duna Trecho inferior da duna Trecho superior da duna Trecho inferior da duna Figura 2. Distribuição em classes de altura do componente arbustivo-arbóreo na restinga da Lagoa do Arroio Corrente, município de Jaguaruna, Santa Catarina, Brasil. O diâmetro médio no trecho superior foi de 4,9 cm e o máximo de 15,5 cm e no trecho inferior o diâmetro médio foi de 7,3 cm e o máximo de 23,9 cm, representados principalmente por indivíduos de Guapira opposita. A maioria dos indivíduos (62%) no trecho superior foi registrada na primeira classe de diâmetro e no trecho inferior a maioria dos indivíduos (34%) foi registrada na segunda classe de diâmetro, seguido pela terceira classe com 22%. A área basal total foi de 4,3 m2.ha-1 e 23,2 m2.ha-1, nos trechos superior e inferior, respectivamente. Figura 3. Distribuição em classes diamétricas do componente arbustivo-arbóreo na restinga da Lagoa do Arroio Corrente, município de Jaguaruna, Santa Catarina, Brasil. 0 10 20 30 40 50 60 70 802,5-4,95-7,47,5-9,910-12,412,5-14,915-17,417,5-19,920-22,422,5-25 Número de indivíduos amostrados Classes de diâmetro Trecho superior da duna Trecho inferior da duna Classes de diâmetro Figura 3. Distribuição em classes diamétricas do componente arbustivo-arbóreo na restinga da Lagoa do Arroio Corrente, município de Jaguaruna, Santa Catarina, Brasil. Rev. Ambient. Água vol. 12 n. 1 Taubaté – Jan. / Feb. Rev. Ambient. Água vol. 12 n. 1 Taubaté – Jan. / Feb. 2017 Figura 4. Distribuição (%) em grupos ecológicos e em síndromes de dispersão das espécies do componente arbustivo-arbóreo na restinga da Lagoa do Arroio Corrente, município de Jaguaruna, Santa Catarina, Brasil. Figura 4. Distribuição (%) em grupos ecológicos e em síndromes de dispersão das espécies do componente arbustivo-arbóreo na restinga da Lagoa do Arroio Corrente, município de Jaguaruna, Santa Catarina, Brasil. Rev. Ambient. Água vol. 12 n. 1 Taubaté – Jan. / Feb. 2017 3. RESULTADOS E DISCUSSÃO 2017 107 Vegetação arbustivo-arbórea em uma restinga de Jaguaruna,… No trecho superior, ocorreu grande quantidade de indivíduos pequenos e finos e com muitas ramificações devido a pressões diferenciadas como luminosidade e ventos em excesso, além de baixos níveis de nutrientes (Sztutman e Rodrigues, 2002). Diferentemente, no trecho inferior, os indivíduos se apresentaram com diâmetros maiores (Figura 3). Analisando o componente arbustivo-arbóreo em relação aos grupos ecológicos, das espécies encontradas no trecho superior, 83% foram classificadas como de início de sucessão (pioneiras + secundárias iniciais) e no trecho inferior foi de 76% (Figura 4). A zoocoria representou, no trecho superior, 78% das espécies amostradas e no trecho inferior, 89%. Esse padrão também foi identificado por outros autores (Scherer et al., 2005; Santos et al., 2012) no componente arbustivo-arbóreo de restinga arenosa. Esses estudos ratificam a importância dos agentes bióticos no fluxo gênico em restingas arenosas, assemelhando-se ao resultado de vários autores, como o mais relevante modo de dispersão das espécies lenhosas na Floresta Atlântica. Figura 4. Distribuição (%) em grupos ecológicos e em síndromes de dispersão das espécies do componente arbustivo-arbóreo na restinga da Lagoa do Arroio Corrente, município de Jaguaruna, Santa Catarina, Brasil. 0 20 40 60 80 100 Anemocoria Autocoria Zoocoria Pioneira Secundária inicial Secundária tardia Clímax Trecho inferior da duna Trecho superior da duna 100 Figura 4. Distribuição (%) em grupos ecológicos e em síndromes de dispersão das espécies do componente arbustivo-arbóreo na restinga da Lagoa do Arroio Corrente, município de Jaguaruna, Santa Catarina, Brasil. Brasil. As informações obtidas podem ser utilizadas para indicar espécies com potencialidades para restauração ecológica da mata ciliar no entorno da Lagoa do Arroio Corrente, já que é utilizada para abastecimento público e, no passado, sofreu impacto pela utilização em sua margem direita, para pastejo de gado bovino e para cultivo agrícola, atividades hoje proibidas. 4. CONCLUSÃO A vegetação da restinga da Lagoa do Arroio Corrente apresentou, no trecho superior, estratificação de baixo porte, destacando-se as espécies arbustivas, apresentando aparência densa devido ao desenvolvimento aglomerado dessas plantas; e no trecho inferior, apresentou espécies arbóreas emergentes, conferindo dois estratos à vegetação, um mais baixo com predomínio de vegetação arbustiva e um segundo, com espécies arbóreas. A heterogeneidade da vegetação de restinga não ficou evidenciada na restinga da Lagoa do Arroio Corrente, mesmo que, aparentemente, se reconheça pela altura dos indivíduos presentes, o reconhecimento de duas fitofisionomias distintas. Tal fato demonstra a complexidade a qual as comunidades vegetais de restinga apresentam, principalmente no que concerne aos diferentes condicionantes ambientais existentes no litoral brasileiro. 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°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«$ 7 # A $ <; 5 ' ' , L4 < . ' C * A * 7 6 = = A 6 6 = M1 < 6 7 = = 3 1M # :< =7 B = C A '? > , % & 7; < 6 < = = 7 < " ! "#"# @ > A 0 & 7; < 6 < = = = = ;= A ' U 2B$ ) ' < U @ = ' = =. 7 * A = M 7 ' ' ) L8 = * < »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«8 / / < P0 =< = : 5 = = = 9 = = 4 6 : / ! : == 7 : / A = = 4 6 K: 9 < C = 6 $; =7 & = 1 - < " = C 4 < ; < 7 % : 0 9 < # / A =< & AN5 A / = 7 4 A ;= 9 = "#"# 6: = 6 = ;= = <; = * & = = = / 9 '# = = C = = & @ = 9 = 6 P0 < 0 = 6 < = B # : < : 4 6 B = = = = = S = = < : = =P 0 AN5 / : A = / = 4 O 9 < = = = A 6 7 9;= = 6 @ " B < @ < = 6 < )' # / / A / : = = " ?< " N = < = =7 = < = 6 = A »1 @ 6 6 % & = = = 8 < 7 : 0 = % < : = 0 B = & C 7 % ;= . < 8 . & 7 ) = 6: % < 6 = 7 B 8 = : C & 6 B 7 = 8 6 = 9 # = % * =6 : & = = = < = / =7 # < := := < = < . A "6 7 /2 > ' 7 ? = 9 > , @ @ 0 9 A $; A =7 : > % ' = = A N/ % < 4 = : : U) V = N/ =; O = : 7 < U @ U % = = < = <; = = : O < / : = AN% 7 % 7 S 6 6 < : 6 @ 0 = = < A A @ E 9 - A C / A < = 6 1 $ := := % = & : % 0 9 < = & 7 2B$ A = = & " 9 . & / A 5 6 < > = = = E A = A < A : ==7 = 9 = A ' > * = < E = 8 = / = 5 7 = < < = @ / = 5 = = < 8 < . 6 E 6 / = 8 & = & , 7 1 86 7 9 6 < 0 = . & < 9 7 = = < .C < = & = 1 : .?< 3 < " = 6 < 7 & ! = "#"# A : = A A 6 / $ = 7 I4 < < 1 < B A = / E : A ?< 3 = 2 = := .$ ANB & ;< 3 $ 9 - : ==7 = 9 0 6 C C > 6 =1 0 0 A = = & 9 < O = < " - / < : '1 = = < 9 A $ 7 7 : = : 6 A < ". = $ ; =7 = ; 6 $ : : = 0 % : = =7 9 < 2> 6 =; 2> A% E 7 9 % A 9 < A N%: / A < .&: A 7 " 0 ; E = = : < % 6: 6 = <; 7 4 = 6 = 4 * < & 8 6: = % / 6: % B * < = = : =6 < 0 6 8 6: 6 = &: = < < = ;= ; < 7 < 7 : A = / A 4 A : < = 6 & = 6 % = = / 6: A A = A 7 % <; < 0 & 6: = / 6: := < < E 1 % C S : ;< 3 :< A A 8 : 6 : <; = = : "#"# < 7 =: = = 6 < = : ! & $ = 6 < G 6 = % = < = 6 = % & % <; < 7 : / 6: = $ % = 4 : = @ =7 :< = ; A 6 < & 4 : 1 = 6 = < 0 C = C . ; / 6: 7 = = < = 6 $ / 6: : = = := <; / 6: 1 1 = < & =7 $ 6: = = : # := # = . < 6 < < 6 S % % = 6 @ E 7 6 . & 8 ! 6 = =7 : 1 = = = 0 = 6 := < = 4 7 % 6:= <; : .& 0 A@ & / = % < = $ 8 6: < 7 :< O : = = & 6 = <; 5 <; / 6: - C A % < = $ = 9 % 7 ;< 3 : = # = A "/ 5 6 & * 7 = 8 7 * < C: < 6 6 < < = = C & & & @ = = = & 7 = & P4 =. & 6 ; # = : ./>41 P $; = & = = 7 =7 = ; 1 = % = ; = : 4 =7 ;< =1 5 = = 7 A = = 6 = = A : 9 < A 7 9 5 = 4 ;= 6 9 A ,)1 = = : .C @ = G . $E B 6 4 := I & E ' 7 P0 < >= : = * & == 7 < P = & <: K 6 = 6 9 7 & $ = 0 = E 5 6 C 6 : C / 6: A : 8 = & < 7 : 0 =7 7 7 7 7 # & 2 6 < = 4 6 = * < = & < & 7 9 =7 < : 7 = % A = ,2 > ' 7 ? : 9 > &, " 7 6 & = = A & @ 0 A& > < 7 6 = 6 2> A% = = 9 C % 7 ;< 3 < ' 2B$ ' T 2B$ ,T T = < , % @ 9 :< = B = C = 6 : - A : = # . 6 =1 .K: - 1 A C E 5 6 6 7 . ?< 3 6 % $ "#"# 0 = < < 7 : 7 6 < ! A ?< 3 6 & 6 & 6 $ .C A =1 == $ = 6 > @ 8 4 : = < = : A ", 7 > % = $ = 6 C 0 6 C .9 C 7 = 6 C = < = 2> A& 6 =7 :< %3 = @ K < .@4 #$ 5 6 K < X?< = " < & % = : = / ;< A @ A = ;< 1 A1 &; : = A = A & A Y1 ' > = : / = 6 = D* 2B$ ' T 2B$ ,F T = 6 & = = : / 0 $: . / 6 = @ K < ./ = & 7 = " 0 9 = ; = : =< 9 = = ; = %3 &' 9 = & < % ( > = @ 8 =7 = = G = = < = ;< = 1 0 " < = 9 A < @ 9 - E 6 C 6 B = > @ A A 2> % ?< 3 # @ = : 6 = @ > = A < A A ;< A < 7 : = 7 % A % E =1 1 A = < < : ; : < .& $511 & 9 . .K: = = = A ' ;= = =7 < = 8< % = 9 = # 6 A < A < = 0 % 9 = =7 < 6 = % 6 = <: 8 : 9 "#"# 3 @ 4 A / 3 < = ! = = 6 = 0 9 9 = @ = = =< < & 7; < A & & A > = =< = % @ - A = ' 9 - A 5 6 4 E 6 C : ==7 = 9 0 A : = A # F * C F, 86 < = < 2 7 0 < = 0 < * C 6 = < 7 = # = = " < = 5 < C = = = B = = <; 7 A 6 = = : 0 <; A / = = 6 6 2 9 <; A = C B A ;< % B = = 7 < = C 0 4 < = % < * 6 < 7 : 4 K B . & 5 $ 7 = = % : = 6 A 4 0 6 9 :1 7 : 6 < = 0 7 9 6 4 : = 8 4 = < :< < ;< = @ < F < 0 = A C < A <; < . & 6 = # 4 = = A = = 1 5 ;< = 8 & 6 & 7; < 8 ; 4 = @ " = 6 0 8 < < 7 1 6 < * " 0 < 2 =7 < : < = & B <; .7 : 6 < = 6 8A < <; : < = = : & C : 7 & C < 9 9 = =. & 1< ; # = 5 A " 7 A 2 & < 7 : & = = 6 7 * < B 7 C = & 6 = 8 = ! "#"# < < 7 : - = .C 4 A * = =1 $ 6 = * A $ / %: R" = & # 6 % 1 C = = /: 6 = < / A;4A 6 = 8 6 < 7 : 6 < @ $ :< < = 4 : = 8 6 < = C = = 6 <; L2 A6 < = * < .8 = 4 0 95 <; < ; = " < C = @ = = . & E 6 = = 7 =7 " 7 " 7 # 6 < 7 @ K < < 6 = B = % < = 8 (, 86 E: = * = B = = 9;= 9 A = 5 " A 7 * = = = = = < =: . C < = % = : < := = = 4 Q 2 % : < ;= = < : 7 6 < : = 6 B; < < : K = < * # 8 % 8= 5 6 = % % < "#"# 7 I % : = C < = = 7 = - 8 ; A I " = 7 & 0 = = = % % B A < < = = = < 7 : 6 7 = 9 = 2 # C = 7 @ = <; 0 < % : % < 6 0 % ! " = < := 7 < 9 : < < 6 7 < % = M1 " ; <; < < < C . "% > 1 % = ,F1 ( % = = / : < 7 : = 8 6 < ;= = = * = 3 0 C B $ = % / F' < 0 < I = 6 8= 6 = & 7 A P C: = = " / : $ .7= A ) ,1 0 7 6 B = 0 = % @ = < % = % = : < ./ 6: < < & I % " =7 - C 7 -, 86 < C = < <A & # < 8 = : * <6 7 < 7 4 = = = = < 2 G < 8 = = A < = < 8,1 9 6 5 A & = < : B 6: = = = = < = : = 4 6 < = A = : == 6 = 6 = < < = * = <6 B ;< = % A 0 < & < - = C & 1 "#"# < : 0 & < ! 6 = .8 8 < = & A < % E; = 6 9 / = A 0 = 1 6 * < 7 = 6 < * = < < <= = % < <7 & < = 1 8 % < ;= 8 & = . & & < = 7 < = 6 , , 1 0 @ A = 0 < % B 6 A = 6 = 7 9 0 = 8 = / 6: % .9 $; = B & < = < = < ='' 3 * = = < / 6: : == 6 # = 3 < 7 : = = =< % / 6: 0 6: = 6 = = < )H2 7 " (' 6 7 1 6 8 = C = @ ( & C 9;= . & / = # / 6: < 7 7 A = 6 = C / 6: . & % = 6: O > = = & C 7 = 7 = % < := < / ; 4 = 6 = % = < 7 : & = 7 A < 7 =< < 03 C 2 7 7 < $ 7 6 < < = = : % = =7 * = 6: <6 A : =6 < < = 6 - & 8 = 7 4 = = = 5 6 6 / 6: ;< = & = =7 G =P 6 0 < < # 6 < 7 6 0 6 = <; 8 = = : = 6 = / = & 7; < = 4 = = 8 6: = = = = = .B 8 6: : 0 6 = O "#"# =A 1 E = < = / % = = = : 0 /C@ C * = <6 7 = . 5 6 1 8 = ./C@1P = : < = A I I ./8@1P 0 / @ = = ! / 7 = 6 / 6 A C <= 7 = / = 4 = / = < A * 9 7 A . 6 % = = 1 = / 86 < 7 = 6 8! = 6 6 C 7 % 491 / C = & 6 = A = 0 = < =7 C = 9 = < 6: C / / 7 = < = / 9 9 . < P 7 % 7 = & 6 C C / / C 7 6 = 7 ,-P 5 = = P = 4 = = @ 6 9 = 0 4 7 % = 0 6 C = = = ; =9 - < = 8 < > = = 8 6: 6 < .7= '# = / A = 0 A : % = = : A ./8@1 = 1 -' B = ;= B = = 0 9 L8 # = = < 7 = % = B 9 > : = > : M #, % < # < . * 6: = C 6 = 7 % < < @ = 1 " = :A = & & ; % C < 8 = 7 < = = 6 = " & 8 6 . & 8 N = C =< 7 = 86 , E < < < < C 6 = = / = < = : = 6: * 0 7 = * % : < C < < =< < $ 6 = = 6 B = A / : 6 < = . & > < 7 : & ! "#"# 6 % 1 < 7 < 7 : & = ; = - = = 6 4 A = @ 6 B < 4 = A 6 % : A : = = & A < ;=A 6 = = 6 6 9 & 4 = A = = = <; & = 7 6 A $: 4 = = E = 6 4 4 # 0 = 8 6: 6 6 = = 0 % A & E % < 9 < / A =7 8! <; 1 * < 7 2 %: 7 = C * 0 = 7 < 6: <; < $ C 4 C 6 < = 6 0 6 = 6 0 A - 1 ; = * 9 , A , ,1 8 = .7= := 6 < .4 < 2 < : = & 8 @ G 6 6 7 = R 7= 9 % = 7 A 6 , , 86 8 = = 4 6 + E 6 9 4 = " 7 = = $ < = $; =< 7 6 = 6 = 7 = C = & = = * < < C 7 $#2 > ' 7 = = 6 = < P0 6 C 7 = " 7 = = & 9 ; < 7 & C A 7 : < = = = % 8 6 < A =7 = ?0 > EC, # C 7 < 7 ' @ > 7 =0 C # = = 7 = 0 = % = 6 % # = < A 6 & 7; A = > & K 4 A # A< < = B 9 - 6 0 6 8 4 = = 6 = 6 % < 8 7 @ 9 E < = < @ 6 6 < C 7 * < = : = # = 6 < E = 6 8 = = # = 6 < 6 < C A A 4 A < ,, / ,, , 8 6 B; < * = <; < =7 * 7 = 1 @ ;= = A 6 6 ;= 7 < A 6 & $ 8 # E = & , ' B " <; < . 6 6: = C " = B 5 = & C 0 : 9 $; E 6 < ! "#"# " < 2 8 6 < =7 < < < ; < < : = < 7 = = 6 . & B = & - = = = = / 7 < 1 % < 6 / = " " < 7 B 7 < : < <; < = : @ & 6 = = = = = = = 7; < = 6: < 6 < & $ = 9 & 7; < ,, = = $ K = = 7 = $ / E % = < 7 5 : % 6 = = A & A <A 8 & 7; < <; A = = = = = 6 ;= / & < = = < A P % = & 7; < = " % = & 7; < = . & < = < ;= 9 = & / = 0 " " 7 < 7 : < ,, ' B 9 A = & 7; < @ :7 = : & = = : =< E < 7 : 9 = 6 = & 0 = 8 6: = : =< = 6 = 7 ; = * & < B = = B < E = 6 = E 8 = 6 : < = 8 <; E = < 6 0 % 6 7 6 < 7 / < 5 = = B < 1 ;= 8 < = @ :7 = = = < < B A = , G , , B & 7 ;= 9 5 : = & = = = = 0 @ 6 = : 6 & < 6 "#"# - = 5 = 7 / ! 9 6 * = = J 9 = A 9 7 = 6 A = 6 = A . 7 $$2 > ' 7 ?E > G, A = = : @ A 8 7 " : ' > < = : * ;= = < * A A A < : = 6 < AU @ = = 9 @ 0 L = : = = : <; M J 6 7 % 6 6 6 9 < = 4 = = 6 5 = = G @ = / A@ 0 = < = 6 "#"# = : = @ = < 6 A 3 = / : 1 A & A ' / % < = = 7 6 < 0 = 6 < 4 : = 7 % < 7 B = 0 7 < = * 7 = 8 . & C& /N& /NC /1 6 6 / = : =< = 6 = 0 O = & : = = / = 5 =7 = % < ! A & 7; < A A > 7 = : : .4 = 6 9 / = = = % " =A 7 0 9 9 < C / < & = 9 = E A 9 % < < 0 8= - A 5 6 C * 6 A 6 / = " 6 9 - < @ 9 - A C 5 6 * E 6 > A A A A : ==7 9 = = / + ' 4 : * = 6 6 6 = 6: = 4 : 7 6 = = # < 6 <; # = = 7 = % < 5 < = 6 8 6: = = = = $ = * & H2 = = 7 6 = < 6 : < < = 8A 7 * 7 : = 6 = 0 = A = C A 6 ' 0 8 < * / = 0 =; @ .7 $; : = / : ./,1 7 ./ 11 9 : < .9,11 9 .7 :< :< : .@ = = 9 : 8 < A9 86 6 < = E <; * A = = = % = < 5 / A : 8A9 : .9, 1 9 = 7 = A A < & * % = :< = 6 = = S B 4 * 8 8 =7 & :1 6 9 7 = =A $; = .E,1 = = 8 = 8 = A = = <A < .E 1 <; A 8 = A 6 =6 = = & A 6 . ,1 7 . 1 0 7 7 4 K C: 9 < 8 < = " :< < & # =7 = C: = = 0 # =7 2 A B < C A % A 6 0 . & B; < 7 $; K: ,) V 2B$ 0 : ' , 1 $ = 8 : = = 7 := 1 6 .* "#"# & := % 6 = < 6 ! 6 < = B = = A = , = 6 C < A < 6 C < = = := 9 = V 2B$ (@ ./ 1 A , 7 $"2 > F ' > /, 4 1 - 4 :< < 8 7 < 1 = =7 .8 = < B 9 / 4 :< 4 9, = < < @ .8 = < 9 < 2B$ ,) ' K: U = $; 8 E E , 6 = A U 2B$ ) V = : 8 % 6 < = A = = = = : == ) * , * , V & 7; < = = = U @ U % * * , 2B$ U @ U % * * , * ' * ' 2B$ ) V , V = A) % = 2B$ ) V , V = A) % = 6 B/A = $; 1 " W, = A < 9 @ : = =@ K < = @ K < : < & @ - 5 6 = E, 1 = : A 8, / 1 / = = = < 8A9 9 1 E 9, = . 6 < = / : /)1 = .8 $; ' = U 2B$ ) ' = U A E 6 A E 6 A 0 A & A A @ * = & 8 = 7 0 = " B & 6 = & : 7 7 * % < E 6 # = < 7 7 E ! "#"# ' 6 = = .B = = C: 6 : = % A > : 1 0 8 6 ?< 3 : & C % = = 2 * = = ?< 3 % < # = 6 < 7 : B 4 : = * : 7 7 7 = A N C <A = 0 <; 6 B AN 6 2 = = 7 A : = 4 = = 7 A # 0 7 = = = 9 = < = K < : 0 = * = 6 < 8 < 7 : < ,' = < 7 < # 6 < = = @ 9 <; .# 1 0 < # = # $ A = = < = = .#'1 = % < < 7 : 6 / = =. = % = < & = = < :A 6 = A " = = = $; & = = 7 A = > K <A = A < 7 * = < = < 4 A =7 % % % = < C: <; ; .C'1 0 < 7 B = < 7 8 = 6 8 ; = 7 < = $; C 7 A 6 = = = A 4 K A 9 " =6 A 7 : A 9 = C 9 = = < 7 .C 1 = * 7 % = 0 < 1 " .&,1 7 6 < = =7 & 7 = 7 .&'1 % ' ?< 3 . & B A 9 & = : =< 5 : % < "#"# * % 7 <; ! A 8 = " = & 7 = = 1 < 0 =7 % =7 .C,1 5 = 4 % = = = A 9 " ?< 3 = : A : 7 8 6 < 0 C: : = & .# 1 > % ;= % . ,1 = A @ < = = : = 7 .#,1 = : 6 0 8 = " @ : = < 8 =7 6 A = = % = < 4 : 9 7 = & = 4 6 " = : = < 0 > < =7 % 9 = # = = : / A $ A A A ?< 3 = 3 = A = % < = ;< A = = A $ 6 & = .& 1 8 & 7 * < 9 * 7 @ 9 = 5 7 9 = = : = .G,1 = 7 .G 1 0 9 < 9 0 2B$ / = : K: 9 # % & : = 6A = : % < = < A : < < 9 A @ = A = A = := 2B$ 0 6 .* = := ,'1 $ 6 (@ A 9 : ' : & = = .EC,1 6 A = ; = 6 > @ = 6 A 0 6 > = < = = O < ' 8 & & <; < > #, <A < J / &' EC, G 1 9 = < ,- % $ 2< 6 6 = 6 6:= 0 7 # =< 0 / .#' 9 = <; V * = < ( < = = * O 7 = 8 = : = 7 = 4 8 = : & < < = 0 = : 0 7 / < = > K < = & < @ - 9 1 ) A1 A, @ $ # = = A * 2B$ ) V = @ $ # = = A * # A & = 6 = A = A 7 #' < @ = * : : C * # 4 =7 8 A : C, " = C = C ! "#"# < 2B$ ) Z = A 2B$ ) V = @ < 7 * C ' * F " > C ' % C' = 2B$ ' V = = C < 6 @ C < 7 < 6 < = @ - 9 1 ) @ = 0 * F * F * ( * ( * - * - * - * , * ,, * ,, C = 2B$ ,T K: = = ) % V C , @ $; N/ = = " U) V 9 =7 : % 2B$ U @ U % = :A A C = = < C &, " 7 & = 2B$ ' T 2B$ ,T T = : ;< = 2B$ ' T A 2B$ ,F T = & 6 A = , % K: C & % / = 6 &' = & 9 EC, % K: = C = & = % = < =7 # =7 = < 7 A E = : A @ 5 6 A C G, G = A A A U @ = = 5 6 / = 6 A 7 A < = 0 7 = < < : 6 = 6 4 : = 4 : = 6 6 <; =; & B = = ! "#"# = .#22% :< < ,(1 $ * :< < 6 9 ' = < = < 7 = 0 < 8< 4 : < A : = C = == A = 6 0 G "#$.2 8 = < ,FI 4 <6 0 ) 0 5 6 "#$+2 & 7 : 6 ; # 8 := & G = : "#$,2 C % = E: E 7 = & = : 4 = 6 A ,) = B K; = ,- "#$,2 = = 4%0 "#$/2 = ' 6 & P9 4 < C % ,( G "#"#2 =; 8 6 < ,( & 5 6 G "#$.2 ; < C A @ / = 8 # ,F P, (% 6 8 * = * E = A =E = & $5 % 6 ,-- I = 2 % : 2 = & $ 6 E %3 # 9 & ,FP 5 6 = & 5 6 A = 9 "#$/2 / : = ,( & 8 = &8 = 9 "#$.2 / : = ,F & 8 = &8 > ' "#$,2 < ,- , A ,( 6 N N< = ,, ,-\ PN666 < [ CC "#$/2 2B ,( A % 6 % "%&#A# 4 0 "#$62 @ R ! RB "#"# 0 6 A< # 2 A A N< A 2 % 7 6 / -F(A'A-) ) ',A A = = C%E & N %R& = @ . & & % = 1 , C 5 6 & $5 & C F% R8 = $ = = C ;= = # 7 6: 6 < = C 7 C & . 6 * $ 24 5 > ,( = 8 6: 7 9 = 6 / 6: % 9 6 7 = = 7 = < 4 : < 6 < : * = % % < , * B A O 9 A ?4 > 7 A @ =7 0 :7 7 9 A & % 7 : C : A = > < :7 " C :7 7 :7 = < : 7 9 :< 6 < $ 7 9 = 9 C : 6 0 : 6 % ; = = 0 ' 2B$ ' V 2B$ , V 7 7 4 C < A A C = B = K: @ 0 = @ 9 - A 9 : # = 6 = 8 @ < > @ $+2 4 5 7 > ' A = = 7 = = A ,F1 <; = ' A ? = C & 7; < A < = A 6 9 = 9 > ' & < @ 4 = = = & = A ' > < = = A < < = 8 = 4 = 6 < = # = 6 & 7 : & =7 / 0 < = ; % ANC 6 < = . & < % ! "#"# & = < = A = 6 @ & $; 9 - = : 5 6 4 A < A C / A A + 7 > 3 7 # <; = = = 6 & 1 0 < & : = 9 4 = 7 $62 4 5 7 > = % = =N > 8 < & @ & A = @ % 7 ' ? : = =7 = & A 9 > & @ A0 A = = = & 6 & > & = & = = ' A 2B$ ) T 2B$ ,' T < < = = 0 & & = / = 6 / = == " $ < = < 6 = 6 = =7 @ & = 8 6: < & := % / 6: E 6 == ; A > < * < A @ & = = C 7 = . & % = % 5 = ; / 7 = = = ;= < < / 6: 3 < 6 = 9 < A = A < U = / 6: 9 : = 8 6: ;< = # 4 = & 6 =/ &: N& K < A % A@ 9 * = C A E - A =C A 5 6 6 > 8 6: A @ @ / 6: <A % 5 ;= C 6 = = 6 < 9 0 9 7 A = : =< / 6: ;< A B = 0 9 K < X?< = " < Y = 7 ; & =7 7 < 1 A ' U 2B$ ) V 6 B = 4 "#"# - 6 A C % K < = % ! 8 := & A = < 8 5 < = A , % 6 A < A @ 9 > / 6: & < = & < 0 < = A K: 6 < = < &) = 6 8 4 # = A / 6: : 6 = = = 6 7 > & @ > 0 ?< 3 = .# # := 1 & = = = 6 / % = : = = = = & / 7 <; = ; ' = A 2B$ T =7 2B$ T < A < = = < Q% 6 < : % = 4 < 7 : 8 6 < & = 6 A A K: = 6 A K: @ 9 - A : = % = < C = & 7 : % A 5 6 / = 6 0 8 6 < = = 6 = 0 < A > / : 0 = 9 % 8 6 < = & 7 : A @ 9 = / : 6 = / : % = = A & = 4 < = 7 # A := 6 & =7 / : 5 = < : 0 9 A & = % 7 > : 4 = 7 : = 8 6: = % = = 7 E S : % = = A 0 9 = / 0 8 6 < = 5 A = 9 = A " 9 7 7 $.2 4 7 5 % :< =7 & = = ; > ' ?7 > = @ /: < , % /: > < ' 2B$ ) Z K: = 6 < 7 = .7 4 : 5 /: N* < 7 " B & == = / 1 = < 6 & = @ A 9 - A * ;= < < < 7 : <; $/2 H F > 5 > ' ! "#"# A 7 " > A = < " : ==7 = 9 6 A = A O 7 V = < = , < '@ = 6 = % < 6 < ' < @ 9 - ) . > C ' ' " 9 C 7 =7 :7 2B$ ' V 2B$ , V 7 % < 7 @ = K: 9 @ = 0 - ) * , * ,) * , * , * , * ,F C : & < = = A A = C = 2B$ ) T 2B$ ,' T K: = / 6: = U 2B$ ) V K: = 2B$ 2B$ & = & & 6 &) 7 = = = , % C U % * = C & / , 6 : % % = = = = 6 '@ : := = ) % ! "#"# = , V % K: C 2B$ ) Z , V K: /: < 0 T T 2B$ '@ A K: $ ') V 9 2B$ 0 6 6 :=
https://openalex.org/W2983281124	https://www.research.unipd.it/bitstream/11577/3316003/1/genes-10-00916.pdf	English	null	The Molecular Determination of Hybridity and Homozygosity Estimates in Breeding Populations of Lettuce (Lactuca sativa L.)	Genes	2,019	cc-by	7,995	Received: 23 September 2019; Accepted: 7 November 2019; Published: 9 November 2019 Received: 23 September 2019; Accepted: 7 November 2019; Published: 9 November 2019 Abstract: The development of new varieties of horticultural crops beneﬁts from the integration of conventional and molecular marker-assisted breeding schemes in order to combine phenotyping and genotyping information. In this study, a selected panel of 16 microsatellite markers were used in diﬀerent steps of a breeding programme of lettuce (Lactuca sativa L., 2 n = 18). Molecular markers were ﬁrst used to genotype 71 putative parental lines and to plan 89 controlled crosses designed to maximise recombination potentials. The resulting 871 progeny plants were then molecularly screened, and their marker allele proﬁles were compared with the proﬁles expected based on the parental lines. The average cross-pollination success rate was 68 ± 33%, so 602 F1 hybrids were completely identiﬁed. Unexpected genotypes were detected in 5% of cases, consistent with this species’ spontaneous out-pollination rate. Finally, in a later step of the breeding programme, 47 diﬀerent F3 progenies, selected by phenotyping for a number of morphological descriptors, were characterised in terms of their observed homozygosity and within-population genetic uniformity and stability. Ten of these populations had a median homozygosity above 90% and a median genetic similarity above 95% and are, therefore, particularly suitable for pre-commercial trials. In conclusion, this study shows the synergistic eﬀects and advantages of conventional and molecular methods of selection applied in diﬀerent steps of a breeding programme aimed at developing new varieties of lettuce. Keywords: pure lines; F1 hybrids; microsatellite markers; marker-assisted breeding; crop improvement; varieties genes G C A T T A C G G C A T genes G C A T T A C G G C A T genes T G T Alice Patella, Fabio Palumbo , Giulio Galla and Gianni Barcaccia Alice Patella, Fabio Palumbo , Giulio Galla and Gianni Barcaccia Department of Agronomy, Food, Natural Resources, Animals and Environment, University of Padova, 35020 Legnaro PD, Italy; alice.patella@phd.unipd.it (A.P.); giulio.galla@unipd.it (G.G.); gianni.barcaccia@unipd.it (G.B.) Department of Agronomy, Food, Natural Resources, Animals and Environment, University of Padova, 35020 Legnaro PD, Italy; alice.patella@phd.unipd.it (A.P.); giulio.galla@unipd.it (G.G.); gianni.barcaccia@unipd.it (G.B.) Department of Agronomy, Food, Natural Resources, Animals and Environment, University of Padova, 35020 Legnaro PD, Italy; alice.patella@phd.unipd.it (A.P.); giulio.galla@unipd.it (G.G.); gianni.barcaccia@unipd.it (G.B.) Correspondence: fabio.palumbo@unipd.it Article Alice Patella, Fabio Palumbo *, Giulio Galla and Gianni Barcaccia 1. Introduction Lettuce (Lactuca sativa L.) is a self-pollinating leafy vegetable species (2 n = 2 x = 18) of the Asteraceae family. It is cultivated on a large scale throughout the world for consumption as a fresh vegetable on its own or in combination with other ready-to-eat vegetable products [1]. Its growing economic importance has led seed companies to regularly develop new varieties with ever higher agronomic traits. However, breeding programmes are highly limited by the reproductive system of this species. The ﬂower structure of lettuce determines a reproductive strategy known as cleistogamy, in which anther dehiscence and subsequent pollination take place before ﬂower opening, resulting in a very high rate of self-pollination, very often equal to or close to 100% [2]. According to recent estimates, out-cross rates are limited to 1–6% [3]. These reproductive barriers mean that in natural conditions the species spontaneously constitutes pure lines, characterised by phenotypic uniformity and genotypic stability, due to their very high homozygosity. In conventional breeding programmes, developing segregating and recombinant F2 populations traditionally requires crosses to be hand pollinated www.mdpi.com/journal/genes www.mdpi.com/journal/genes Genes 2019, 10, 916; doi:10.3390/genes10110916 2 of 12 Genes 2019, 10, 916 while self-pollination is prevented by emasculating the ﬂowers. The most popular emasculation and hand-pollination technique is that described by Olivier [4]. Known as the “wash method”, it involves hand-spraying the inﬂorescence with water during pistil emergence to remove the pollen attached to the female part of the ﬂower. The inﬂorescence is then left to dry for a short period, after which it is rubbed with a ripe ﬂower of the pollinating variety [5]. A slightly diﬀerent, but also widely used, technique is the “clip-and-wash method”, which involves clipping the tip of the corolla before spraying with water. This guarantees more eﬃcient pollen removal and cross rates close to 100% from the subsequent manual pollination [2]. However, these breeding techniques are time-consuming and technically highly demanding, and are only really eﬀective if coupled with molecular analyses aimed at screening progeny plants and assessing their hybridity. In recent years, many seed ﬁrms have begun using molecular markers to carry out assisted selection schemes and to speed up varietal development programmes [3]. 1. Introduction Simple Sequence Repeat (SSR) markers are, so far, the most commonly used markers for these purposes [6–8] as they are codominant, have high reproducibility and multi-allelism, and can be detected at any stage of plant development, without being inﬂuenced by the environment [9]. There are a considerable number of SSR markers for lettuce in the literature [10]. Truco, et al. [11] produced an integrated genome map from 7 diﬀerent linkage maps, which included 130 SSR loci organised in 9 linkage groups. Rauscher and Simko [10] augmented this genomic map with 54 genomic SSR and 52 EST-SSR (Expressed Sequence Tag) loci. Finally, with the publication of the L. sativa genome draft [12], tens of thousands of new SSR regions have become available for testing and use. Given the availability of markers in lettuce, Marker-Assisted Selection (MAS) has started to be adopted in plant breeding programmes for various purposes, including identiﬁcation of resistance genes [13,14] or Quantitative Trait Loci (QTLs) of phytopathogens [15,16], the study of QTLs controlling complex traits [17,18], and investigation of the genetic identity and purity of either experimental or commercial lines [19]. On the other hand, very few attempts have been made to prove the eﬃciency of molecular markers in Marker-Assisted Breeding (MAB) activities, where the genotypic background is molecularly investigated to complement traditional phenotypic selection [20]. In this work, SSR markers were used in three diﬀerent steps of a conventional breeding scheme aimed at developing new varieties characterised by distinctiveness, uniformity, and stability (Figure 1). Genes 2019, 10, x FOR PEER REVIEW 3 of 13 84 Figure 1. Simplified overview of a lettuce breeding scheme in which selection is based on both plant 85 phenotyping and genotyping. 86 2 Materials and Methods 87 Figure 1. Simpliﬁed overview of a lettuce breeding scheme in which selection is based on both plant phenotyping and genotyping. Figure 1. Simplified overview of a lettuce breeding scheme in which selection is based on both plant 85 phenotyping and genotyping. 86 Figure 1. Simpliﬁed overview of a lettuce breeding scheme in which selection is based on both plant phenotyping and genotyping. Genes 2019, 10, 916 3 of 12 We ﬁrst examined the genetic background of a number of superior pure lines in order to plan experimental matings to produce F1 hybrids and then derived F2 progenies manifesting morphological variability as a result of genetic segregation and recombination (Figure 1). 2.1. Plant Materials and Breeding Techniques Plant materials were developed and provided by Blumen Group SpA, Italy and belonged to ﬁve diﬀerent lettuce cultivar types (Table S1). Seventy-one parental lines (germplasm composed of experimental, pre-commercial and commercial lines) were involved in 89 combinations of crosses, in which each progeny consisted of 6–12 individuals (871 progeny samples). Parental lines were grown in the spring of 2015, and the 89 programmed crosses were carried out in the summer using the clip-and-wash method [2]. This involved making an incision in the calyx and corolla and washing the anthers in the early morning before the pollen grains could settle naturally on the outermost stigmatic surface of pistils. The plants were then manually pollinated by rubbing anthers of the pollen donor on the stigma of the seed parent. For each planned cross, a bulk of 4/5 ﬂowers from a pollinator parent was used to pollinate as many ﬂowers of a seed plant. Seeds were collected from the seed plant and sown in early autumn for genotyping selection and agronomic evaluation (spring 2016). Finally, to assess the uniformity of the 47 experimental lines, previously chosen for morpho-phenological traits and pathogen resistances, 940 samples belonging to the 47 F3 populations (labelled 1 to 47) were collected in the spring of 2018. Each experimental line comprised 20 individuals. 1. Introduction Each oﬀspring in the F1 generation was analysed to distinguish the individuals resulting from planned out-crosses from those resulting from accidental selﬁng (Figure 1). After genotyping, the S1 individuals were discarded, and the F1 individuals were self-pollinated. In the F3 generations (Figure 1); experimental populations, previously selected according to their morphological traits, were also characterised by molecular markers due to the need to assess their stability and uniformity in order to run pre-commercial trials. 2.2. DNA Isolation A total of 100 mg of fresh leaves was collected from each of the 1882 lettuce samples (71 parents, 871 progeny and 940 F3) and ground to a ﬁne powder using Tissue Lyser II (Qiagen, Valencia, CA, USA). Genomic DNA (gDNA) was extracted with the Dneasy® 96 Plant Kit (Qiagen), according to the manufacturer’s protocols. After extraction, the integrity of the gDNA was assessed by electrophoresis on 1% (w/v) agarose gel stained with SYBR Safe® 1 × DNA Gel Stain (Life Technologies, Carlsbad, CA, USA) in Tris-Acetate-EDTA (TAE) running buﬀer. Both the yield and purity of the extracted gDNA samples were evaluated using a NanoDrop 2000c UV-Vis Spectrophotometer (Thermo Scientiﬁc, Carlsbad, CA, USA). Following DNA quantiﬁcation, all DNA samples were diluted to a ﬁnal concentration of 20 ng/µL. 2.3. Primer Design and Testing of SSR Marker Ampliﬁcation Marker Name ID LG Motif Primer Sequence Dye Multiplex LSSA27-2 [10] Lsat1 1 (AC)7 For [M13]CACACTACCACCCAACACG 6-FAM 1 Rev ACCCTCTTCGCTTCTTCTT SML-045 [21] Lsat2 2 (AAG)9/12 For ACAAAACCGTTTCACCCAAA 6-FAM 1 Rev [M13]AGCCCTGTCCTCTTCAGGAT LSSB54 [10] Lsat3 8 (GT)10 For [PAN1]CTTGAGAGTGCTTGGAGAGGAT VIC 1 Rev CACATACAACAAGACAAGTCCCA LSSA05 [10] Lsat4 8 (TC)18 For AGAACAACGGTAGCTTGTTAAATTG VIC 1 Rev [PAN1]ATCGTCGGTTAATCTTCGTCG LSSA04 [10] Lsat5 4 (TC)14 For [PAN2]AAGGAAAGGAAGGGTTGACTTGT NED 1 Rev TTGGTGAAGAAAAGAGAGAGTTT LSSA11 [10] Lsat6 3 (CT)20 For [PAN2]ACTCCCACTATCCTCTTTGCAT NED 1 Rev GCCCACATTCTTAATCTTGTCC LSSA14 [10] Lsat7 9 (AG)18 For [PAN3]TGATGACTCCAGTCTTAGATACCA PET 1 Rev AGTCCCCGACTATCAGTCTCA LSSB09 [10] Lsat8 2 (TG)8 For AGAATGAGAAGGATGAAATGGCTG 6-FAM 2 Rev [M13]AAACACCTTTAGCATCAAAATACCC SML-029 [21] Lsat9 9 (GAG) 7/8 For [M13]AGCCCAGAAGAGCGTGATTA 6-FAM 2 Rev TGCAGGGCTCCTTGATCTAC LSSB17-1 [10] Lsat10 7 (GT)11 For ACTAGGGCTCTAATACAACTTGT VIC 2 Rev [PAN1]TTGGCTTACAGTTATGGATTAAATG LSSA17 [21] Lsat11 3 (AG)21 For [PAN1]AATGTGCGTGAGAGTTTCCTTT VIC 2 Rev CAAGAAGGCAGTGATGAAGTTG LSSA12 [10] Lsat12 5 (GT)11 For [PAN2]ACAAGGCCCAATCCTTTTCT NED 2 Rev TCGAAAATTTGGAGAGAGTTTCTT LSSA15 [10] Lsat13 1 (AC)11 For GCCCAACCCAAGAAGAGGAG PET 2 Rev [PAN3]TGGAGAGGAGTGGAGAGTGTT LSSA28-1 [10] Lsat14 4 (GA)28 For TTCATCTCTCTCCTCCTTCAGC 6-FAM 3 Rev [M13]ATCCCCATTGTCCTCCC LSSA21-1 [10] Lsat15 8 (TC)19 For [PAN2]TTGTACCCAGTTGTCCAAACAG NED 3 Rev CAGATTGTTGCAGATTTCTTCG LSSB68 [10] Lsat16 na (CT)20 For GTCTGTGTGGTTTTGGT PET 3 Rev [PAN3]TGTGGTGGAGTGTGATTT The 16 primer pairs were ﬁrst tested individually (singleplex reactions) using three randomly chosen lettuce gDNA to evaluate primer eﬃciency and to check the correspondence between expected and actual size of the bands; they were then evaluated in multiplex PCRs to assess possible primer interactions. p All ampliﬁcation reactions (both singleplex and multiplex) were performed in a 10 µL reaction volume containing 1× Platinum® Multiplex PCR Master Mix (Thermo Scientiﬁc), 10% GC Enhancer (Thermo Scientiﬁc), 0.25 µM of non-tailed primer, 0.75 µM of tailed primer, 0.50 µM of ﬂuorophore-labelled primer (universal primer) and 20 ng of genomic DNA. Thermal cycling conditions were as follows for multiplex 1 and 2:94 ◦C for 5 minutes followed by 6 cycles of 94 ◦C for 30 seconds, 61 ◦C for 30 seconds, 72 ◦C for 45 seconds, with a 1 ◦C annealing temperature stepdown per cycle (from 61 ◦C to 56 ◦C). The annealing temperature for the following 35 cycles was set at 56 ◦C, with denaturation and extension phases as above and a ﬁnal extension at 60 ◦C for 30 minutes. 2.3. Primer Design and Testing of SSR Marker Ampliﬁcation Sixteen SSR marker loci were selected from those available in the scientiﬁc literature [10,21], according to (i) chromosomal location; (ii) polymorphism rate, expressed as PIC (Polymorphism Information Content); (iii) allele size range; (iv) annealing temperature of the locus-speciﬁc primers. Ampliﬁcations were performed according to the method previously described by Schuelke [22], with some minor modiﬁcations. Brieﬂy, three primers were used to amplify each microsatellite locus: a pair of locus-speciﬁc primers, one with an oligonucleotide tail at the 5′ end (M13, PAN-1, PAN-2 or PAN-3, Table S2), and a third universal primer complementary to the tail and labelled with a ﬂuorescent dye (6-FAM, VIC, NED, or PET). Primer pair eﬃciency was tested in silico using the PRaTo [23] web-tool and were organised in three multiplex reactions, as shown in Table 1. 4 of 12 Genes 2019, 10, 916 Table 1. Microsatellite loci information. For each primer pair, the original simple sequence repeat (SSR) name, ID used in this work, linkage group [10,21], SSR motif, primer sequences (PAN1, PAN2, PAN3, or M13 tails at the 5’ end are indicated in square brackets; for further details see Table S2), dye and the multiplex to which the SSR marker locus belongs is shown. 2.4. Genotyping and Data Analysis The 71 potential parents were genotyped at 16 SSR loci and statistical analyses were performed using NTSYS (Numerical Taxonomy and Multivariate Analysis System) version 2.2 (Exeter Software) [24]. Rohlf’s (or the simple matching) coeﬃcient was used to calculate pairwise genetic similarity (GS) in all possible comparisons and to construct a genetic similarity matrix, according to the formula: (1) GSij = m/(m + n) (1) where “i” and “j” are two diﬀerent individuals, while “m” and “n” represent the number of matching and non-matching attributes, respectively. An unweighted pair group method with an arithmetic mean (UPGMA) dendrogram and a Principal Coordinates Analysis (PCoA) of parental lines were carried out using the Jaccard coeﬃcient in the PAST software v. 3.14 with 10,000 bootstrap repetitions [25]. The genetic structure of the lines was modelled using a Bayesian clustering algorithm implemented in STRUCTURE v. 2.2 [26]. Since no a priori knowledge of the origin of the populations under study was available, the admixture model and then the correlated allele frequencies model were used. Ten replicate simulations were conducted for each value of K, with the number of founding groups ranging from 1 to 8, using a burn-in of 200,000 and 1,000,000 iterations. The most likely K Estimates were determined using the method described by Evanno et al. [26]. Estimates of membership were plotted as a histogram in an Excel spreadsheet. Finally, observed homozygosity (Ho) was determined with the POPGENE software [27]. The 89 subsequent crosses were planned according to the following criteria: (i) high genetic dissimilarity values among parents within the same lettuce cultivar type and between them, (ii) availability of informative loci able to distinguish between the resulting oﬀspring and individuals resulting from accidental self-pollitated. Only homozygous loci for diﬀerent alleles were considered informative, whereas heterozygous loci were taken into account only if the origin of the parental alleles could be clearly discerned in the progenies. The resulting oﬀspring (871 samples) were then screened, with the analysis restricted to those SSR loci which had previously proven to be informative for hybrid detection. This made it possible to determine whether individuals belonging to a given F1 population originated from cross-pollination or self-pollination. The successful crosses (S.C.) rate of 89 was calculated as follows: S.C. = (No F1 × 100)/(No Tot −No U.G.) (2) S.C. 2.3. Primer Design and Testing of SSR Marker Ampliﬁcation The multiplex 3 thermal cycling conditions were instead: 94 ◦C for 5 minutes followed by 6 cycles of 94 ◦C for 30 seconds, 56 ◦C for 30 seconds, 72 ◦C for 45 seconds, with a 1 ◦C annealing temperature stepdown per cycle (from 56 ◦C to 51 ◦C). The annealing temperature for the following 35 cycles was set at 51 ◦C with denaturation and extension phases as above and a ﬁnal extension at 60 ◦C for 30 minutes. All ampliﬁcations were performed in a GeneAmp® PCR 9700 thermal cycler (Applied Biosystems, Carlsbad, CA, USA). PCR products were ﬁrst checked on gel electrophoresis (2% Ultrapure™Agarose in TAE 1×, SYBR Safe® 1×, Life Technologies) then run on capillary electrophoresis with ABI 3730 DNA Analyzer (Applied Biosystem), using LIZ500 as the molecular weight standard. The size of each peak was determined with the Peak Scanner 1.0 software (Applied Biosystems). Genes 2019, 10, 916 5 of 12 3.1. Parental Lines 3.1. Parental Lines The 16 SSR markers, organised in three multi-locus PCRs, were used ﬁrstly to amplify and score the 71 parental lines. Fourteen of the 16 SSR markers proved to be polymorphic among plant accessions. The similarity matrix constructed using Rohlf’s coeﬃcient revealed genetic similarity values ranging from 53% to 100% (Figure S1). The resulting unweighted pair group method with an arithmetic mean (UPGMA) dendrogram showed the samples clustering into two main sub-groups. Eighteen parental lines were not fully distinguishable, while the remaining 53 had unique genotypic proﬁles. The ﬁrst principal coordinate from the PCoA accounted for 22% of the total variation and divided the samples into two groups, analogous to the clustering in the tree. The second principal coordinate accounted for 12% of the total variation. These results were conﬁrmed by investigation of the genetic structure of the 71 parental lettuce lines based on allele frequencies; the best estimate of population size was K = 2 (Figure S2), such that the samples were grouped into two genetically distinct clusters (Figure 2). The lettuce cultivar types were reported in Table S1, but they did not correspond to diﬀerent clusters in the UPGMA tree. The 16 SSR markers, organised in three multi-locus PCRs, were used firstly to amplify and score the 71 parental lines. Fourteen of the 16 SSR markers proved to be polymorphic among plant accessions. The similarity matrix constructed using Rohlf’s coefficient revealed genetic similarity values ranging from 53% to 100% (Figure S1). The resulting unweighted pair group method with an arithmetic mean (UPGMA) dendrogram showed the samples clustering into two main sub-groups. Eighteen parental lines were not fully distinguishable, while the remaining 53 had unique genotypic profiles. The first principal coordinate from the PCoA accounted for 22% of the total variation and divided the samples into two groups, analogous to the clustering in the tree. The second principal coordinate accounted for 12% of the total variation. These results were confirmed by investigation of the genetic structure of the 71 parental lettuce lines based on allele frequencies; the best estimate of population size was K = 2 (Figure S2), such that the samples were grouped into two genetically distinct clusters (Figure 2). The lettuce cultivar types were reported in Table S1, but they did not correspond to different clusters in the UPGMA tree. Figure 2. 3.1. Parental Lines 3.1. Parental Lines (a) Genetic similarity-based unweighted pair group method with an arithmetic mean (UPGMA) dendrogram of 71 parental lines calculated using the Jaccard coefficient. Bootstrap estimates ≥30% are reported next to the nodes (red and blue branches indicate the two clusters identified). (b) Principal coordinate analysis (PCoA). The 71 samples are shown in red or blue Figure 2. (a) Genetic similarity-based unweighted pair group method with an arithmetic mean (UPGMA) dendrogram of 71 parental lines calculated using the Jaccard coeﬃcient. Bootstrap estimates ≥30% are reported next to the nodes (red and blue branches indicate the two clusters identiﬁed). (b) Principal coordinate analysis (PCoA). The 71 samples are shown in red or blue according to the clustering shown in the UPGMA tree. (c) The population genetic structure of the 71 lines as estimated by STRUCTURE. Each sample is represented by a vertical histogram partitioned into K = 2 coloured segments (red or blue, in accordance with (a,b)) representing the estimated membership. The proportion of subgroup membership (%) is reported on the ordinate axis, and the identiﬁcation number of each accession is reported below each histogram. Figure 2. (a) Genetic similarity-based unweighted pair group method with an arithmetic mean (UPGMA) dendrogram of 71 parental lines calculated using the Jaccard coefficient. Bootstrap estimates ≥30% are reported next to the nodes (red and blue branches indicate the two clusters identified). (b) Principal coordinate analysis (PCoA). The 71 samples are shown in red or blue Figure 2. (a) Genetic similarity-based unweighted pair group method with an arithmetic mean (UPGMA) dendrogram of 71 parental lines calculated using the Jaccard coeﬃcient. Bootstrap estimates ≥30% are reported next to the nodes (red and blue branches indicate the two clusters identiﬁed). (b) Principal coordinate analysis (PCoA). The 71 samples are shown in red or blue according to the clustering shown in the UPGMA tree. (c) The population genetic structure of the 71 lines as estimated by STRUCTURE. Each sample is represented by a vertical histogram partitioned into K = 2 coloured segments (red or blue, in accordance with (a,b)) representing the estimated membership. The proportion of subgroup membership (%) is reported on the ordinate axis, and the identiﬁcation number of each accession is reported below each histogram. The mean observed homozygosity was 82%, with a minimum value of 69% and a maximum of 100%. 2.4. Genotyping and Data Analysis = (No F1 × 100)/(No Tot −No U.G.) (2 (2) where “No F1” is the number of hybrid individuals, “No Tot” is the number of all individuals in a progeny population (No tot = No F1 + No U.G. + No SP) and “No U.G.” is the number of unexpected genotypes deriving from unplanned crosses. Finally, 940 samples from the 47 F3 populations were genotyped using the previously-described panel of SSR markers. The POPGENE software [27] was used to compute the mean values of observed homozygosity for each population (3), where n is the total number of samples). In addition, the median of genetic similarity between the 47 lines was calculated using Rohlf’s coeﬃcient, which was designed for codominant molecular markers [28,29]. Comparison of genetic similarity among ten selected populations was instead calculated using the Jaccard coeﬃcient, in accordance with the literature [30]. Genetic similarity matrices were generated in the NTSYS software [24]. Ho = X n Ho/n (3) (3) 6 of 12 (3) Genes 2019, 10, 916 3.1. Parental Lines 3.1. Parental Lines It is worth noting that 19 of the 71 parental lines (27%) had observed homozygosity values greater than 90%, while 30 of the 71 (42%) had a medium-high observed homozygosity (Ho) between Genes 2019, 10, 916 were all the 0 hybridization 1 7 of 12 81% and 90%. Fourteen of the 71 parental lines (20%) had observed homozygosity ranging from 71% to 80%, and only 8 individuals had values lower than 70% (Figure 3a and Figure S1). 46 individuals (5% of the total) had a unexpected genotypes (U.G.) compared with their putative 3 parents (Table S3). 4 Figure 3. (a) Observed homozygosity of 71 lettuce parental individuals belonging to as many pure lines. (b) Histogram of discriminating loci in 89 cross combinations (in percentages). (c) Histogram of Figure 3. (a) Observed homozygosity of 71 lettuce parental individuals belonging to as many pure lines. (b) Histogram of discriminating loci in 89 cross combinations (in percentages). (c) Histogram of the percentages of pollination success in 89 programmed lettuce crosses. Figure 3. (a) Observed homozygosity of 71 lettuce parental individuals belonging to as many pure lines. (b) Histogram of discriminating loci in 89 cross combinations (in percentages). (c) Histogram of Figure 3. (a) Observed homozygosity of 71 lettuce parental individuals belonging to as many pure lines. (b) Histogram of discriminating loci in 89 cross combinations (in percentages). (c) Histogram of the percentages of pollination success in 89 programmed lettuce crosses. the percentages of pollinatio 8 3.2. Determination of Hybridity 9 Using a combination of genotypic and phenotypic data, 89 cross combinations were planned (Table S3). Before proceeding, we also checked the availability of informative loci able to distinguish between oﬀspring resulting from out-cross and those obtained by accidental self-pollination. Screening identiﬁed 1 discriminant locus in 16% of cases, 2 informative loci in 36% of cases, and 3 to 7 informative molecular markers in 48% of the crosses (Figure 3b). The three most informative loci were Lsat3, Lsat7, and Lsat6, while Lsat4 and Lsat13 were monomorphic in almost all parental groups. It is worth noting that the Lsat8 marker was in a heterozygous state in all but four parental genotypes (7, 45, 58, and 60). We were able to take advantage of these informative loci to calculate the success rate of each cross. In 30% of manual pollinations (27 out of 89), a success rate of 100% was achieved (i.e., all the oﬀspring were hybrids), whereas in 18% of crosses (16 out of 89), the S.C. ranged from 71% to 90%. A hybridity rate ﬂuctuating between 51% and 70% was reported in 15% of cases (13 out of 89), while 26% of the crosses produced fewer than 50% hybrids each. Finally, in only 7% of crosses (6 out of 89) were all the oﬀspring the result of self-pollination (Figure 3c and Table S3). Overall, the mean hybridization rate (the average number of hybrids per crosses) was 68 ± 33%, and out of a total of 871 individuals, 602 (69%) were hybrids, and 556 were derived from programmed crosses. The remaining 46 individuals (5% of the total) had a unexpected genotypes (U.G.) compared with their putative parents (Table S3). 3.3. Lettuce Breeding Populations The 47 F3 experimental lines were genotyped using the same set of 16 SSR loci as for the previous analyses. The homozygosity estimates of all samples (940) ranged from 67% to 93% (Figure 4a). Ten experimental populations had a median observed homozygosity ≥90%. Outliers—with homozygosity values consistently deviating from the median—were present in only three experimental populations (11, 14, and 32). The median genetic similarity observed within each line was always greater than 90% (Figure 4b), and 37 experimental populations had a median genetic similarity ≥95%. Outliers were present in 21 of the 47 lines (Figure 4b). After assembling the data, we found 10 breeding populations, belonging to butterhead type (Table S4), to have Ho values ≥90%, and a median genetic similarity ≥95%; the box-plots of these populations are labelled in red in Figure 4a,b. Finally, in the genetic similarity matrix calculated from all pairwise comparisons of these ten populations, the Jaccard’s index ranged from 44% ± 3% (between populations 3 and 18) to 96 ± 5% (between populations 45 and 47, Figure S3). Moreover, the populations called 45 and 47 were constituted starting from the same parents (2 × 6, Table S4). 8 of 12 ver, the ) 8 of 12 ver, the ) Genes 2019, 10, 916 (between popul l l Figure 4. Statistics relating to the observed homozygosity and genetic similarity among lines. (a) Box- plot of the median observed homozygosity (in percentages) in each of the 47 populations. The red dotted line represents the homozygosity threshold set at 90%. (b) Box plot of the median genetic similarity in each experimental population (in percentages). The red dotted line represents the genetic similarity threshold set at 95%. The red box-plots represent the ten best experimental populations (observed homozygosity ≥90% and genetic similarity values ≥95%). The second and third quartiles are marked inside the square and are divided by a bold bar (median). Dots show outlier samples. Figure 4. Statistics relating to the observed homozygosity and genetic similarity among lines. (a) Box-plot of the median observed homozygosity (in percentages) in each of the 47 populations. The red dotted line represents the homozygosity threshold set at 90%. (b) Box plot of the median genetic similarity in each experimental population (in percentages). The red dotted line represents the genetic similarity threshold set at 95%. The red box-plots represent the ten best experimental populations (observed homozygosity ≥90% and genetic similarity values ≥95%). 3.3. Lettuce Breeding Populations The second and third quartiles are marked inside the square and are divided by a bold bar (median). Dots show outlier samples. Di i Figure 4. Statistics relating to the observed homozygosity and genetic similarity among lines. (a) Box- plot of the median observed homozygosity (in percentages) in each of the 47 populations. The red dotted line represents the homozygosity threshold set at 90%. (b) Box plot of the median genetic similarity in each experimental population (in percentages). The red dotted line represents the genetic similarity threshold set at 95%. The red box-plots represent the ten best experimental populations (observed homozygosity ≥90% and genetic similarity values ≥95%). The second and third quartiles are marked inside the square and are divided by a bold bar (median). Dots show outlier samples. Figure 4. Statistics relating to the observed homozygosity and genetic similarity among lines. (a) Box-plot of the median observed homozygosity (in percentages) in each of the 47 populations. The red dotted line represents the homozygosity threshold set at 90%. (b) Box plot of the median genetic similarity in each experimental population (in percentages). The red dotted line represents the genetic similarity threshold set at 95%. The red box-plots represent the ten best experimental populations (observed homozygosity ≥90% and genetic similarity values ≥95%). The second and third quartiles are marked inside the square and are divided by a bold bar (median). Dots show outlier samples. 4. Discussion The last decade has seen major advances in the acquisition of knowledge concerning the genetics of lettuce and, in particular, the development of molecular markers [1,11,21]. This has facilitated marker-assisted selection programmes, especially those aimed at countering the onset of new diseases. For example, several studies have dealt with identifying the QTLs associated with biotic and abiotic stress resistance [17,31,32]. Molecular markers have also been extensively used to assess genetic variation and relationships in lettuce germplasm [19] and to identify possible duplicate varieties [33]. However, although the beneﬁts derived from exploitation of these molecular tools have also been discussed in marker-assisted breeding programmes [34] and demonstrated in several species [7,35], there are only a few studies on this type in lettuce [20]. The aim of our work, therefore, was to integrate conventional and biotechnological methods in three diﬀerent steps of a breeding programme to show that this strategy is also eﬀective in L. sativa (Figure 1). This is of pivotal importance if we consider the economic impact of lettuce (the world production of lettuce and chicory in 2017 was 26.8 million tons [36]) and the need to regularly develop new varieties. Commercial lettuce varieties are usually characterised by pure lines due to the autogamous nature of this species. In order to introduce variability, manual pollination is usually carried out to cross genetically stable parent lines with agronomic traits of interest. Progeny selection is a crucial step, but despite the eﬃciency of some emasculation and hand pollination methods developed over the years [2], a major problem—distinguishing unequivocally and rapidly F1 individuals from self-pollinated progeny—still remains. The use of molecular assays to quickly and accurately screen progeny makes it possible to overcome most of the conventional breeding limits in this species. p g y p g p In this context, our SSR-based analysis has (i) facilitated selection of the best parents to cross in order to maximise the variability of the progeny both within the same cultivar type and among Genes 2019, 10, 916 9 of 12 them, (ii) allowed accurate evaluation of the resulting oﬀspring, and (iii) sped up the screening of experimental F3 lines for their stability and uniformity. The ﬁrst part of our work focused on pre-screening 71 parental lines for crossing with the aim of maximising the gains obtained from each out-pollination within cultivar type and, in some cases, among them. 4. Discussion As expected, the similarity matrix and the unweighted pair group method with an arithmetic mean (UPGMA) dendrogram showed varying levels of similarity among the diﬀerent parental genotypes. Parental germplasm appeared to divide into two diﬀerent groups, as revealed by the Principal Coordinates Analysis (PCoA) results and particularly by the genetic structure analysis. However, samples did not separate in UPGMA tree and PCoA according to the cultivar type, but we may assume that increasing the number of markers it could be possible to clarify this clustering. Although 53 parental lines were found to be fully distinguishable, with similarity values ranging from 53% to 98% and characterised by a unique genotypic proﬁle, it was impossible to identify unequivocally the remaining 18. This is not surprising if we consider that some of the parental lines were closely related. We may speculate that increasing the number of SSRs would allow us to address these remaining issues. Given the aim of this study, these data were useful to avoid crosses between parents with 100% similarity. To introduce variability according to the phenotype and the lowest similarity values, we carried out 89 crossing combinations. Another aspect that needs to be considered when planning crosses is the stability of the parental line in terms of homozygosity. In our study, the median observed homozygosity of the parental lines was lower than expected (82%), especially in light of the strictly autogamous reproductive system of lettuce [37]. Overall, the fact that only one individual in four had homozygosity values greater than 90% showed that some of these lines were not entirely stable. However, it must be borne in mind that, although the observed homozygosity was not optimal, some of these lines, experimental lines, were chosen to produce F1 partly because they displayed resistance to multiple pathogens and had interesting phenotypic traits. Before proceeding with hand pollination, in order to distinguish between F1 individuals resulting from cross-pollination and those resulting from self-pollination, we ﬁrst examined the informative loci among the parental lines used in the crosses. Only homozygous loci for diﬀerent alleles in parental lines were considered informative. Our analysis showed at least 2 informative loci in 84% of the programmed crosses. It is worth pointing out that restricting the analysis to the informative loci brought us considerable savings in terms of time and costs. 5. Conclusions The results of this study demonstrate the advantages of mutual integration of traditional and biotechnological methods and show the added value that molecular markers can give to breeding programmes. We used microsatellite markers in three diﬀerent steps of a conventional lettuce breeding program (see Figure 1) and demonstrated, ﬁrstly, the eﬃciency of SSR markers not only in selecting the best parental plants for crossing based on their observed homozygosity and dissimilarity values, but also in screening the resulting F1 progeny to distinguish between the oﬀspring resulting from cross-pollination and those resulting from self-pollination. Furthermore, using the same SSR panel, we were able to act downstream of the breeding scheme to assess the uniformity of some pre-commercial cultivars. Our molecular assay could therefore also be used by seed ﬁrms to assess newly developed varieties for distinctiveness, uniformity and stability (DUS test), three major requirements for registering plant materials [6]. Finally, molecular characterisation of a new variety could also be used to register it in national or international varietal catalogues. In fact, the genotype or molecular proﬁle of a registered variety can be crucial in solving cases of fraudulent practices, and in curbing plagiarism and unfair free-riding on the original plant breeder’s time and investment [30]. Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4425/10/11/916/s1: Figure S1: Pairwise genetic similarity matrix of the 71 individuals analysed (in percentages) based on Rohlf’s genetic similarity coeﬃcient. High genetic similarity values are labelled in green, the low values in red, and intermediate values are coloured on a scale from green to red. The observed homozygosity values of the 71 putative parental lines are reported to the left of each ID name. Figure S2: Deﬁnition of the subgroup number of parental lines based on the SSR marker dataset. Mean ∆K is calculated as \|L” (K)\|/(SD(L(K)), following Evanno et al. [23]. The blue line represents the ∆K values; Figure S3: Pairwise genetic similarity matrix of ten selected populations (in percentages) based on the Jaccard coeﬃcient. The high genetic similarity values are labelled in green, the low values in red, and intermediate values are coloured on a scale from green to red; Table S1: Lettuce parental lines information, including ID of accessions, cultivar type of materials and subpopulation classiﬁcation based on STRUCTURE analysis (1 = blue and 2 = red).; Table S2: SSR primer tails and dyes. 4. Discussion Overall, the molecular determination of hybridity was successful: F1 individuals represented at least 51% of the oﬀspring in 67% of the manual crosses, and 100% of the oﬀspring in 30% of the crosses (100% success rate), in agreement with the estimates originally reported by the developer of the pollination technique [2]. Unexpected genotypes (U.G.) were identiﬁed in 5% of the individual progeny. In these cases, the progeny genotypes appeared to diverge from what would be expected given the parents. This percentage is consistent with the spontaneous or undesired occurrences of cross-pollination (1–6%) reported in the literature for this species [3], mainly due to pollinator insects. However, we cannot exclude human error during manual pollination or seed collection. Finally, at an advanced step of the breeding programme, we genetically assessed 47 diﬀerent experimental F3 populations (940 samples), previously selected for their morpho-phenotypic traits and resistances of interest (Figure 1). Interestingly, the ﬁndings in terms of both homozygosity and intra-line similarity were very good. This would suggest that in strictly autogamous species, such as lettuce, three cycles of self-pollination may already be suﬃcient to reach desired outcomes in terms of genetic uniformity and homozygosity. This also conﬁrms that the use of molecular markers could speed up the process by making it possible to select the best individuals on the basis of their genotype, thereby reducing the number of generations needed to develop new varieties. The ten experimental populations with the highest homozygosity estimates (≥90%) and the highest intra-genetic similarity values (≥95%) were considered suitable for pre-commercial trials (red box plot, Figure 4). However, a pairwise comparison of two of them (identiﬁed as 45 and 47) showed them to be genetically too similar (96% ± 5% genetic similarity, Figure S3), in agreement with phenotypic data and 10 of 12 10 of 12 Genes 2019, 10, 916 their common origin (Table S4), to be registered and marketed as distinct varieties. According to the most recent guidelines concerning the protection of new plant varieties, the similarity threshold to deﬁne two lettuce varieties as distinct is set at 96% [30]. The next step will be to integrate molecular data and morphological observations in order to the select the best genotypes (positive selection) for evaluation as pre-commercial varieties. 4. Discussion In particular, the eligible genotypes will be self-pollinated to multiply the seed so that their agronomic performance can be compared in diﬀerent locations and periods of the year, and with the best commercial varieties already on the market. y y For the remaining experimental populations (white box blot, Figure 4), an attempt could be made to increase their genetic uniformity through negative selection to remove the most genetically divergent individuals (i.e., outlier samples). Moreover, if necessary, the remaining genotypes can undergo a further selﬁng cycle aimed at reaching optimum values of homozygosity. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Wang, S.; Wang, B.; Liu, J.; Ren, J.; Huang, X.; Zhou, G.; Wang, A. 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Conclusions List of the primer tails used with their sequences and corresponding dyes; Table S3: Lettuce plant material information, including ID of accessions used in the crosses, total number of plants per programmed cross, number of informative marker loci, hybrid plants, selfed plants and unexpected genotypes, and the mean hybridisation values (in percentages) for all the programmed crosses; Table S4: Information about ten F3 selected populations, including ID of parental lines used in the crosses. Author Contributions: Conceptualization, F.P. and G.B.; formal analysis, A.P. and G.G.; investigation, A.P. and G.G.; methodology, F.P. and G.B.; resources, A.P.; software, A.P. and G.G.; supervision, G.B.; Writing—Original Draft, A.P. and F.P.; Writing—Review and Editing, F.P. and G.B. Author Contributions: Conceptualization, F.P. and G.B.; formal analysis, A.P. and G.G.; investigation, A.P. and G.G.; methodology, F.P. and G.B.; resources, A.P.; software, A.P. and G.G.; supervision, G.B.; Writing—Original Draft, A.P. and F.P.; Writing—Review and Editing, F.P. and G.B. Funding: This project was ﬁnancially supported by Blumen Group SpA (Bologna, Italy), within a Research Contract stipulated with DAFNAE, University of Padova (Italy): “Development of new varieties in horticultural species using molecular marker-assisted breeding methods” PI: Gianni Barcaccia. Acknowledgments: This research was carried out in partial fulﬁllment of the Ph.D. Program of Alice Patella by taking advantage of a Doctoral Research Fellowship provided by Blumen Group SpA (Bologna, Italy). Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 11 of 12 11 of 12 Genes 2019, 10, 916 1. Simko, I. 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https://openalex.org/W2677601691	https://www.shs-conferences.org/10.1051/shsconf/20173501022/pdf	English	null	A mechanism for encouraging active consumers to optimize the operations of power supply systems	SHS web of conferences	2,017	cc-by	4,869	1 Introduction constraints binding these two variables by natural requirements imposed on the values of the consumer utility functions. Consideration is given to the interaction among electricity consumers and an electric power retailer in which each consumer tries to reduce the cost of electricity by redistributing (shifting) its demand (or load) between peak hours and low-demanded ones, whereas the power retailer tries to reduce its expenses associated with storing electricity, especially, at peak hours [1, 2]. We propose to analyze such an interaction as a game in which the retailer’s goal is to maximize its profit, and the consumer’s goal is to maximize its utility. The utility function of each consumer is described proceeding from the form of the loss function of the consumer, and one of our goals in the submitted paper consists of finding how to describe this load function mathematically. However, the major goal is to develop an economic mechanism that would help the power retailer (considered as a monopoly in the marketplace) maximize its profit based upon the known loads of all the consumers. We consider a finite number of different types of the so-called “active” (load-controlled) consumers based upon their incentives to optimize their loads. Each type of the incentives (and, consequently, consumers) is defined by a utility function of the consumer, which is the function of the hour load volumes within a 24-hour segment. We offer an alternative utility function to describe the behaviour of a consumer who is not an “active” one (since this consumer does not optimize the consumption of electricity by means of shifting its loads from the peak hours to low-cost ones). We formulate the problem of finding an optimal strategy for the power retailer as the one of maximizing the profit that the retailer can make with respect to each type of the “active” consumers on the set of possible loads and tariffs that satisfy a set of The use of market mechanisms and development of information and communication technologies encourage active behaviour of another participant of the electricity supply process, i.e. power supply company, as well. The company becomes capable of encouraging consumers to actively manage their electricity consumption [3-8] by establishing price options in real time. The interaction between the power supply company and consumers requires coordination of their operating conditions. A mechanism for encouraging active consumers to optimize the operations of power supply systems 1Melentiev Energy Systems Institute of SB RAS, Irkutsk, Russia 2National Research Irkutsk State Technical University, Irkutsk, Russia Abstract. We propose to analyze an interaction of power supply company and consumers as a game in which the retailer’s goal is to maximize its profit, and the consumer’s goal is to maximize its utility. The utility function of each consumer is described proceeding from the form of the loss function of the consumer, and one of our goals in the submitted paper is to find how to describe this load function mathematically. We want to develop an economic mechanism that help the power retailer to find an optimal strategy on the set of possible loads and tariffs. They should satisfy a set of constraints binding these two variables by natural requirements imposed on the values of the consumer utility functions. Corresponding author: ayzenberg.nata@gmail.com © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). , 01022 (2017) SHS Web of Conferences 35 ICIE-2017 , 01022 (2017) SHS Web of Conferences 35 ICIE-2017 DOI: 10.1051/ 73501022 shsconf/201 © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). 1 Introduction Until now the potentialities of the coordination have been studied on the basis of mathematical programming methods, including two-stage stochastic programming [8, 9], a multi-agent [10-14] and a game-theoretic [9, 10, 12-14] approaches. The behaviour and strategies of load-controlled consumers have been studied by many researchers in the field. The authors of [15] study one such problem in which for any consumer of the power retailer of electricity, a disclosure of any information on its load is not mandatory is considered, and the power retailer tries to offer stimulating tariffs to all the consumers while even determining sources of saving for the consumers. Situations in which the prices for electricity are determined in advance so that each consumer actively changes the load in order to reduce its expenses as much as possible are considered in [12, 13]. Variants of a two- level formulation of the problem in which the interaction in considered for a set of vertically integrated legal entities such as a generating company, a retailer, a network and a consumer, which is represented in an aggregate form, without division into types, are presented in [5]. In some statements of the problem, consumers are allowed to supply any electricity surplus , 01022 (2017) SHS Web of Conferences 35 ICIE-2017 , 01022 (2017) SHS Web of Conferences 35 DOI: 10.1051/ 73501022 shsconf/201 back to the network [16]. There are statements dealing with an additional generation of electricity, when consumers can only shift load from the peak time via changing their production process, in particular, by using electricity storage systems or by automating the production process [4]. The number of -type consumer is m and K m . The power supply company appoints the tariff for -type of consumer tR using the total load m k kt q 1 and its cost function m k kt q C 1 . The cost function is convex with respect to m k kt q 1 . The main purpose of the power supply company is to maximize profits of tariffs tR : The subject of consideration in the present paper differs from those in the enumerated studies. 2 The Model The variables are the load of the individual consumer tk q and the tariffs value tR which is offered by the power supply company. We consider the example of the two types of consumer: with full rationality and with bounded rationality. Let describe utility function of - type consumers. . , ,1 ,0 , max , 1 K k q R q u q U kt q T t kt k The function of utility usually is monotonically increasing and concave. It should be performed additional property of Spence-Mirrlees: 1 Introduction That is, we consider consumers of several types each having different incentives to optimize their load, and we show how to determine the tariffs harmonized for both fully rational and bounded rational consumers. Here, we consider a customer to be fully rational if it does not vary its load, whereas we consider a customer to be bounded rational if it may change its load curve by shifting part of its load during peak hours to low-demand ones. The latter is offered a tariff stimulating load optimization. Standard solution to the problem of profit maximization of an electricity supply organization under the presence of asymmetric information on the agent type can lead to the formation of conditions for the adverse selection. We form constraints to determine the contracts (tariffs) that make it possible to search for a separating equilibrium where each type of consumers selects “their” tariff and adjusts their consumption as is necessary for the system. The research formulates the incentive rationality constraints and incentive compatibility constraints for the time-of-use and two-rate tariffs. function is convex with respect to m k kt q 1 . The main purpose of the power supply company is to maximize profits of tariffs tR : .0 , max 1 1 R q C R R T t m k kt We can decide the problem of select the tariff menu provided optimization the load curve through the coordination of interests of all stakeholders (1) and (2). We will update the form of utility functions and rewrite a problem through the theory of contracts. The problem (1) is rewritten in terms of the conditions of participation and of individual rationality constraints for consumer types, which mean that the selection of “their” tariff proves more beneficial than the selection of a tariff of another consumer type. Moreover, we will assume that there is always some basic tariff which determines some alternative utility of k -th consumer - k U . This alternative utility will be derived by a consumer without participating in the selection of an optimal consumption pattern. 2 The Model There are a finite number of different types of the so- called “active” (load-controlled) consumers who have the ability to adjust own load, and consumer who does not optimize the electricity consumption by means of shifting its loads. is the set of N types of consumers . There are K consumers in model. Each consumer derives some utility from receiving a certain amount of electricity. Denote this utility through the function tk k q u where the load at a certain t -zone of the day is tk q , 0 tk q . tR is a tariff value at hour t , T t ,1 for type . The problem of each k consumer is the maximizing of gains from electricity buying with respect to tk q , K k ,1 : p Then the general problem can be formulated as follows. .0 ,0 ;0 ,0 , , ]; ,1[ , , ; ], ,1[ , ; max 1 1 1 1 1 R R q q K j k R q u R q u m k U R q u q C R kt kt T t T t kt k kt k T t k kt k q R T t m k kt kt ; max 1 1 q C R q R T t m k kt kt .0 ,0 ;0 ,0 , , R R q q kt kt The variables are the load of the individual consumer tk q and the tariffs value tR which is offered by the power supply company. We consider the example of the two types of consumer: with full rationality and with bounded rationality. Let describe utility function of - type consumers. 2.1 The determination of optimal tariff for the two types of consumers II j j II I j j III q T x q T U 2 2 2 x f x q T III j j III ! $ 2 where peak A 1 is entrance fee or the price of the maximum power, 1 tT is the tariff value at hour t , 24 ,1 t . If the consumer selects the tariff 1 R and optimize the load, then she wants the expenses less than the costs for her consumption unchanged. Then the condition for the consumer’s participation with full rationality is U R 1 . where peak A 1 is entrance fee or the price of the maximum power, 1 tT is the tariff value at hour t , 24 ,1 t . If the consumer selects the tariff 1 R and optimize the load, then she wants the expenses less than the costs for her consumption unchanged. Then the condition for the consumer’s participation with full rationality is U R 1 . Consumer determines the load to be shifted to maximize her payoff 2 U . For example ' $ ( / 1 log x x f ) ) The power supply company use the possibility of withdrawal the consumer surplus as much as possible. Therefore, we get that power supply company increases the tariff (under the entrance fee) until the condition of participation allows. Thus the consumer load does not change because of her rationality. The power supply company use the possibility of withdrawal the consumer surplus as much as possible. Each parameter can be interpreted in the following way. Parameter ' (asymptote) is a maximum possible load that can be shifted by the consumer from the peak time. Parameter ( controls the level of costs expressed in some money equivalent. Here this parameter is estimated on the basis of empiric data on the possibilities of load change for the considered categories of consumers. Let us take for an example the function which can describe potential consumer expenses caused by the load shift. The measurement units can be represented by money units, but here we assume that these units are an equivalent of the consumer efforts to change her load curves. 2.1 The determination of optimal tariff for the two types of consumers The base consumer problem is to maximize the utility: It means that condition of participation for non- active participant fulfils as equality in the problem (3). II. Now let us write the utility function for consumer with bounded rationality. The costs of this consumer are defined via differentiated tariff. Let 2 jT is the tariff of j- th time zone, " # III II I j , , .Then the differentiated tariff is $ ! $ ! $ III j j III II j j II I j j I q T q T q T R 2 2 2 2 x U max 2 Let's formulate the problem of maximizing the consumer profits when the load shifts from a time zone to another one. The retail price in the spot market is S tP , where t defines the purchase zone; B tP is the price in the balancing market; x is a potential scope of load optimizing by consumer (here we consider the scope of load shift from the peak time to the night time), 0 x . Then the payoff of the power supply company from the load shift can be written in the form The solution is x f ' % . Let the function of costs for power supply company be a concave function of x . To find the Nash equilibrium and to match the interests of all the stakeholders, it is necessary to find a point such that the maximum profit for the power supply company and the maximum payoff for the consumer are reached at the same time. ; max , % % x S x f ' % & .0 ,0 % x ; max , % % x S x x P P x S S I B III $ % $ %, & x f ' % & .0 ,0 % x where % is step of tariff tT which are established by the retail company at different hours of the day. For simplicity we can assume that III I T T % . Here we represent a cost function of power shortage in a simplified form. 2.1 The determination of optimal tariff for the two types of consumers I. At first the problem of utility maximization for the participation of the rational consumer 1 is described. The constraints that include the conditions of a positive utility in selecting the tariff are the conditions of participation. In our research we believe that such a 0 ) , ( ,0 ) , ( ,0 ) , ( 2 2 2 dq d q U d dq q U d dq q dU 2 DOI: 10.1051/ 73501022 shsconf/201 , 01022 (2017) SHS Web of Conferences 35 , 01022 (2017) SHS Web of Conferences 35 ICIE-2017 price difference in the balancing and spot markets. There can be additional costs not related to the price difference, to eliminate the shortage. consumer can use a two-part tariff. In this tariff is assumed that the electricity unit price corresponds to the marginal cost of a electricity unit, and fixed costs are covered through an entrance fee (or fee peak power). Should take into account that with such scheme due to the absence of constraints the monopolist can confiscate the full consumer surplus, the value of which may exceed the fixed costs of power supply company. The government should regulate the entrance fee. We assume that the consumer costs related to the load shift is the function of the load x f , 2 C x f . Here, we suppose that some load will be shifted by consumer without any special efforts. For example, this may concern the use of washing and dishwashing machines. At the same time, there is a load whose shift can cause greater inconveniences, for example, a shift of the food cooking time or additional installation of energy saving equipment. The function x f reflects an extent to which the consumer is ready to such costs. The total payoff of the consumer will be represented as The utility function of the consumer with the full rationality is written via consumer’s loss function or as the tariff 1 1 R U of 1 -type offered by the power supply company. 1 R is two-part tariff. ! 24 1 t 1 1 1 1 t t peak q T A R $ $ % ! Using (4) we have that S III B I P P ( % . Using (4) we have that S III B I P P ( % . 2 Example: the search of optimal power supply company strategy in the case of two types of consumers 24 1 t 2 2 2 t t peak q T A U , + ! 24 1 t 2 2 2 t t peak q T A U , , It’s easy to prove by the methods of the contract theory that the restriction (7) is active. Here, we do not consider the case, when consumers with bounded rationality choose a two-part tariff, and at the same time shift their loads, since the statement of the problem in this situation changes. Our objective is just to encourage consumers with bounded rationality to optimize their load curves. If any consumer can optimize its load curve without an additional action from the power supply company, it refers to the rational type 1 . It’s easy to prove by the methods of the contract theory that the restriction (7) is active. Here, we do not consider the case, when consumers with bounded rationality choose a two-part tariff, and at the same time shift their loads, since the statement of the problem in this situation changes. Our objective is just to encourage consumers with bounded rationality to optimize their load curves. If any consumer can optimize its load curve without an additional action from the power supply company, it refers to the rational type 1 . Table 1 represents the results of the application of tariffs imposed by the state (see [17], the starting point). These results define the company's income, the consumer spending and the possible shortage of power. All consumers choose identical tariff (indicated in bold), this is a situation of adverse selection with high shortage. The function of the power supply company profit is concave. Table 2. The results of the interaction of the power supply company and three customers after optimization of tariffs. Table 2. The results of the interaction of the power supply company and three customers after optimization of tariffs. Consumer spending, rub/day 1 2 2 2 1 1 R 29744,4 16777,4 13027,5 2 R 28476,2 16361,2 14265,7 Shortage of power, kWt 340 Company's income, rub/day 10090.3 1 2 1 2 1 , ,max , , % % ! ! 2 Example: the search of optimal power supply company strategy in the case of two types of consumers Let an alternative utility is determined by means a basic linear (i.e., constant in each time of the day) tariff L T . We want that the consumer of rationality type will prefer two-part tariff, while the consumer of bounded rationality type will prefer differentiated tariff. Let us write the conditions of participation. The several types of consumers is considered in the example of the campus. The set of consumers forms the load curve of a power system. The consumers are hostels, educational buildings, student’s catering facilities, municipal health centre and prophylactic sanatorium. p p For the consumer of full rationality type: 24 1 1 1 t t L q T U (5) We consider the health centre and prophylactic sanatorium as the unique consumer of rationality type, consumer 1 , her load is not changed. She should choose a two-rate tariff 1 R with large payment for connection (power) and non-differentiated small payment per unit of electricity. Another group are consumers with bounded rationality, their loads are changed: 1 2 for hostels, educational buildings and students’ catering facilities; and ( 2 2 ) for cafe). They should choose a differentiated tariff 2 R per electricity unit at a zero rate for power. (5) For the consumer of bounded rationality type: For the consumer of bounded rationality type: 24 1 2 2 t t L q T U * * We already know that restriction (5) in the problem (3) is active for the consumer of full rationality type. The individual rationality constraints for the consumer of this type means that the choice of this tariff is more beneficial for the consumer, than choosing another types of consumer tariff. Table 1. The results of the interaction of the power supply company and three customers of power tariffs established by the state. Consumer spending, rub/day 1 2 2 2 1 1 R 29744.4 18847.8 13027.5 2 R 32418.9 19202.2 15326.7 Shortage of power, kWt 1065 Company's income, rub/day 8767,5 Table 1. The results of the interaction of the power supply company and three customers of power tariffs established by the state. $ } , , { 1 1 III II I t t t q T U & + ! 2.1 The determination of optimal tariff for the two types of consumers The function takes into account only the The conditions mean that the consumer will shift a certain load at different tariff difference % : 0 , max ' ' 1 1 % % $ % % $ % f f P P S III B I 3 , 01022 (2017) SHS Web of Conferences 35 DOI: 10.1051/ 73501022 shsconf/201 , 01022 (2017) SHS Web of Conferences 35 ICIE-2017 the equilibrium, i.e., the optimal tariffs. These tariffs match the interests of different types of consumers and suppliers. Using (4) we have that S III B I P P ( % . 2 Example: the search of optimal power supply company strategy in the case of two types of consumers 2. N. Aizenberg, E. Stashkevich, N. Voropai, Proceedings of the Russian Academy of Sciences. Power engineering, 3, 15-25 (2016) The proposed methodology of the tariff determination allows both categories of consumers interacting with a power retailer — fully rational (who do not vary their load) and bounded rational (who change their load curve by shifting part of peak load) — to optimize the HOHFWULFLW\ supply to them under tariffs stimulating their load optimization. The profit maximization problem to be solved by the power retailer under the presence of asymmetric information on the consumer type is formulated in the paper in such a manner that its set of constraints contains the incentive rationality constraints and those of incentive compatibility. This allows consumers of each type to select “their” tariffs and adjust their consumption as it is necessary for the system as a whole. 3. G.G. Grebenyuk, M.M. Soloviev, Remote Control, 5, 166-173, (2004) 4. I.O. Volkova, M.V. Gubko, E.A. Salnikova, Control Sciences, 6, 53-61 (2013) 5. E. Pettersen, A.B. Philpott, S.W. Wallace, Decision support systems, 40(3), 427-438 (2005) 6. N. Aizenberg, E. Stashkevich, Proc. Int. Conf. on Problems of Critical Infrastructures, 25-27 (2015) 7. K. Sharma, C. Bhattacharya, IEEE Trans. on Smart Grid, 6, 2, 795-807 (2015) 8. Proc. ICEE (2015) 9. N. Good, E. Karangelos, A. Navarro-Espinosa, P. Mancarella, IEEE Trans. on Smart Grid, 6(3), 2333- 2342 (2015) For an industrial area (the city of Irkutsk), different consumer types have been identified, and utility functions for all the consumer types have been proposed. The choice of these proposed functions has helped implement the system of stimuli for the load optimization (in the sense of shifting the load from peak hours to off-peak ones) by all the consumers with bounded rationality of their preferences. The equilibrium tariffs have been calculated, whereas the tariffs approved by the legislature have been tested. The (tested) inefficiency of the latter follows, in particular, from their unprofitability for the power retailer. Indeed, since as long as part of load is not shifted from peak hours to off-peak ones, the shortage of electricity becomes inevitable, and the retailer expenses associated with shorting electricity increase substantially. References 1. N.I. Voropai, Z.A. Styczynski, E.V. Kozlova, V.S. Stepanov, K.V. Suslov, Proceedings of the Russian Academy of Sciences. Power engineering, 1, 84-90 (2014) This work was supported Russian Foundation for Basic Research-06-00071. 2 Example: the search of optimal power supply company strategy in the case of two types of consumers R x x S q q C R R & - - where 2 1 , q q C is the total cost function of the power supply company for power purchase at the market and its delivery to a consumer. x S , . is the profitability from the possible shift to another time zone. The variables are the step values in the differentiated tariff and prices in the two-part tariff. Restrictions (5) and (8) are active, (7) run as strict inequalities, (6) is superfluous. Further, we are determined the type of consumers that can be interested in choosing “not their tariff”, then we solve the main problem (8) (see table 2). This way we By solving the problem of power supply company profit maximization (8) subject to constraints (4)-(7), we get 4 DOI: 10.1051/ 73501022 shsconf/201 , 01022 (2017) SHS Web of Conferences 35 , 01022 (2017) SHS Web of Conferences 35 ICIE-2017 find the conditions for the mixed equilibrium and for the equilibrium differentiating the types. find the conditions for the mixed equilibrium and for the equilibrium differentiating the types. 2 Example: the search of optimal power supply company strategy in the case of two types of consumers In contrast, the tariffs calculated on the basis of the proposed approach can make the above- mentioned system of stimuli for optimizing the consumer loads working, which reduces the probability of emerging shortages in electricity supply. 10. R. Olfati-Saber, J. Fax, Proceedings of the IEEE, 95(1), 215-233 (2007) 11. M.R. Sheikhi, Sh. Bahrami, A.M. Ranjbar, IEEE Trans. on Smart Grid, 6(2), 675-683 (2015) 12. A.H. Mohsenian-Rad, V.W. Wong, J. Jatskevich, R.Schober, A. Leon-Garcia, IEEE Trans. on Smart Grid, 1(3), 320-331 (2010) 13. H. Chen, Y. Li, R.H. Louie, B. Vucetic, Autonomous IEEE Trans. on Smart Grid, 5(4), 1744-1754 (2014) 14. J. Chai, Z. Chen, Y. Yang, Zhang, IEEE Trans. on Smart Grid, 5(2), 1340-1350 (2014) 15. Z.M. Fadlullah, D.M. Quan, N. Kato, I. Stojmenovic, IEEE Systems journal, 8(2) (2014) 16. V.S. Stepanov, K.V. Suslov, E.V. Kozlova, Prom. Energetika, 6, 2-7 (2013) 17. Resolution of the Government of the RU of 2012 no.442.http://www.sbyt.irkutskenergo.ru/qa/2103.ht 17. Resolution of the Government of the RU of 2012 no.442.http://www.sbyt.irkutskenergo.ru/qa/2103.ht ml 5
https://openalex.org/W3115529021	https://bmcvetres.biomedcentral.com/track/pdf/10.1186/s12917-020-02719-3	English	null	Development and evaluation of recombinant GRA8 protein for the serodiagnosis of Toxoplasma gondii infection in goats	BMC veterinary research	2,021	cc-by	6,461	Jirapattharasate et al. BMC Veterinary Research (2021) 17:27 https://doi.org/10.1186/s12917-020-02719-3 Jirapattharasate et al. BMC Veterinary Research (2021) 17:27 https://doi.org/10.1186/s12917-020-02719-3 Development and evaluation of recombinant GRA8 protein for the serodiagnosis of Toxoplasma gondii infection in goats Charoonluk Jirapattharasate1, Ruenruetai Udonsom2, Apichai Prachasuphap3, Kodcharad Jongpitisub3 and Panadda Dhepakson3 Abstract Background: The development of sensitive and specific methods for detecting Toxoplasma gondii infection is critical for preventing and controlling toxoplasmosis in humans and other animals. Recently, various recombinant proteins have been used in serological tests for diagnosing toxoplasmosis. The production of these antigens is associated with live tachyzoites obtained from cell cultures or laboratory animals for genomic extraction to amplify target genes. Synthetic genes have gained a key role in recombinant protein production. For the first time, we demonstrated the production of the recombinant protein of the T. gondii dense granular antigen 8 (TgGRA8) gene based on commercial gene synthesis. Recombinant TgGRA8 plasmids were successfully expressed in an Escherichia coli system. The recombinant protein was affinity-purified and characterized via sodium dodecyl sulfate- polyacrylamide gel electrophoresis and Western blotting. Furthermore, the diagnostic potential of the recombinant protein was assessed using 306 field serum samples from goats via indirect enzyme-linked immunosorbent assay (iELISA) and the latex agglutination test (LAT). Results: Western blotting using known positive serum samples from goats identified a single antigen at the expected molecular weight of TgGRA8 (27 kDa). iELISA illustrated that 15.40% of goat samples were positive for T. gondii-specific IgG antibodies. In addition, TgGRA8 provided high sensitivity and specificity, with significant concordance (91.83) and kappa values (0.69) compared with the results obtained using LAT. Conclusion: Our findings highlight the production of a recombinant protein from a synthetic TgGRA8 gene and the ability to detect T. gondii infection in field samples. The sensitivity and specificity of TgGRA8 demonstrated that this protein could be a good serological marker for detecting specific IgG in goat sera. Keywords: Toxoplasma gondii, GRA8, Serodiagnosis, Goat, Gene synthesis Correspondence: Charoonluk.jir@mahidol.edu * Correspondence: Charoonluk.jir@mahidol.edu 1Department of Preclinic and Applied Animal Science, Faculty of Veterinary Science, Mahidol University, 999 Phutthamonthon sai 4 Rd, Salaya, Nakhonpathom 73170, Thailand 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/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. * Correspondence: Charoonluk.jir@mahidol.edu 1Department of Preclinic and Applied Animal Science, Faculty of Veterinary Science, Mahidol University, 999 Phutthamonthon sai 4 Rd, Salaya, Nakhonpathom 73170, Thailand Full list of author information is available at the end of the article Results Construction of the recombinant TgGRA8 plasmids The 582-bp GRA8 gene was PCR-amplified from syn- thetic TgGRA8 (Fig. 1). The PCR product was purified and double digested with NdeI and AgeI. The digested product (25 ng/ml) was used for ligation. E. coli DH5α- competent cells were transformed with recombinant pET-21a vectors and cultured with 2XTY agar contain- ing ampicillin. Positive colonies were identified by col- ony PCR. Sequence analysis of the clone revealed 100% homology with the sequence of recombinant TgGRA8. Serological methods play a major role in the diagnosis of T. gondii infection in humans and animals [5, 6]. Sev- eral serological tests have been developed using either live tachyzoites or native soluble antigens; however, they are expensive, laborious, and nonspecific [7]. Recently, recombinant T. gondii antigens were identified as good candidates for replacing native antigens because they are easily produced in large volumes using standardized methods [8, 9]. Dense granule antigens (GRAs) of T. gondii are secreted in the parasitophorous vacuole (PV), and they are involved in survival and virulence of the parasite [10]. Several studies demonstrated the diagnos- tic potential of numerous GRAs such as GRA2 [11], GRA5 [12], GRA6 [13], and GRA7 [14, 15]. GRA8 is a 38-kDa praline-rich (24%) protein that is released from PVs shortly after invasion. GRA8 is a 269-amino acid polypeptide with a terminal signal peptide, three degen- erate proline-rich repeats in the central region, and a po- tential transmembrane domain near the carboxy- terminal region [16]. Previous studies used the recom- binant GRA8 protein in specific IgM and IgG enzyme- linked immunosorbent assay (ELISA) in humans [17– 19]. However, little information is available concerning the use of recombinant GRA8 protein-based ELISA for the serodiagnosis of T. gondii infection in animals. Fig. 1 Screening of the PCR product of the TgGRA8 gene. Lane M, 100-bp DNA ladder; Lane 1, 582-bp PCR product after digestion with NdeI and AgeI Regarding recombinant protein production in T. gon- dii, the mRNA of target genes is extracted from live tachyzoites and recombinant plasmid is transformed to bacteria for protein expression. However, the transfer of gene sequences between organisms may not be success- ful, leading to low level protein expression because of differences in codon usage [20, 21]. Recently, synthetic gene synthesis has been used to design and create genes without an existing DNA template [22]. Background template for recombinant protein production. Further- more, the purified protein was used in specific IgG indir- ect ELISA (iELISA) in the diagnosis of T. gondii infection using goat sera. The latex agglutination test (LAT) was used to validate the detection system in this study. g Toxoplasmosis is caused by the protozoan parasite Toxo- plasma gondii, and this infection is widespread in humans and animals, occurring in approximately 25– 30% of the human population [1]. Most people infected with T. gondii are asymptomatic; however, fatal enceph- alitis caused by this protozoan can be observed in im- munocompromised patients [2]. Infection in humans and animals, as the intermediate hosts, occurs mainly through the ingestion of raw or undercooked meat con- taining viable tissue cysts or through exposure to soil, food, or water contaminated with oocysts passed in the feces of infected cats or other felines [3]. Normally, farm animals display no clinical symptoms, although T. gondii infection may induce abortion, leading to reproductive losses in the livestock industry [4]. © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Jirapattharasate et al. BMC Veterinary Research (2021) 17:27 Page 2 of 9 Page 2 of 9 Confirmation of TgGRA8 protein The identity of protein expressed and purified recombin- ant protein was confirmed by mass spectrometry (MS) analysis. The partial sequence of TgGRA8 in this study shared 98.95–100% identities with database sequences (XP002369526, KFG46645, RQX68523 and AAD55381). Therefore, we confirmed that our expressed recombin- ant protein was T. gondii GRA8. Production of recombinant TgGRA8 was optimized by altering various incubation periods, and expression levels were analyzed by SDS-PAGE as shown in Fig. 2. A 27 kDa band was observed in the induced bacteria. Expres- sion of this protein increased up to 2 h after induction and remained constant after overnight. To confirm the protein expression, the induced bacteria exhibited a pro- tein expression band of 27 kDa in size after purification using anti-FLAG tag affinity resin (Fig. 3a). Results In addition, gene synthesis tools do not require access to a pathogen, thus preventing the exposure of research staff to harmful living parasites [23]. In this study, we used a synthesized T. gondii GRA8 gene (designated TgGRA8) as a DNA Fig. 1 Screening of the PCR product of the TgGRA8 gene. Lane M, 100-bp DNA ladder; Lane 1, 582-bp PCR product after digestion with NdeI and AgeI Jirapattharasate et al. BMC Veterinary Research (2021) 17:27 Page 3 of 9 Fig. 2 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis on the optimized expression of TgGRA8 in E. coli strain Rosetta (DE3), Coomassie blue stained. Lane M: protein molecular weight marker. Lane 1 to 3: pellet fractions of cells grown at 20 °C after induction with 1.0 mM IPTG (4,8 h and overnight). Lane 4 to 6: uninduced total cell lysate of E. coli strain Rosetta (DE3)-pET21a-TgGRA8 (4,8 h and overnight) Fig. 2 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis on the optimized expression of TgGRA8 in E. coli strain Rosetta (DE3), Coomassie blue stained. Lane M: protein molecular weight marker. Lane 1 to 3: pellet fractions of cells grown at 20 °C after induction with 1.0 mM IPTG (4,8 h and overnight). Lane 4 to 6: uninduced total cell lysate of E. coli strain Rosetta (DE3)-pET21a-TgGRA8 (4,8 h and overnight) Evaluation of the serodiagnostic potential of recombinant TgGRA8 by indirect ELISA (iELISA) The serodiagnotic potential of recombinant TgGRA8- iELISA was evaluated for its potential utility in sero- logical testing using known positive (N = 10) and nega- tive (N = 21) goat sera. The cut-off value was calculated as the average OD450 plus three standard deviations of standard T. gondii-negative control goat sera. The cut- off value for goats in this study was determined as 0.61 (Fig. 5). Base on recombinant TgGRA8-iELISA, 15.35% goat sera samples were positive for T. gondii-specific IgG antibodies. The purified protein was analyzed by Western blot- ting using peroxidase-conjugated anti-FLAG tag anti- body (GenScript, USA) diluted 1:1000 in blocking buffer. The result illustrated that the TgGRA8 fusion protein was specifically recognized by anti-FLAG tag antibody (Fig. 3b). However, the specific band size was slightly larger than the estimated size of 22.55 kDa (amino acids 24–217 plus the 2X FLAG tag). The concentration of the protein was measured as 1.26 mg/ml by BSA assay (Pierce Biotechnology, Inc., USA). The specific reactivity and purity of TgGRA8 was checked using known positive and negative serum samples from goats. Western blotting revealed that the TgGRA8 fusion protein was recognized by the known positive serum (Fig. 4). Comparison of iELISA and LAT The diagnostic performance of recombinant TgGRA8- iELISA was evaluated with reference to LAT [14]. The sensitivity and specificity of the recombinant protein and Jirapattharasate et al. BMC Veterinary Research (2021) 17:27 Page 4 of 9 Fig. 3 a Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of expression of recombinant Toxoplasma gondii dense granular antigen 8 (TgGRA8) protein. Lane M, protein molecular weight marker; Lane 1, the soluble recombinant TgGRA8 protein was purified using anti- FLAG tag affinity resin. b Western blot analysis of purified recombinant TgGRA8. Lane M, protein molecular weight marker; Lane 1, the purified 27-kDa TgGRA8 protein was detected using an anti-FLAG tag antibody Fig. 3 a Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of expression of recombinant Toxoplasma gondii dense granular antigen 8 (TgGRA8) protein. Lane M, protein molecular weight marker; Lane 1, the soluble recombinant TgGRA8 protein was purified using anti- FLAG tag affinity resin. b Western blot analysis of purified recombinant TgGRA8. Lane M, protein molecular weight marker; Lane 1, the purified 27-kDa TgGRA8 protein was detected using an anti-FLAG tag antibody kappa values at 95% confidence interval (95% CI) were calculated. The seropositivity rate of goat samples in LAT was 17.0%. The sensitivity and specificity of LAT for the recombinant protein were 71.1 and 96.0%, re- spectively. Substantial agreement between the two methods was indicated by κ = 0.69 (Table 1). natural gene sequences, including no or low expression, inclusion body formation, and protein inactivity, has been described [25]. To overcome these problems, we demonstrated the production of recombinant protein from a synthetic TgGRA8 gene and tested the immuno- diagnostic potential of the produced protein via iELISA. Although codon optimization was used to optimize and enhance protein expression in the present study, we failed to produce the recombinant protein using the full-length TgGRA8 gene. Previous studies described a transmem- brane region of the GRA8 gene encoding amino acids 223–242 using bioinformatic prediction [16] and reported that the region can affect host cell growth and decrease protein yield [26]. Therefore, we selected the specific re- gion of the TgGRA8 protein based on the prediction of transmembrane helices in proteins using an online pro- gram (http://www.cbs.dtu.dk/services/TMHMM/). After removing the transmembrane region, the gene fragment encoding amino acids 24–217 was used to express the protein, and a specific 27-kDa band was observed on SDS- PAGE. Our result was similar to that of Babaie et al. Comparison of iELISA and LAT [18], who designed and expressed a recombinant protein from Discussion Recently, recombinant DNA technology and synthetic DNA have played important roles in high-quality recom- binant antigenic protein production for the serological diagnosis of T. gondii infection. Several recombinant proteins have been produced and applied for the detec- tion of T. gondii infection. These proteins include rhop- try proteins, matrix proteins, microneme proteins, surface antigens, and GRAs [8]. Among them, the GRA proteins have been considered potential diagnostic anti- gens and have been used to differentiate the stages of in- fection [24]. Generally, the method of recombinant protein production requires cDNA extracted from live pathogens as a template to amplify target genes. How- ever, unsuccessful recombinant protein production using Jirapattharasate et al. BMC Veterinary Research (2021) 17:27 Page 5 of 9 Page 5 of 9 Jirapattharasate et al. BMC Veterinary Research Fig. 4 Western blot analysis. Purified proteins were separated via 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and then probed with known positive and negative goat sera. a Lane M, protein molecular weight marker; Lane 1, strong reactivity with known positive serum; b Lane M, protein molecular weight marker; Lane 1, result for negative serum protein-based ELISA has been used in seroprevalence studies of farm animals in Egypt [29] and Thailand [30]. To date, only one study describing the use of TgGRA8 together with recombinant GRA7 to detect specific IgG antibodies against the parasite was published in domestic turkeys [31]. The potential utility of recombinant TgGRA8 protein in serodiagnosis was assessed using known positive and negative goat sera. The result of Western blotting indi- cated that the protein is a potential marker for detecting T. gondii infection in goats. A previous immunochemical evaluation of TgGRA8 using ELISA recorded high re- activity for the recombinant protein using human sera [19], in line with the present result. A possible explan- ation for the high OD in this study could be the unspe- cific epitopes of this antigen in the amino-terminal region [32]. The infection rate in our study was lower than that of 27.9% in a previous report on goats in Satun province, Thailand [33]. The difference of the seroprevalence rate may be attributable to the use of different serological diagnosis techniques (iELISA and LAT) and different sampling areas. The sensitivity and specificity obtained using recombinant TgGRA8 in this study indicated that the recombinant protein could be used as an antigen for serological tests of T. gondii. Discussion However, the use of recom- binant proteins for the serodetection of animal toxoplas- mosis may be affected by the immune system in different animal species. Therefore, the antibody re- sponse in various animals and the epitope structures of recombinant TgGRA8 should be confirmed. Fig. 4 Western blot analysis. Purified proteins were separated via 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and then probed with known positive and negative goat sera. a Lane M, protein molecular weight marker; Lane 1, strong reactivity with known positive serum; b Lane M, protein molecular weight marker; Lane 1, result for negative serum Conclusion Our study produced a recombinant protein from a syn- thetic TgGRA8 gene. The sensitivity and specificity of TgGRA8 demonstrated that this protein could be a good serological marker for detecting specific IgG in goat sera. Commercial gene synthesis is an alternative tool to sup- port recombinant protein expression in the absence of pathogen access. the GRA8 gene fragment corresponding to amino acids 23–169. However, a difference in size between the appar- ent band and the calculated molecular weight of TgGRA8 (22.55 kDa) was observed in the present study. The pre- dicted protein encoded by the TgGRA8 gene featured high proline content (54 amino acids). The presence of ex- cessive proline residues in proteins causes structural rigid- ity in the primary sequence, thereby decreasing the electrophoretic mobility [16]. Regarding the yield of TgGRA8, our production gained lower yields than the method of Babaie and colleague [18]. Therefore, the at- tempt of using codon-optimisation DNA is not considered advantageous for recombinant GRA8 expression. Gene synthesis of TgGRA8 The complete GRA8 coding sequence (accession num- ber: TGME49_054720) was obtained from an online database (http://ToxoDB.org). The TgGRA8 sequence consists of 810 nucleotides that encode a 269-amino acid protein. A signal peptide (SPs) of GRA8 was determined using online program, SignalP 4.1 (http://www.cbs.dtu. dk/services/SignalP/). The results showed that small fragments of amino acids 1–23 were expressed as a sig- nal sequence. Therefore, encoding amino acid 24–269 was constructed and inserted into a pET-21a vector Numerous GRA proteins, both single and combina- tions of proteins, have been applied for the serodetection of animal toxoplasmosis. In cats, a single GRA7 recom- binant protein [27] and a mixture of recombinant GRA2, GRA6, GRA7, and GRA15 [28] were used to de- termine the prevalence of T. gondii infection in China and Japan, respectively. Moreover, recombinant GRA7 Jirapattharasate et al. BMC Veterinary Research (2021) 17:27 Page 6 of 9 Fig. 5 Antibody response to Toxoplasma gondii in field sera from goats using recombinant T. gondii dense granular antigen 8 protein-based indirect enzyme-linked immunosorbent assays Fig. 5 Antibody response to Toxoplasma gondii in field sera from goats using recombinant T. gondii dense granular antigen 8 protein-based indirect enzyme-linked immunosorbent assays using NdeI and XhoI as the cloning sites (General Bio- systems, USA). protein linker (GGGS) and 2X FLAG tag (DYKDDDDK DYKDDDDK) (General Biosystems, USA) and trans- formed into Escherichia coli DH5α-competent cells. Construction of recombinant TgGRA8 Ten colonies were selected and expanded in overnight cultures, and DNA was extracted using a QIAprep Spin Miniprep Kit (Qiagen, Germany). The insert of TgGRA8 in the purified plasmid was sequenced using a Dye Ter- minator Cycle Sequencing Kit (Applied Biosystems, USA) and the 3500xL genetic analyzer (Applied Biosys- tems). The TgGRA8 sequences were determined using Bioedit version 7.2.5 (Tom Hall Ibis Biosciences, USA). The potential transmembrane regions (TMs) of TgGRA8 were predicted by online server (http://www.cbs.dtu.dk/ services/TMHMM/). The encoding amino acids 218– 269 was transmembrane region. Therefore, only an anti- genic fragment of recombinant TgGRA8 encoding amino acids 24–217 was PCR-amplified (Fig. 6). The primers used for amplification of the sequence by PCR was T7 promoter-(FW), 5′-TAA TACG ACT CAC TAT AG-3′ (New England Biolabs, UK); and TgGRA8-RW, 5′-AGT acc ggt GGT GGC GGT TGC CGG CTG-3′. The reverse primer was designed to contain the AgeI re- striction site. PCR was performed using PCR Q5® High- Fidelity DNA Polymerase (New England Biolabs) using the following program: 98 °C for 1 min, followed by 30 cycles of 98 °C for 10 s, 58 °C for 20 s, and 72 °C for 20 s, and final extension at 72 °C for 2 min. Expression of TgGRA8 The recombinant TgGRA8 plasmids were transformed into E. coli strain Rosetta (DE3) cells and cultivated in 2XTY supplemented with 1% glucose and 200 ng/ml ampicillin at 37 °C with shaking at 200 rpm. E. coli carry- ing recombinant TgGRA8 was measured at an optimal density at 600 nm (OD600) of 0.5 and induced with isopropyl-β-D-thiogalactopyranoside at a final concen- tration of 1 mM 20 °C for various incubation periods (2, 4 and overnight) with shaking at 250 rpm. The induced bacteria were harvested via centrifugation at 4400×g for The PCR amplicon was digested using NdeI and AgeI. After digestion, the PCR product was ligated into the modified pET-21a vector harboring a C-terminal fusion Table 1 Comparison of LAT and TgGRA8 recombinant protein-based iELISA for the detection IgG antibodies against Toxoplasma gondii infection TgGRA8 iELISA LAT Sensitivity (95% CI) Specificity (95% CI) Kappa value Positive Negative Total Positive 37 10 47 71.17 96.06 0.69 Negative 15 244 259 (56.72–82.45) (92.65–97.98) Total 52 254 306 LAT Latex agglutination test, TgGRA8 T. gondii dense granular antigen 8, iELISA Indirect enzyme-linked immunosorbent assay, CI Confidence interval LAT and TgGRA8 recombinant protein-based iELISA for the detection IgG antibodies against Toxoplasma Table 1 Comparison of LAT and TgGRA8 recombinant protein-based iELISA for the detection IgG antibod gondii infection Jirapattharasate et al. BMC Veterinary Research (2021) 17:27 Page 7 of 9 Fig. 6 The complete nucleotide and amino acid sequence of T. gondii GRA8 (accession number: TGME49_054720). The expression region of GRA8 in this study is delineated by shading (encoding a 194-residual peptide) Fig. 6 The complete nucleotide and amino acid sequence of T. gondii GRA8 (accession number: TGME49_054720). The expression region of GRA8 in this study is delineated by shading (encoding a 194-residual peptide) centrifugation at 10,000×g for 5 min at 4 °C, and the concentration of the purified recombinant protein was assayed using both SDS-PAGE and a Coomassie protein assay reagent kit using BSA according to the manufac- turer’s protocol (Pierce Biotechnology, Inc., USA). 20 min at 4 °C, and the bacterial pellet was resuspended in 20 ml of pre-chilled lysis buffer (150 mM NaCl, 50 mM Tris-HCL [pH 9.5], 1% Triton X-100, 1 mM EDTA [pH 8.0], and 1% NP 40) and then incubated at 4 °C for 30 min. Expression of TgGRA8 After incubation, the bacterial cells were lysed via sonication on ice for 10 min, and 1 ml of 1× Protease Inhibitor Cocktail was added (Promega, USA). TgGRA8 expression was analyzed using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). To confirm the sequence of the recombinant protein, the mass spectrometry (MS) analysis was carried out by Proteomics Services Center, Faculty of Medical Technol- ogy, Mahidol University. Author details 1 1Department of Preclinic and Applied Animal Science, Faculty of Veterinary Science, Mahidol University, 999 Phutthamonthon sai 4 Rd, Salaya, Nakhonpathom 73170, Thailand. 2Department of Protozoology, Faculty of Tropical Medicine, Mahidol University, 420/6 Ratchawithi Road, Ratchathewi, Bangkok 10400, Thailand. 3Department of Medical Sciences, Medical Life Sciences Institute, 88/7 Tiwanon Road, Talad Kwan Subdistrict, Muang District, Nonthaburi 11000, Thailand. Abbreviation Abbreviation ELISA: Enzyme-linked immunosorbent assay; LAT: Latex agglutination test; PBS: Phosphate-buffered saline; PV: Parasitophorous vacuole Funding Thi This research is supported by Mahidol University (grant number: A30/5261). The funding body has not participated in the research design, collection, analysis, interpretation of data and writing the manuscript. Authors’ contributions CJ i d th t g Purified recombinant TgGRA8 was diluted at a final concentration of 0.1 μg/ml in coating buffer (50 mM bi- carbonate, pH 9.6) and added to separate wells of the ELISA plates (Nunc, Denmark). The coated plates were incubated overnight at 4 °C. The next day, the plates were washed five times with PBS-T and blocked with 5% PBS-skimmed milk (PBS-SM) for 1 h at 37 °C. After washing with PBS-T, duplicate serum samples were di- luted 1:250 in PBS-SM, and 50 μl of diluted serum were added to each well. The plates were incubated at 37 °C for 1 h and washed with PBS-T five times. Specific IgG antibody was detected using horseradish-peroxidase- conjugated anti-goat IgG antibodies (Invitrogen, USA). The conjugate was diluted 1:5000 with PBS, and 50 μl of diluted conjugates were added. After incubation at 37 °C for 1 h, the plates were washed five times with PBS-T, and then 3,3′,5,5′-tetramethylbenzidine (Invitrogen, USA) was added to develop the color. After 15 min, the reaction was stopped by adding 50 μl of 0.1 M HCl. OD450 was read using a microplate reader (model ELx808, Biotex, VT, USA). CJ conceived the present idea in this paper, artwork, perform data analysis and drafted the first manuscript. RU, AP, KJ carried out the laboratory work. PD approved the manuscript. All authors read and approved the final version of the manuscript. Competing interests Competing interests The authors declare that they have no competing interests. The negative and positive control sera were confirmed using MAST® TOXOREAGENT (Mast Group, Liverpool, UK). Positive samples were considered when agglutin- ation was observed at a dilution of 1:32 or greater. Western blotting F Five micrograms of recombinant TgGRA8 was resolved by 12% SDS-PAGE and then electrotransferred (Trans- blot, Bio-Rad) onto a nitrocellulose membrane (Milli- pore, USA). The membrane was washed three times with PBS, blocked with 5% skim milk, and then incubated at 37 °C for 1 h with constant shaking. After incubation, the membrane was washed three times with PBS con- taining 0.01% Tween 20 (PBS-T) and rinsed with PBS. The TgGRA8 protein in nitrocellulose membrane was probed using antibody or known reference positive and negative goat sera (diluted 1:250 in 5% skim milk) kept in our laboratory and incubated at 37 °C for 1 h with constant shaking. The monoclonal antibody (mAb) against Flag-tag (GenScript, USA) was diluted 1:1000, while polyclonal mouse anti-goat immunoglobulin/HRP (Dako, Denmark) was diluted 1:2000 in blocking buffer. Anti-DYKDDDDK G1 affinity resin (GenScript, USA) was used for protein purification. The debris was centri- fuged at 10,000×g for 30 min at 4 °C, after which the supernatant was transferred to a clean tube. The resin suspension (600 μl) was loaded into an empty gravity flow column (Bio-Rad, USA) and washed with Tris- buffered saline (50 mM Tris-HCl, 150 mM NaCl, pH 7.4). Protein was eluted from the resin using alkaline elution buffer (0.1 M Tris, 0.5 M NaCl, pH 12.0) and neutralized with 1 M HCl. The protein concentration was measured using NanoDrop ND-1000 UV/Vis spec- trophotometer (Thermo Fisher Scientific, USA). The eluted fractions were dialyzed using SnakeSkin Dialysis Tubing, 10 kDa cut-off (Thermo Fisher Scien- tific) against phosphate-buffered saline (PBS, pH 7.2) at 4 °C. The debris formed during dialysis was removed via Page 8 of 9 Jirapattharasate et al. BMC Veterinary Research (2021) 17:27 After incubation, the membrane was washed three times with PBS-T. The protein band was developed according to peroxidase activity using 3,3′,5,5′-tetramethylbenzi- dine (KPL, Gaithersburg, MD, USA). percentage of agreement, sensitivity, specificity, and the kappa values with 95% confidence intervals. The strength of agreement was graded as fair (κ = 0.21–0.40), moderate (κ = 0.41–0.60), and substantial (κ =0.61–0.80). Acknowledgements We thank Dr. Sarawut Taksinoros and Dr. Raweenipa Tohkwankeaw, for their significant contribution during sample collection, and veterinarians from Kanchanaburi provincial livestock office for their general support. The authors thanks Dr. Phirom Prompiram for technical assistance. In addition, we would like to express our appreciation to Asst. Prof. Dr., Onrapak Reamtong for mass spectrometry analysis. The authors would like to thank Enago (https:// www.enago.com) for the professional English language review. Supplementary Information The process of sample collection was reviewed and ap- proved by the Animal Care and Use Committee of the Faculty of Veterinary Science, Mahidol University, Thailand (Approval No. MUVS-2018-03-09). A total of 306 serum samples were obtained from a goat farm in Kanchanaburi province, Thailand. The goats were re- strained by holding the base of the horn and blood was collected from the jugular vein and immediately trans- ferred into 10 ml vacuum blood tubes without anticoagu- lant. The animals were not allowed returned to their cage until complete hemostasis has been achieved. All blood samples were kept in cooled box with ice pack and sent to the laboratory at Faculty of Veterinary Science, Mahidol University. The sera were separated after sedimentation of blood cells and stored at −20 °C until examination. The online version contains supplementary material available at https://doi. org/10.1186/s12917-020-02719-3. Additional file 1. Additional file 2. Additional file 1. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Consent for publication Not applicable. Ethics approval and consent to participate The process of sample collection was reviewed and approved by the Animal Care and Use Committee of the Faculty of Veterinary Science, Mahidol University, Thailand (approval no. MUVS-2018-03-09). The animals were han- dled humanely in strict accordance with the requirements of the Animal Eth- ics Procedures and Guidelines of Institute of Animals for Scientific purpose Development (IAD), Thailand. Before signing a consent, the owners of the se- lected farms were informed of the study and provided their approval for sampling of the goats. All samples collection process was conducted by local authorities and veterinarians. 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https://openalex.org/W1520961623	https://www.cadernos.prodisa.fiocruz.br/index.php/cadernos/article/download/107/149	Portuguese	null	A regulação do planejamento público e o conselho municipal de saúde	Cadernos Ibero-Americanos de Direito Sanitário	2,013	cc-by	5,904	Cad. IberAmer. Direito. Sanit., Brasília, v.2, n.2, jul./dez. 2013 Anais dos III Congresso Iberoamericano de Direito Sanitário / II Congresso Brasileiro de Direito Sanitário ISSN 2317-8396 5.08 5.08 Key-words: Health Law; local health council; Health Plan; regulation of health planning. Oswaldo José Barbosa Silva Graduado em Direito, Especialista em Direito Sanitário. Subprocurador-Geral da República, Cidade, Brasil. Resumo: Apresenta-se uma visão das dificuldades que sofre o modelo de estruturação do controle social na saúde pública, por meio dos conselhos de saúde, que exsurgem de seu dever de deliberar sobre o planejamento e fiscalizar a execução das ações e serviços públicos de saúde no âmbito municipal, estabelecido que o plano de saúde e os programas anuais de saúde que lhe seguem materializam a organização que se espera para a prestação desse serviço público, que atende um direito público subjetivo de matriz constitucional. Para tanto foi feita uma revisão de literatura e uma pesquisa documental. Observou-se que falta qualificação técnica dos conselheiros para discutir assuntos relacionados com planejamento de saúde, financeiro e orçamentário como se pode observar em pesquisas de campo realizadas em Municípios do Nordeste Brasileiro, dos Estados de Mato Grosso e Goiás e no município de Belo Horizonte (MG). Todavia, essas deficiências não diminuem a importância do modelo de participação social adotado por meio dos conselhos de saúde, devendo ser construídas estratégias para enfrentá-las. Palavras-chave: Direito Sanitário; conselho municipal de saúde; Plano de Saúde; regulação do planejamento. Key-words: Health Law; local health council; Health Plan; regulation of health planning. 595 Cad. IberAmer. Direito. Sanit., Brasília, v.2, n.2, jul./dez. 2013 Anais dos III Congresso Iberoamericano de Direito Sanitário / II Congresso Brasileiro de Direito Sanitário ISSN 2317-8396 ISSN 2317-8396 Introdução O controle social na saúde pública foi resultado dos esforços políticos do assim denominado movimento da reforma sanitária brasileira que, expressos na VIII Conferência Nacional de Saúde (1986), esse esforço foi recompensado pelos Constituintes de 1988, de forma que o controle social foi previsto na atual Constituição quando esta determinou a participação da comunidade como uma das diretrizes do Sistema Único de Saúde (SUS). No entanto, somente dois anos depois, com o advento das Leis nos 8.080/90 e 8.142/90, foram formalmente instituídos e tiveram sua constituição regulada e fomentada pelo Ministério da Saúde por meio de Portarias que instituíram as Normas Operacionais Básicas (NOB) do SUS, expedidas, sucessivamente, em 1991, 1993 e 1998. Registre-se que criação dos conselhos municipais de saúde depende de lei municipal, contudo seu funcionamento deve observar as diretrizes do SUS. A atribuição legal dos conselhos de saúde está prescrita no art. 1º, inciso II, da Lei nº 8.142/90, que lhe confere caráter permanente e deliberativo para atuar na formulação de estratégias e no controle da execução da política de saúde na instância correspondente, inclusive nos aspectos econômicos e financeiros. Assim, é sua função inarredável deliberar sobre o plano de saúde, sobre a programação anual de saúde e, depois, fiscalizar-lhes a execução. Importa saber o quão importante é para os conselheiros municipais de saúde o conhecimento técnico e prévio do planejamento público em geral, do planejamento específico para a saúde. Este conhecimento, como pretende o presente artigo revelar, é complexo. No que tange ao planejamento público em geral existem normas de estatura constitucional e legal a regê-lo. A Constituição Federal, em seu art. 165 institui como instrumentos do planejamento público o Plano Plurianual, a Lei de Diretriz Orçamentária e a Lei Orçamentária Anual. Estabelece, também, o financiamento da saúde pública, nos termos da Emenda Constitucional n° 29/ 2000. No plano legislativo, o planejamento público deve observar os já citados instrumentos, mas ainda, a Lei de Responsabilidade Fiscal (Lei Complementar nº 101/2000) e a Lei de Finanças Públicas (Lei nº 4.320/64). Mais recentemente a Lei Complementar nº 141/2011, 596 Cad. IberAmer. Direito. Sanit., Brasília, v.2, n.2, jul./dez. 2013 Anais dos III Congresso Iberoamericano de Direito Sanitário / II Congresso Brasileiro de Direito Sanitário ISSN 2317-8396 ISSN 2317-8396 impôs regras de elaboração orçamentária em face dos aportes financeiros para a saúde. No âmbito da saúde pública, propriamente dita, o planejamento municipal encontra matriz legal na Lei nº 8.080/90, arts. Metodologia Foi feita uma revisão de literatura sobre o tema, em especial, trabalhos científicos que abordaram as dificuldades enfrentadas pelos Conselhos Municipais de Saúde para a realização de suas competências. A seleção desses artigos foi realizada sob o critério de que "efetividade "e "capacitação deveriam ser as palavras- chave para a pesquisa, porquanto seu objeto era mensurar o grau de dificuldade que os conselheiros municipais de saúde suportam em face de um eventual débito de capacitação técnica em especial diante da complexidade do arcabouço normativo. Procurou-se listar artigos não muito antigos, limitando a pesquisa a artigos publicados após o ano de 2006. Para a localização dos artigos foi utilizada a Livraria Científica Eletrônica Online, projeto da FAPESP - Fundação de Amparo à Pesquisa do Estado de São Paulo, em parceria com a BIREME - Centro Latino-Americano e do Caribe de Informação em Ciências da Saúde com o apoio do CNPq- Conselho Nacional de Desenvolvimento Científico e Tecnológico (www.scielo.br). Também foi realizada pesquisa documental no âmbito dos documentos públicos do PlanejaSUS, bem como textos legais pertinentes ao tema, todos localizados no sítio do Ministério da Saúde e da Presidência da República, na rede mundial de computadores. Introdução 15, inciso VIII e 18, incisos I e II. No plano infralegal, o Ministério da Saúde instituiu o Sistema de Planejamento do SUS (PlanejaSUS) e das orientações gerais acerca dos seus instrumentos, pactuadas na Comissão Intergestores Tripartite e aprovadas pelas Portarias Nº 3.085/GM e Nº 3.332/GM, do Ministério da Saúde, ambas de 2006, donde se extrai: “o Plano de Saúde (PS) é o instrumento básico que, em cada esfera, norteia a definição da Programação Anual das ações e serviços de saúde prestados, assim como da gestão do SUS”. Segundo a Portaria 3.332/GM/2006 citada, o Plano “apresenta as intenções e os resultados a serem buscados no período de quatro anos, expressos em objetivos, diretrizes e metas” (§1º do art. 2º) (Brasil, 2009). O plano de saúde é a base, portanto, para a definição e a implementação de todas as iniciativas no âmbito da saúde. Ou seja: é a referência para os processos de planejamento regional e formulação de programações, projetos, entre outros. Assim, deve ser valorizado como o instrumento central de planejamento, sendo necessário que todas as iniciativas estejam nele contidas, a partir dos seus diversos componentes. Trata-se, enfim, de instrumento no qual precisam estar refletidas as necessidades e peculiaridades próprias de cada esfera, constituindo referencial para a execução, o acompanhamento, a avaliação e a gestão do sistema de saúde (Brasil, 2009). O pleno e eficaz funcionamento dos Conselhos Municipais de Saúde é um objetivo ainda não alcançado (Martins et al. 2008, e Labra, 2006) e que impõe um contínuo e trabalhoso esforço de seu aprimoramento, com o qual este artigo pretende contribuir. As dificuldades deste modelo de controle social e participação societária em uma incumbência tipicamente governamental são, no plano municipal, há muito conhecidas: adesão escassa ou inexistente da comunidade; ausência de publicidade de sua existência e de suas atividades; apequenamento de seu papel institucional; 597 Cad. IberAmer. Direito. Sanit., Brasília, v.2, n.2, jul./dez. 2013 Anais dos III Congresso Iberoamericano de Direito Sanitário / II Congresso Brasileiro de Direito Sanitário ISSN 2317-8396 ISSN 2317-8396 dificuldades no funcionamento interno; hipertrofia do papel dos gestores de saúde; irregularidades na composição, representação e representatividade (Labra, 2006). Nesse sentido, o presente artigo pretende investigar as dificuldades que o modelo de participação social estruturado nos Conselhos Municipais de Saúde enfrenta para realizar a sua atividade de deliberar sobre o planejamento e fiscalizar a execução das ações e serviços públicos de saúde no âmbito municipal. Plano de Saúde e controle social Não é dever do Conselho Municipal de Saúde elaborar o Plano de Saúde, mas da gestão municipal de saúde, de acordo com a conformação que lhes dá a 598 Cad. IberAmer. Direito. Sanit., Brasília, v.2, n.2, jul./dez. 2013 Anais dos III Congresso Iberoamericano de Direito Sanitário / II Congresso Brasileiro de Direito Sanitário ISSN 2317-8396 Cad. IberAmer. Direito. Sanit., Brasília, v.2, n.2, jul./dez. 2013 ISSN 2317-8396 Anais dos III Congresso Iberoamericano de Direito Sanitário / II Congresso Brasileiro de Direito administração pública municipal. O plano de saúde é uma exigência legal (art. 15, VIII, da Lei 8.080/90), informa a elaboração da proposta orçamentária do município (art. 15, X, da Lei 8.080), deve compatibilizar-se com a disponibilidade de recursos, seu financiamento será previsto na proposta orçamentária e não haverá transferência de recursos para ações que não estejam previstas em seu bojo (art. 36 e 37 da Lei 8.080). Mais recentemente o plano de saúde foi objeto da Lei Complementar nº 141/2012, que estabeleceu novas prescrições para fortalecer a sua importância, de modo que, para o fim de apuração da aplicação do mínimo dos valores que, constitucionalmente (art. 77, do ADCT) e nessa própria lei, devem ser aplicados em ações e serviços públicos de saúde, essas ações devem atender, dentre outras diretrizes, a que determina que estejam em conformidade com objetivos e metas explicitados no Plano de Saúde (art. 2º, inciso II). Mais grave é a prescrição que o rateio dos recursos da União (e dos Estados) em favor dos municípios pode ser condicionado à apresentação do Plano de Saúde. Sua elaboração, mesmo para o gestor de saúde experiente, não é tarefa fácil. Mesmo para um pequeno município, sua formulação deve conter o planejamento para o prazo de quatro anos e se divide em dois momentos: a análise situacional e a formulação de objetivos, diretrizes e metas. Resumidamente, a análise situacional deve considerar os dados para a identificação do município e da própria secretaria municipal de saúde; a situação de saúde do município; a informação de toda a estrutura da rede de atenção do município (atenção integral à saúde) bem como sobre a produção dos serviços de saúde; a análise da gestão municipal da saúde e a definição dos problemas prioritários observando-se, neste último caso três eixos: as condições de saúde da população; os determinantes e condicionantes da saúde, sob a ótica de sua magnitude, transcendência, magnitude e custos (Brasil, 2009). Plano de Saúde e controle social A formulação de objetivos, diretrizes e metas estão vinculados à análise situacional e devem ser orientados pelos princípios que regem o SUS: universalidade, integralidade e gratuidade, de modo a propiciar as ações e serviços de saúde propriamente dito. Por fim, devem ser construídos os indicadores de resultados, instrumento necessário à medição do cumprimento das metas. 599 Cad. IberAmer. Direito. Sanit., Brasília, v.2, n.2, jul./dez. 2013 Anais dos III Congresso Iberoamericano de Direito Sanitário / II Congresso Brasileiro de Direito Sanitário ISSN 2317-8396 Cad. IberAmer. Direito. Sanit., Brasília, v.2, n.2, jul./dez. 2013 ISSN 2317-8396 erAmer. Direito. Sanit., Brasília, v.2, n.2, jul./dez. 2013 s III Congresso Iberoamericano de Direito Sanitário / II Congresso Brasileiro de Direito Sanitário ISSN 2317-8396 dos III Congresso Iberoamericano de Direito Sanitário / II Congresso Brasileiro de Direito Sanitá Resulta óbvio que uma tarefa de tal magnitude é um desafio mesmo para os grandes municípios do país. Ainda na instância dos formuladores dos planos de saúde, Sulpino (2008, p. 1570) dá uma noção da dificuldade de seu planejamento: A discussão do planejamento em saúde no SUS parece já ter ultrapassado as questões metodológicas, quanto à definição de instrumentos para sua realização, evidenciando-se na atualidade a necessidade de definição de fluxos e mecanismos de interligação entre os diversos atores, tanto do ambiente interno quanto externo a cada esfera de governo. Quando a questão tange ao planejamento no ambiente intra-organizacional (Secretarias de Saúde e Ministério da Saúde), geralmente o tema é tratado como assunto de um setor específico, responsável pelo planejamento da instituição. Nesta lógica, há escasso envolvimento dos profissionais de saúde, que seriam responsáveis pelo alcance dos objetivos e metas propostos. Como estes não fazem parte do processo de sua definição, invariavelmente se observa o distanciamento entre o plano estabelecido e os resultados alcançados. O plano constitui-se apenas de um conjunto de intenções que figuram em um documento, mas que não levam a resultados práticos. O planejamento acaba sendo feito para o cumprimento de exigência legal, em vez de instrumento para a implementação da política de saúde ou como base para a alocação de recursos. Plano de Saúde e controle social Percebe-se que, mesmo na instância elaborativa do Plano de Saúde, cujo produto deve ser levado ao Conselho de Saúde, existem problemas que podem contaminar sua eficácia e, mesmo neste quadro, o Plano de Saúde deve ser objeto de apreciação e deliberação pelo órgão do controle social, que por sua vez padece de enormes dificuldades para seu próprio funcionamento. Basta passar os olhos pela Resolução nº 333, de 4 de novembro de 2003, do Conselho Nacional e Saúde, onde, em sua Quinta Diretriz, são enumerados vinte e quatro competências e atribuições para os Conselhos e considerar que sua composição é voluntária, sendo considerada de relevância pública apenas para justificar faltas ao trabalho, como se vê na terceira diretriz do mesmo documento. Essas dificuldades acima insinuadas têm sido alvo de numerosos estudos, dos quais transcrevem-se, abaixo, alguns excertos. Abertamente, aqui se trata da dificuldade dos Conselheiros Municipais de Saúde lidarem e intervirem propositivamente na deliberação, tendo por objeto o Plano Municipal de Saúde. No campo da pesquisa empírica, podemos citar alguns autores que debruçaram-se sobre a matéria. Uma análise sobre a efetividade deliberativa dos Conselhos Municipais de Saúde no nordeste assevera: 600 Cad. IberAmer. Direito. Sanit., Brasília, v.2, n.2, jul./dez. 2013 Anais dos III Congresso Iberoamericano de Direito Sanitário / II Congresso Brasileiro de Direito Sanitári ISSN 2317-8396 Cad. IberAmer. Direito. Sanit., Brasília, v.2, n.2, jul./dez. 2013 ISSN 2317-8396 Anais dos III Congresso Iberoamericano de Direito Sanitário / II Congresso Brasileiro de Direito Há também um outro aspecto que não pode ser ignorado e que diz respeito a alguma dificuldade manifesta pelos conselheiros quanto à intervenção propositiva sobre a política e sobre o orçamento. A criação de câmaras técnicas na estrutura dos conselhos tem o potencial de produzir alterações nesta situação, mas os resultados de sua existência ainda precisam ser verificados. O que se tem de concreto são demandas constantes por processos de capacitação dos conselheiros, não só nas reuniões dos conselhos, mas também em suas Conferências municipais, estaduais e nacionais, que têm deliberado no sentido de que sejam criadas condições para que seja reduzida a assimetria informacional entre os diversos segmentos que têm assento nos conselhos e apontado o processo de formação de conselheiros como uma estratégia para isso. Sabe-se que têm sido empreendidas diversas iniciativas no sentido de formação de conselheiros e multiplicam-se ações educativas voltadas para suprir esta demanda. Plano de Saúde e controle social 629) positivas), melhora a eficiência da gestão pública (seis respostas positivas) e a relação entre a Secretaria de Saúde e a comunidade (quatro respostas positivas). (Van Stralen et alii, 2006, p. 629) Mesmo em um grande centro urbano, como Belo Horizonte (MG), de modo a demonstrar que este não é um problema dos pequenos municípios, constatou-se que: A questão da informação, como ponto crucial para se efetivar a participação de todos os envolvidos, é muito bem discutida por Guizardi e Machado (2005) quando esclarecem que realmente fica muito difícil participar em quaisquer instâncias da qual o sujeito não se percebe 'agente de constituição e produção da política'. Assim, os modos pelos quais essas informações são produzidas e disseminadas têm sido ao longo da nossa história política território avesso à participação da população. Quem detém o poder de construir a informação e divulgá-la também tem o poder de considerá-la verdade, à revelia das demandas para quem foram construídas. No caso da saúde, as políticas acabam sendo criadas PARA e não COM os sujeitos, comprometendo todo o processo. Os conceitos são sempre carregados de noções técnicas, e há uma dificuldade muito grande na apropriação do conteúdo das políticas públicas. (Santos et al., 2011, p. 490) Noutro campo, o da literatura, a dificuldade acima constatada é afirmada por Labra (2006, p. 210), quando sintetiza, como um dos problemas que fustigam os Conselhos de Saúde, o desvio de seu papel institucional: Conselhos de Saúde, o desvio de seu papel institucional: O CS é muito valorizado por todos aqueles que o conhecem ou dele participam. Entretanto, predomina a impressão de que é um espaço para reivindicações específicas ou denúncias pontuais. A maior parte do tempo de cada reunião mensal é gasta na discussão de assuntos internos, sendo raros os debates de temas substantivos. Quanto ao Plano de Saúde, ao Orçamento e ao Relatório de Gestão, os conselheiros não têm papel relevante na discussão, convertendo-se em um mero ritual a aprovação dessas importantes peças da gestão. O CS é muito valorizado por todos aqueles que o conhecem ou dele participam. Entretanto, predomina a impressão de que é um espaço para reivindicações específicas ou denúncias pontuais. A maior parte do tempo de cada reunião mensal é gasta na discussão de assuntos internos, sendo raros os debates de temas substantivos. Plano de Saúde e controle social No entanto, ainda se percebe muito pouca intervenção dos conselheiros na proposição ou na alteração da política e do orçamento para as áreas, mesmo quando as normas operacionais condicionam o repasse de recursos financeiros do governo federal aos governos municipais à aprovação tanto de planos de gestão da política (aí incluída a proposta orçamentária), quanto de prestações de conta (das ações desenvolvidas e dos recursos utilizados – Relatórios de Gestão). Isso faz com que, obrigatoriamente, esses temas sejam tratados nos conselhos e ganhem visibilidade, mas ainda assim, tem conselhos que parecem apenas cumprir as formalidades de aprovação desses documentos, pois são temas muito pouco deliberados em suas reuniões. A prevalência do exercício do controle indica que os conselhos despendem mais energia sobre decisões já tomadas e em execução, muitas delas não compartilhadas pelos governos com os conselhos. Isso pode significar que os conselhos estão tendo muito pouca capacidade de intervenção nos rumos da política em si, que é definida nas fases de elaboração dos orçamentos e dos planos municipais de saúde e da criança e do adolescente. O estudo possibilitou constatar, inclusive, a pouca discussão que existe nos conselhos sobre o financiamento da política e os critérios de repartição dos recursos públicos geridos por eles. Neste sentido, entende-se que os conselhos têm menor efetividade deliberativa quando prevalece a função de controle sobre a função propositiva, o que se manifestou em todos os casos estudados.” (Cunha, 2007, p. 21) Enquanto em outro estudo, este abarcando municípios de Goiás e Mato Grosso do Sul, informa, significativamente que: As deliberações parecem pautar-se mais por um discurso em defesa dos princípios do SUS do que por considerações técnicas ou orçamentárias, como sugere o fato de que apenas um informante soube participar o percentual dos gastos municipais aplicados à saúde e nenhum sabia o percentual dos gastos com atenção básica. Mesmo assim, produzem efeitos. Na visão dos informantes, o conselho contribui para a melhoria da atenção básica, redireciona prioridades e torna mais transparente a alocação de recursos (sete respostas 601 Cad. IberAmer. Direito. Sanit., Brasília, v.2, n.2, jul./dez. 2013 ISSN 2317-8396 Anais dos III Congresso Iberoamericano de Direito Sanitário / II Congresso Brasileiro de Direito positivas), melhora a eficiência da gestão pública (seis respostas positivas) e a relação entre a Secretaria de Saúde e a comunidade (quatro respostas positivas). (Van Stralen et alii, 2006, p. Plano de Saúde e controle social Quanto ao Plano de Saúde, ao Orçamento e ao Relatório de Gestão, os conselheiros não têm papel relevante na discussão, convertendo-se em um mero ritual a aprovação dessas importantes peças da gestão. Esse desvio, o de se converter o Conselho de Saúde em um mero homologador das peças de planejamento da saúde que lhe são submetidas, decorre do fato de que os conselheiros, especialmente os representantes dos usuários, são tecnicamente despreparados para perceberem a lógica de funcionamento e as necessidades do sistema de saúde, razão pela qual os gestores, normalmente apoiados por quadros especializados, exercem hegemonia nos conselhos, definindo- lhes as agendas e induzindo os conselheiros à aprovação das decisões do próprio gestor, à míngua da capacidade dos conselheiros de opor-lhes argumentos técnicos ou jurídicos (Martins et al., 2007). Acresço à dificuldade de compreender os argumentos técnicos de saúde pública o cipoal legislativo que regula o planejamento 602 Cad. IberAmer. Direito. Sanit., Brasília, v.2, n.2, jul./dez. 2013 Anais dos III Congresso Iberoamericano de Direito Sanitário / II Congresso Brasileiro de Direito Sanitário ISSN 2317-8396 ISSN 2317-8396 público e que sujeita, não só os representantes dos usuários, mas todos os demais conselheiros. E é esse Conselho Municipal de Saúde que, diante de seu dever de apreciar e deliberar sobre o Plano Municipal de Saúde e, em decorrência, sobre a Programação Anual de Saúde e o Relatório Anual de Gestão, esbarra na complexidade da regulação do planejamento público, mormente sem poder, de regra, contar com o apoio técnico imprescindível para o entendimento da matéria. A primeira necessidade é o conhecimento do financiamento da saúde, hoje regulado pelo art. 198, da Constituição Federal e pela Lei Complementar nº 141/2011, que, a partir de sua vigência, afastou a aplicação do art. 77, do ADCT (conferir o § 4º, desse artigo). Não é possível planejar sem a certeza que as ações planejadas encontrarão recursos financeiros para sua execução. Da mesma forma e reciprocamente a execução dos recursos financeiros somente atenderá aos ditames do interesse público se submetida a planejamento prévio. No entanto, o financiamento da saúde, por si só, não significa injetar incondicionalmente recursos na saúde. Diversas condições e requisitos foram impostos por lei para a aplicação das verbas públicas na saúde. A primeira condição era a definição do que vem a ser ações e serviços públicos de saúde, o que somente aconteceu recentemente, com o advento da Lei Complementar acima citada (arts. Plano de Saúde e controle social 2º, 3º e 4º). Antes disso, batalhas jurídicas se travaram porque os gestores de saúde, especialmente no palco de conflitos interfederativos, não reconheciam obrigatoriedade (art. 5º, inciso 2º da CF), na Resolução nº 322, de 8 de maio de 2003, quando esta, em suas quarta, quinta e sexta diretrizes, declaravam o que era e o que não era ação e serviço público de saúde. Além desses requisitos há outras condições que o conselheiro precisa conhecer, especialmente aquela que diz respeito às exigências para a consecução das transferências obrigatórias da União ou dos Estados para o município, previstas nas seções II a V, do Capítulo III, da Lei Complementar nº 141/2011. Colhem-se, dali, alguns exemplos que demonstram a enorme complexidade da questão do financiamento da saúde e que precede e se entretece, ainda, com outras questões tão complexas como as exigências orçamentárias e de execução financeira, dos quais pode-se enumerar: a constatação da aplicação dos recursos mínimos para a saúde; a manutenção de contas específicas para o recebimento dos repasses, no 603 Cad. IberAmer. Direito. Sanit., Brasília, v.2, n.2, jul./dez. 2013 Anais dos III Congresso Iberoamericano de Direito Sanitário / II Congresso Brasileiro de Direito Sanitário ISSN 2317-8396 Cad. IberAmer. Direito. Sanit., Brasília, v.2, n.2, jul./dez. 2013 ISSN 2317-8396 erAmer. Direito. Sanit., Brasília, v.2, n.2, jul./dez. 2013 s III Congresso Iberoamericano de Direito Sanitário / II Congresso Brasileiro de Direito Sanitário ISSN 2317-8396 dos III Congresso Iberoamericano de Direito Sanitário / II Congresso Brasileiro de Direito Sanitá fundo municipal de saúde; o conhecimento prévio dos critérios de rateio das verbas federais a serem repassadas ao ente municipal; o conhecimento prévio do montante das verbas federais e estaduais a serem repassados anualmente ao município. Segue-se a necessidade de se conhecer o sistema de regulação do planejamento, instituído no art. 165 da Constituição Federal, que impõe aos entes federativos e, portanto aos municípios, leis que estabeleçam o plano plurianual, as diretrizes orçamentárias e os orçamentos anuais. Plano de Saúde e controle social Tanto o plano plurianual quanto as diretrizes orçamentárias encontram-se na esfera do poder político, mas a lei orçamentária deve observar as normas gerais de direito financeiro para elaboração e controle dos orçamentos e balanços da União, dos Estados, dos Municípios e do Distrito Federal, previstos na Lei nº 4.320/64 (recepcionada com status de Lei Complementar pela CF/88), bem assim a Lei de Responsabilidade Fiscal (Lei Complementar nº 101/2000 e, por fim, a recente Lei Complementar 141/2011, que, por sua especificidade e importância para o exame do Plano de Saúde, merece ser transcrito: Art. 30. Os planos plurianuais, as leis de diretrizes orçamentárias, as leis orçamentárias e os planos de aplicação dos recursos dos fundos de saúde da União, dos Estados, do Distrito Federal e dos Municípios serão elaborados de modo a dar cumprimento ao disposto nesta Lei Complementar. Art. 30. Os planos plurianuais, as leis de diretrizes orçamentárias, as leis orçamentárias e os planos de aplicação dos recursos dos fundos de saúde da União, dos Estados, do Distrito Federal e dos Municípios serão elaborados de modo a dar cumprimento ao disposto nesta Lei Complementar. § 1º O processo de planejamento e orçamento será ascendente e deverá partir das necessidades de saúde da população em cada região, com base no perfil epidemiológico, demográfico e socioeconômico, para definir as metas anuais de atenção integral à saúde e estimar os respectivos custos. § 2º Os planos e metas regionais resultantes das pactuações intermunicipais constituirão a base para os planos e metas estaduais, que promoverão a equidade interregional. § 3º Os planos e metas estaduais constituirão a base para o plano e metas nacionais, que promoverão a equidade interestadual. § 4º Caberá aos Conselhos de Saúde deliberar sobre as diretrizes para o estabelecimento de prioridades. Este substantivo influxo de normas legais que se impõe à formulação do plano municipal de saúde, tanto na perspectiva do financiamento quanto na observância das regras de planejamento público e financiamento, para além, como foi dito, das ações e serviços públicos de saúde objeto do plano propriamente dito, constituem 604 Cad. IberAmer. Direito. Sanit., Brasília, v.2, n.2, jul./dez. 1 cf. em http://www.ead.fiocruz.br/curso/index.cfm?cursoid=676, acesso em 31/03/2013 Plano de Saúde e controle social 2013 ISSN 2317-8396 dos III Congresso Iberoamericano de Direito Sanitário / II Congresso Brasileiro de Direito Sanitá Tal é a realidade do país. O esforço incessante de capacitação de conselheiros não atingirá e nem pretende atingir o objetivo de conferir-lhes plena habilitação para lidar com a inarredável e complexa regulação jurídica que informa a elaboração dos planos de saúde e seus consectários, mas pode prepará-los, dentro de um limite mínimo de aprendizado, para, quando for necessário, dialogar com técnicos que lhes forem designados para auxiliá-los e alertá-los quanto à incidência das normas jurídicas e contábeis em determinada avaliação de plano de saúde. Fung e Cifuentes, citados em Coelho (2007, p. 81), nesse sentido, o de estabelecer incentivos estruturais e metodologias participativas, argumentam que: a capacidade dos foros participativos de contribuir para definir agendas que expressem os interesses dos mais pobres poderia ser favorecida por mecanismos institucionais e procedimentais que promovam tanto a inclusão de participantes com menor capacitação técnica e escassos recursos de comunicação, como também sua capacidade de se posicionar diante dos temas em discussão. Tais autores sugerem incentivos estruturais, metodologias participativas e abordagens deliberativas como meio de aperfeiçoar a inclusão e a qualidade dos processos endógenos de formação de preferências. a capacidade dos foros participativos de contribuir para definir agendas que expressem os interesses dos mais pobres poderia ser favorecida por mecanismos institucionais e procedimentais que promovam tanto a inclusão de participantes com menor capacitação técnica e escassos recursos de comunicação, como também sua capacidade de se posicionar diante dos temas em discussão. Tais autores sugerem incentivos estruturais, metodologias participativas e abordagens deliberativas como meio de aperfeiçoar a inclusão e a qualidade dos processos endógenos de formação de preferências. Assim, ao que parece, a par dos esforços de capacitação acima apontados, outro caminho a ser trilhado é o de fornecer estrutura técnica aos Conselhos de Saúde, mediante o oferecimento de técnicos na área jurídica e contábil para que eles possam informar-se no processo deliberativo. 2 cf. em http://cms.pmf.sc.gov.br/?p=lei_3291, acesso em 31/03/2013 Plano de Saúde e controle social 2013 Anais dos III Congresso Iberoamericano de Direito Sanitário / II Congresso Brasileiro de Direito Sanitário ISSN 2317-8396 ISSN 2317-8396 mais um desafio para a subsistência do modelo de participação e controle social, porquanto a incapacidade dos conselheiros municipais de lidar com esse arcabouço jurídico resulta na deliberação e aprovação de planos de saúde que não resistem à submissão às regras acima apresentadas tornando-os passíveis de alterações posteriores, para conformá-los às leis, sem a intervenção do controle social, quando não simplesmente os tornam inexequíveis. São muitos os esforços de tornar os conselheiros municipais (e estaduais) capazes de exercerem suas funções mediante o acréscimo de conteúdos técnicos às suas experiências pessoais. Destaque-se a atuação da Escola Nacional de Saúde Pública Sérgio Arouca, da Fundação Oswaldo Cruz que, por meio do ensino à distância (EAD), oferece curso de Capacitação de Conselheiros Municipais e Estaduais de Saúde, em cujo programa se destaca as seguintes unidades de aprendizagem que se referem ao assunto aqui tratado: Planejamento de Saúde: Agenda, Plano de Saúde, Quadro de Metas; e Planejamento em Saúde: orçamento.1 Já em 2006 esse esforço era registrado por Labra (2006, p. 208): Várias outras iniciativas têm sido empreendidas na esfera do Ministério da Saúde para reforçar o Controle Social, das quais ressalta o Programa de Capacitação para Conselheiros levando adiante pelo Ministério da Saúde em conjunto com a Escola Nacional de Saúde Pública/Fiocruz e outras instituições, que envolveu 35 mil participantes de todo o país. Esta bem-sucedida experiência, atualmente em fase de multiplicação mediante Pólos de Educação Permanente, teve como objetivo central fornecer aos conselheiros conhecimentos e instrumentos indispensáveis para o aprimoramento de seu desempenho nos CS e para o exercício do controle social de maneira informada e assertiva. Parece, no entanto, que não é o caso de transformar os conselheiros municipais de saúde em juristas ou contadores para que possam exercer suas atribuições. O modelo participativo do controle social deve encontrar outras soluções que permitam a inclusão de conselheiros de menor capacitação técnico-jurídica mas que, no entanto, tenham experiência pessoal para deliberar sobre a oportunidade e conveniência das propostas de ação e serviços públicos de saúde que constituem o plano de saúde e a programação anual de saúde. 605 Cad. IberAmer. Direito. Sanit., Brasília, v.2, n.2, jul./dez. 2013 Anais dos III Congresso Iberoamericano de Direito Sanitário / II Congresso Brasileiro de Direito Sanitário ISSN 2317-8396 Cad. IberAmer. Direito. Sanit., Brasília, v.2, n.2, jul./dez. Plano de Saúde e controle social Para citar um exemplo, a Lei que instituiu o Conselho Municipal de Saúde de Florianópolis (SC), prescreveu que esse conselho terá a Assessoria Técnica dos Profissionais da Secretaria Municipal da Saúde e Desenvolvimento Social e da Comissão Interinstitucional Municipal de Saúde (Santa Catarina, 1989).2 Por outro lado, os usuários que compõem o Conselho são representantes de diversas organizações da sociedade civil com as quais, evidentemente, podem contar, no sentido de obterem, dali, tal assessoria técnica, o que, aliás, seria mais desejável, de modo a garantir a imparcialidade no assessoramento, evitando a prevalência do poder público que ordinariamente hegemoniza os conselhos municipais de saúde, mormente nos pequenos municípios. 2 cf. em http://cms.pmf.sc.gov.br/?p=lei_3291, acesso em 31/03/2013 606 Cad. IberAmer. Direito. Sanit., Brasília, v.2, n.2, jul./dez. 2013 Anais dos III Congresso Iberoamericano de Direito Sanitário / II Congresso Brasileiro de Direito Sanitário ISSN 2317-8396 ISSN 2317-8396 Conclusão O cotejo das diversas percepções acerca das dificuldades do modelo de participação social no planejamento da saúde pública, aliado ao complexo modelo jurídico-contábil do planejamento público é fator inibitório da atuação deliberativa e fiscalizatória dos Conselhos Municipais de Saúde, mercê da natural falta de capacitação dos conselheiros municipais, em especial, nos menores municípios do país. No entanto, essa deficiência não justifica solapar o modelo de participação social e democrática conquistado pelo movimento da reforma sanitária e insculpida na Constituição Federal de 1988 de que se constituem os conselhos de saúde. Afinal, todo o processo de produção normativa que envolve o financiamento, o planejamento público e a execução orçamentária, sobre ser realmente complexo é uma garantia de aplicação regular e apropriada das verbas públicas originárias ou transferidas de um ente federativo para outro. Enfrentar esta dicotomia que põe de um lado essas garantias de boa aplicação do dinheiro público e de outro a inviabilidade de se apreender todo esse complexo arcabouço legal instituído em favor dessas garantias, justamente para realizá-las, não prescinde de capacitação dos conselheiros, mas não prescinde, também, de estruturação dos Conselhos Municipais de Saúde mediante a possibilidade de os conselheiros contarem com assessoria jurídico-contábil à sua disposição. SIL. Lei Complementar nº 101, de 4 de maio de 2000. Disponível na internet BRASIL. Lei Federal nº 8.080, de 19 de setembro de 1980. Disponível na internet na URL: http://www.planalto.gov.br/ccivil_03/leis/l8080.htm, acesso em: 28.mar.2013. BRASIL. Lei Federal nº 8.142, 28 de dezembro de 1980,. Disponível na internet na URL: http://www.planalto.gov.br/ccivil_03/leis/l8142.htm, acesso em: 28.mar.2013. BRASIL. Emenda Constitucional nº 29, de 13 de setembro de 2000. Disponível na internet na URL: http://www.planalto.gov.br/ccivil_03/constituicao/Emendas/Emc/emc29.htm, acesso: em 28.mar.2013. BRASIL L i C l t º 101 d 4 d i d 2000 Di í l i t t Referências BRASIL. Constituição da República Federativa do Brasil (1988). Disponível na internet na URL: <http://www.planalto.gov.br/ccivil_03/constituicao/ConstituicaoCompilado.htm#adct> Acesso em: 28.mar.2013. http://www.planalto.gov.br/ccivil_03/constituicao/Emendas/Emc/emc29.htm, acesso: em 28.mar.2013. 607 Cad. IberAmer. Direito. Sanit., Brasília, v.2, n.2, jul./dez. 2013 Cad. IberAmer. Direito. Sanit., Brasília, v.2, n.2, jul./dez. 2013 ISSN 2317-8396 ISSN 2317-8396 Anais dos III Congresso Iberoamericano de Direito Sanitário / II Congresso Brasileiro de Direito VIEIRA, FS. Avanços e Desafios do Planejamento do Sistema Único de Saúde. Ciência & Saúde Coletiva; 14(supl. 1): 1.565-1.577, 2009. URL: http://www.planalto.gov.br/ccivil_03/leis/lcp/lcp101.htm, 28.mar.2013. em: BRASIL. Lei Federal nº 4.320, de 17 de março de 1964. Disponível na internet na URL: http://www.planalto.gov.br/ccivil_03/leis/l4320.htm, acesso em: 28.mar.2013. BRASIL. Lei Federal nº 4.320, de 17 de março de 1964. Disponível na internet na URL: http://www.planalto.gov.br/ccivil_03/leis/l4320.htm, acesso em: 28.mar.2013. BRASIL. Lei Complementar nº 141, de 13 de janeiro de 2012. Disponível na internet na URL: http://www.planalto.gov.br/ccivil_03/leis/lcp/Lcp141.htm, acesso em: 28.mar.2013. BRASIL. Ministério da Saúde. Sistema de Planejamento do SUS: Uma Construção Coletiva. Série B, Textos Básicos de Saúde, Série Cadernos de Planejamento, vol. 6. Brasília : Ministério da Saúde, 2009. BRASIL, Resolução nº 333, de 4 de novembro de 2003. Ministério da Saúde, Conselho Nacional de Saúde, disponível na internet na URL: http://conselho.saude.gov.br/biblioteca/livros/resolucao_333.pdf, acesso em 31.mar.2013. BRASIL. Resolução nº 322, de 8 de maio de 2003. Ministério da Saúde, Conselho Nacional de Saúde, disponível na internet na URL: http://portalsaude.saude.gov.br/portalsaude/arquivos/pdf/2012/Set/26/resolucao_CNS _322.pdf acesso em 31.mar.2013 COELHO, VSP, Democratização dos Conselhos de Saúde. Revista Novos Estudos, CEBRAPA, (78):77-92, 2007. CUNHA, E.S.M. A efetividade deliberativa dos conselhos municipais de saúde e de criança e adolescente no Nordeste. In: AVRITZER, Leonardo (Org.). A participação social no Nordeste. (1 ed). Belo Horizonte : Ed. UFMG, 2007. p. 135-162. LABRA, M.E., Conselho de Saúde Visões “macro” e “Micro”. Civitas - Revista de Ciências Sociais; 6(1):199:221, 2006. MARTINS, P.C., et alii, Conselhos de Saúde e a Participação Social no Brasil: Matizes da Utopia. Physis, Revista de Saúde Coletiva, Rio de Janeiro; 18(1):105-121, 2008. SANTOS, S.F., et alii, Conselheiros Usuários do Conselho Municipal de Saúde de Belo Horizonte: características sociais e representatividade. Saúde Soc. São Paulo; 20(2): 483-495, 2011. VAN STRALEN, C. J., et alii. Conselhos de Saúde: efetividade do controle social em municípios de Goiás e Mato Grosso do Sul. Ciência & Saúde Coletiva; 11(3):621-632, 2006. VIEIRA, FS. Avanços e Desafios do Planejamento do Sistema Único de Saúde. Ciência & Saúde Coletiva; 14(supl. 1): 1.565-1.577, 2009. VIEIRA, FS. Avanços e Desafios do Planejamento do Sistema Único de Saúde. Ciência & Saúde Coletiva; 14(supl. 1): 1.565-1.577, 2009. 608
https://openalex.org/W4387528314	https://paris.ipb-intl.ac.id/index.php/paris/article/download/492/371	Malay	null	Perbedaan Kualitas Keripik Bengkuang dan Keripik Kentang	Jurnal Ilmiah Pariwisata dan Bisnis	2,023	cc-by	2,467	DOI: https://doi.org/10.22334/paris.v2i7. 492 * Corresponding Author: I Dewa Gede Unesa Megantara: unesamegantara510@gmail.com A R T I C L E I N F O A R T I C L E I N F O Article History: Submitted 17th July 2023 Revised 23th July 2023 Accepted 26th July 2023 Available online 30th July 2023 Kata Kunci: Kualitas keripik; Bengkuang; Kentang Keywords: Chips quality; Jicama; Potato Article History: Submitted 17th July 2023 Revised 23th July 2023 Accepted 26th July 2023 Available online 30th July 2023 Article History: Submitted 17th July 2023 Revised 23th July 2023 Accepted 26th July 2023 Available online 30th July 2023 Penelitian ini diupayakan untuk menjadikan bengkuang sebagai keripik. Penelitian ini bertujuan untuk mengetahui kualitas dari keripik bengkung yang dibandingkan dengan kualitas keripik kentang. Penelitian ini diharapkan dapat meningkatkan nilai ekonomis dari bengkuang dan dapat menghasilkan produk keripik bengkuang yang mempunyai sifat fisik dan organoleptik yang dapat diterima oleh konsumen. Dalam 7 hari, keripik bengkuang dan keripik kentang yang disimpan di wadah tertutup dan disuhu ruang tidak mengalami perubahan mulai dari segi tekstur, aroma, rasa, dan warna. Berdasarkan hasil dari penelitian yang telah dilaksanakan, dapat dinyatakan bahwa keripik bengkuang bisa dijadikan sebagai alternatif pengganti kentang dalam pembuatan keripik. Yang mana, data hasil penelitian yang diperoleh menunjukkan tidak ada perbedaan yang signifikan terkait dengan kualitas antara keripik bengkuang dengan keripik kentang. PARIS (Jurnal Pariwisata dan Bisnis) Vol 02 No 7, 2023: 1679-1684 https://dx.doi.org/10.22334/paris.v2i7 EISSN: 2828-3325 Open Access: https://paris.ipb-intl.ac.id/ PARIS (Jurnal Pariwisata dan Bisnis) Vol 02 No 7, 2023: 1679-1684 https://dx.doi.org/10.22334/paris.v2i7 EISSN: 2828-3325 Open Access: https://paris.ipb-intl.ac.id/ Differences in the Quality of Jicama Chips and Potato Chips A B S T R A C T This research attempted to make yam as chips. This study aims to determine the quality of yam chips compared to the quality of potato chips. This research is expected to increase the economic value of yam and can produce yam chips which have physical and organoleptic properties that are acceptable to consumers. Within 7 days, yam chips and potato chips stored in closed containers and at room temperature did not change in terms of texture, aroma, taste, and color. Based on the results of the research that has been carried out, it can be stated that yam chips can be used as an alternative to potato in making chips. In which, the research data obtained showed no significant difference related to the quality between yam chips and potato chips. 1679 EISSN: 2828-3325 EISSN: 2828-3325 Jurnal Ilmiah Pariwisata dan Bisnis Vol 02 No 7, 2023: 1679-1684 https://dx.doi.org/10.22334/paris.v2i7 https://paris.ipb-intl.ac.id/ Except where otherwise noted, content on this site is licensed under a Creative Commons Attribution 4.0 International license. (CC BY 4.0) 1. PENDAHULUAN Melalui penelitian ini, diharapkan keripik ini mampu menjadi produk yang berkualitas dan bermanfaat bagi 1680 Jurnal Ilmiah Pariwisata dan Bisnis Vol 02 No 7, 2023: 1679-1684 https://dx.doi.org/10.22334/paris.v2i7 EISSN: 2828-3325 EISSN: 2828-3325 pecinta cemilan, maupun menjadi referensi bagi pengembangan ilmu pengetahuan, khususnya bidang pangan di masa-masa mendatang. pecinta cemilan, maupun menjadi referensi bagi pengembangan ilmu pengetahuan, khususnya bidang pangan di masa-masa mendatang. 2. METODE zPENELITIAN Penelitian ini dilaksanakan di rumah peneliti yang berada Jalan Gunung Salak Gang Tegal Ayu No.3 Padangsambian Kelod, Denpasar Barat. Lokasi ini zditentukan berdasarkan beberapa alasan akademis yaitu fleksibilitas dan efisiensi waktu dalam pelaksanaan penelitian. Waktu yang dibutuhkan dalam melakukan penelitian ini dari tahap persiapan, pembuatan, hingga ujian dari bulan Desember 2021 zsampai Juli 2022. Dalam melakukan penelitian untuk eksperimen bagaimana zcara memanfaatkan bengkuang sebagai pengganti kentang dalam pembuatan keripik dengan zmelakukan zpengetesan zmelalui zuji zorganoleptik, zdan zuji zdaya ztahan zproduk. Jumlah zpanelis zdalam zpenelitian zini zberjumlah z20 zorang zresponden zuntuk zmenilai kualitas uji tes organoleptik (rasa, aroma, tekstur dan warna) panelis tersebut diantaranya orang tua kandung, adik kandung, keluarga sekitar, dan anak-anak sekolah. Jenis data yang digunakan adalah data kualitatif dan sumber data yang digunakan data primer, yaitu data yang diperoleh langsung dari hasil eksperiment dan observasi yang dilakukan oleh peneliti. Penulisan eksperimen ini menggunakan kuisioner dan juga observasi. 1. PENDAHULUAN Keripik atau kripik adalah sejenis makanan ringan berupa irisan tipis dari umbi- umbian, buah-buahan, atau sayuran yang digoreng di dalam minyak nabati. Untuk menghasilkan rasa yang gurih dan renyah biasanya dicampur dengan adonan tepung yang diberi bumbu rempah tertentu. Secara zumum zkeripik zdibuat zmelalui ztahap zpenggorengan, ztetapi zada zpula zdengan zhanya zmelalui zpenjemuran, zatau zpengeringan. zKeripik zdapat zberasa zdominan zasin, zpedas, zmanis, zasam, zgurih, zatau zpaduan zdari zkesemuanya. zKeripik zyang zbiasa zkita zjumpai zsalah zsatunya zyakni zkeripik zkentang. zDi zIndonesia, ztanaman zkentang zdapat zdikatakan zsangat zmudah zdijumpai, zterutama zdi zdaerah zdataran ztinggi zseperti zpegunungan. zKeripik zkentang zternyata zdibuat zpertama zkali zkarena zketidaksengajaan. Hal ztersebut zberbeda zdengan zbengkuang. zYang zmana, zbengkuang z(Pachyrhizus zerosus) zmerupakan zsalah zsatu zumbi zyang zbanyak zdigemari zkarena ztahan zcukup zlama zdalam zkeadaan zsegar, zenak zdimakan zmentah, zberaroma zkhas zdan zterasa zmanis z(Susanto, z2011). zKebanyakan zumbi zbengkuang zdikonsumsi zmasyarakat zdalam zkeadaan zsegar. zAkan ztetapi, zbengkuang zdulu zkurang zterkenal zdan ztidak zmemiliki znilai zekonomi zyang ztinggi. zMasalah zyang zdihadapi zpada zpenanganan zumbi zbengkuang zadalah zhasil zpanen zyang zmelimpah zdan zbelum zbegitu zdimanfaatkan zoleh zpengusaha zsehingga zpemasarannya zdalam zbentuk zproduk zolahan zmasih zterbatas. zOrang zmengenal zbengkuang zsebagai zumbi zyang zbentuknya zseperti zgasing. zPengolahan zumbi zbengkuang zbelum zbanyak zragamnya zkarena zmasih zterbatas zpenggunaannya zdan zkarena zitu zpenting zartinya zbila zdilakukan zpengolahan zsehingga zdapat zmeningkatkan zkegunaan, zserta znilai zekonomi zdari zumbi zbengkuang. Menurut zSusanto z(2011), zKandungan znutrisi zumbi zbengkuang zbermanfaat zdan zberkhasiat zuntuk zkesehatan. zKandungan zvitamin zB1 zumbi zbengkuang zdapat zmencegah zpenyakit zbiri-biri. zKandungan zvitamin zC zbengkuang zsangat zbaik zuntuk zmeningkatkan zdaya ztahan ztubuh zterhadap zserangan zpenyakit, zmencegah zsariawan zdan zpanas zdalam, zsekaligus zberfungsi zsebagai zantioksidan zyang zsangat zbaik zuntuk zmemperbaiki zjaringan zsel zyang zrusak. zUmbi zbengkuang zjuga zsangat zampuh zuntuk zmencegah zproduksi zasam zlambung zberlebih zyang zbisa zmenyebabkan zmaag. zDisamping zcukup zefektif zuntuk zmenurunkan zdemam, ztanaman zbengkuang zsangat zpenting zsebagai zbahan zherbal. zUmbi zbengkuang zjuga zdapat zdigunakan zindustri zpangan zuntuk zdiolah zmenjadi zbentuk zolahan, zmisalnya zkeripik, zmanisan zkering, zmanisan zbasah, zasinan zkering, zasinan zbasah, zaneka zminuman zsegar, ztepung zdan zbedak. Berdasarkan pertimbangan tersebut perlu adanya suatu proses agar bengkuang dapat dimanfaatkan sebagai salah satu alternatif bahan pangan yang dapat berkontribusi dalam kesehatan masyarakat sehingga dapat menjadi makanan ringan sehat bagi masyarakat. Salah satu cara pemanfaatan bengkuang adalah dengan menjadikannya keripik. Hal tersebut membuat peneliti tertarik untuk menggunakan bengkuang sebagai bahan pokok dalam membuat keripik. Hasil zpenelitian Penelitian ini dilakukan dengan membandingkan antara kualitas keripik bangkuang dengan keripik kentang. Adapun hasil yang diamati adalah skor yang di uji dari keseluruhan dari rasa, warna, tekstur, dan aroma. Eksperimen yang dilakukan menghasilkan 2 (dua) specimen, yaitu (P0) keripik kentang, (P1) keripik bengkuang. Hasil eksperimen ini kemudian di uji oleh 20 panelis untuk menilai kualitas uji tes organoleptik (rasa, aroma, tekstur dan warna) panelis tersebut di antaranya orang tua kandung, adik kandung, keluarga sekitar, dan anak-anak sekolah. 1681 https://paris.ipb-intl.ac.id/ Except where otherwise noted, content on this site is licensed under a Creative Commons Attribution 4.0 International license. (CC BY 4.0) Tabel z1. zRekap zPerbandingan zSkor zKeripik zKentang z(P0) zdan zKeripik zBengkuang z(P1)(Sumber:penulis,2022) No Indikator Eksperimen (P0) z (P1) z 1 Rasa 83 80 2 Warna 75 86 Tabel z1. zRekap zPerbandingan zSkor zKeripik zKentang z(P0) zdan zKeripik zBengkuang z(P1)(Sumber:penulis,2022) No Indikator Eksperimen (P0) z (P1) z 1 Rasa 83 80 2 Warna 75 86 l z1. zRekap zPerbandingan zSkor zKeripik zKentang z(P0) zdan zKeripik zBengkuang z(P1)(Sumber:penulis,2022) 1681 https://paris.ipb-intl.ac.id/ Except where otherwise noted, content on this site is licensed under a Creative Commons Attribution 4 0 International license (CC BY 4 0) 1681 https://paris.ipb-intl.ac.id/ Except where otherwise noted, content on this site is licensed under a Creative Commons Attribution 4.0 International license. (CC BY 4.0) 1681 https://paris.ipb-intl.ac.id/ Except where otherwise noted, content on this site is licensed under a Creative Commons Attribution 4.0 International license. (CC BY 4.0) Jurnal Ilmiah Pariwisata dan Bisnis EISSN: 282 Vol 02 No 7, 2023: 1679-1684 https://dx.doi.org/10.22334/paris.v2i7 3 Tekstur 85 80 4 Aroma 78 80 Total 321 326 Jurnal Ilmiah Pariwisata dan Bisnis Vol 02 No 7, 2023: 1679-1684 https://dx.doi.org/10.22334/paris.v2i7 EISSN: 2828-3325 EISSN: 2828-3325 Berdasarkan zdari zhasil zdiatas zmenunjukkan zbahwa zperbedaan zkualitas zdari zkeripik zbengkuang zdengan zkeripik zkentang ztidak zjauh zberbeda zdikarenakan zkeripik zbengkuang zmemiliki zkualitas zyang zhampir zsama zdengan zkeripik zkentang. Berdasarkan zdari zhasil zdiatas zmenunjukkan zbahwa zperbedaan zkualitas zdari zkeripik zbengkuang zdengan zkeripik zkentang ztidak zjauh zberbeda zdikarenakan zkeripik zbengkuang zmemiliki zkualitas zyang zhampir zsama zdengan zkeripik zkentang. Selanjutnya zberdasarkan zuji ztahan zlama zuntuk zmengetahui zmasa zkadaluarsa zproduk zyang zditinjau zdari zmutu zproduk zkeripik zbengkuang zperlu zdilakukan zpenelitian zdan zmenjelaskan zmasa zsimpan zyang ztepat zsebelum zmutunya zmenurun. zMetode zyang zdigunakan zuntuk zmenguji zdaya ztahan zpada zkeripik zbengkuang zyaitu zdengan zmenyimpan zdi zwadah ztertutup zdan zdisimpan zpada zsuhu zruangan. zHasil zdari zpengamatan zyang zdiperoleh zsebagai zberikut: Tabel z2. Hasil zpenelitian zRekap zData zDaya zTahan zKeripik zBengkuang zpada zsuhu zruangan (Sumber:penulis,2022) KRITERIA PERIODE zWAKTU HARI zKE z-1 HARI zKE z- z2 HARI zKE z– z3 HARI zKE z- z4 HARI zKE z-5 HARI zKE z- z6 HARI zKE z- z7 Warna Coklat zCerah Coklat zCerah Coklat zCerah Coklat zCerah Coklat zCerah Coklat zCerah Coklat zCerah Aroma Harum Harum Harum Harum Harum Harum Harum Rasa Enak Enak Enak Enak Enak Enak Enak Tekstur Renyah Renyah Renyah Renyah Renyah Renyah Renyah 1682 https://paris.ipb-intl.ac.id/ Except where otherwise noted, content on this site is licensed under a Creative Commons Attribution 4.0 International license. (CC BY 4.0) Tabel z2. zRekap zData zDaya zTahan zKeripik zBengkuang zpada zsuhu zruangan (Sumber:penulis,2022) KRITERIA PERIODE zWAKTU HARI zKE z-1 HARI zKE z- z2 HARI zKE z– z3 HARI zKE z- z4 HARI zKE z-5 HARI zKE z- z6 HARI zKE z- z7 Warna Coklat zCerah Coklat zCerah Coklat zCerah Coklat zCerah Coklat zCerah Coklat zCerah Coklat zCerah Aroma Harum Harum Harum Harum Harum Harum Harum Rasa Enak Enak Enak Enak Enak Enak Enak Tekstur Renyah Renyah Renyah Renyah Renyah Renyah Renyah Tabel z3. zRekap zData zDaya zTahan zKeripik zKentang zpada zsuhu zruangan (Sumber:penulis,2022). KRITERIA PERIODE zWAKTU HARI zKE z-1 HARI zKE z - z2 HARI zKE z– z3 HARI zKE z - z4 HARI zKE z -5 HARI zKE z- z6 HARI zKE z- z7 Warna Coklat zKekuningan Coklat zKekuningan Coklat zKekuningan Coklat zKekuningan Coklat zKekuningan Coklat zKekuningan Coklat zKekuningan Aroma Harum Harum Harum Harum Harum Harum Harum Rasa Enak Enak Enak Enak Enak Enak Enak Tabel z2. zRekap zData zDaya zTahan zKeripik zBengkuang zpada zsuhu zruangan (Sumber:penulis,2022) 1682 https://paris.ipb-intl.ac.id/ Except where otherwise noted, content on this site is licensed under a Creative Commons Attribution 4.0 International license. (CC BY 4.0) Tabel z3. zRekap zData zDaya zTahan zKeripik zKentang zpada zsuhu zruangan (Sumber:penulis,2022). KRITERIA PERIODE zWAKTU HARI zKE z-1 HARI zKE z - z2 HARI zKE z– z3 HARI zKE z - z4 HARI zKE z -5 HARI zKE z- z6 HARI zKE z- z7 Warna Coklat zKekuningan Coklat zKekuningan Coklat zKekuningan Coklat zKekuningan Coklat zKekuningan Coklat zKekuningan Coklat zKekuningan Aroma Harum Harum Harum Harum Harum Harum Harum Rasa Enak Enak Enak Enak Enak Enak Enak Tabel z3. zRekap zData zDaya zTahan zKeripik zKentang zpada zsuhu zruangan (Sumber:penulis,2022). Hasil zpenelitian 1682 Jurnal Ilmiah Pariwisata dan Bisnis EISSN: 2828-3325 Vol 02 No 7, 2023: 1679-1684 https://dx.doi.org/10.22334/paris.v2i7 Tekstur Sangat zRenyah Sangat zRenyah Sangat zRenyah Sangat zRenyah Sangat zRenyah Sangat zRenyah Sangat zRenyah EISSN: 2828-3325 Berdasarkan zTabel z2 zdan ztabel z3 ztersebut zjika zditinjau zberdasarkan zuji zdaya ztahan zatau zmasa zkadaluarsa zpada zproduk zkeripik zbengkuang z zdan zkeripik zkentang zyaitu zsetelah zkeripik zbengkuang zdan zkeripik zkentang zditempatkan zpada zwadah ztertutup zdan zdisimpan zpada zsuhu zruangan ztidak zmenimbulkan zdampak zselama z7 zhari. Pemberdayaan zBengkuang zmenjadi zKeripik Dewasa zini, zsuatu zide zcerdas zagar zbengkuang zdapat zdiolah zmenjadi zproduk zyang zlebih zbervariasi zdan zmempunyai znilai zjual zserta zekonomis zlebih ztinggi zsangat zdiperlukan. zSehingga, zminat zmasyarakat zterhadap zbengkuang zkembali ztergugah. zOleh zsebab zitu, zdilakukan zusaha zuji zcoba zmengolah zbengkuang zmenjadi zkeripik. zUji zcoba zini zdiharapkan zbisa zmemunculkan zalternatif zbaru zdalam zpemanfaatan zbengkuang. Berdasarkan zhasil zdari zpenelitian zyang ztelah zdilaksanakan, zdapat zdinyatakan zbahwa zkeripik zbengkuang zbisa zdijadikan zsebagai zalternatif zpengganti zkentang zdalam zpembuatan zkeripik. zYang zmana, zdata zhasil zpenelitian zyang zdiperoleh zmenunjukkan ztidak zada zperbedaan zyang zsignifikan zterkait zdengan zkualitas zantara zkeripik zbengkuang zdengan zkeripik zkentang. zSelain zitu, zbengkuang zsebagai zbahan zpokok zpembuatan zkeripik zsangat zmudah zdidapatkan. zJika zdilihat zdari zsegi zmanfaatnya, zbengkuang ztidak zkalah zsehatnya zdibandingkan zdengan zkentang. https://paris.ipb-intl.ac.id/ Except where otherwise noted, content on this site is licensed under a Creative Commons Attribution 4.0 International license. (CC BY 4.0) 4. SIMPULAN Penelitian ini menunjukkan bahwa perbedaan kualitas dari keripik bengkuang dengan keripik kentang tidak jauh berbeda dikarenakan keripik bengkuang memiliki proses pengolahannya hampir sama dengan keripik kentang. Berdasarkan uji daya tahan atau masa kadaluarsa keripik bengkuang dengan keripik kentang dapat bertahan selama 10 hari dalam suhu ruangan dan setelah itu di hari keberikutnya akan muncul beberapa tanda – tanda seperti kurangnya tekstur renyah pada keripik bengkuang dan kentang. Berdasarkan hasil dari penelitian yang telah dilaksanakan, dapat dinyatakan bahwa keripik bengkuang bisa dijadikan sebagai alternatif pengganti kentang dalam pembuatan keripik. Yang mana, data hasil penelitian yang diperoleh menunjukkan tidak ada perbedaan yang signifikan terkait dengan kualitas antara keripik bengkuang dengan keripik kentang. Selain itu, bengkuang sebagai bahan pokok pembuatan keripik sangat mudah didapatkan. Jika dilihat dari segi manfaatnya, bengkuang tidak kalah sehatnya dibandingkan dengan kentang. 1683 https://paris.ipb-intl.ac.id/ Except where otherwise noted, content on this site is licensed under a Creative Commons Attribution 4.0 International license. (CC BY 4.0) 1683 EISSN: 2828-3325 EISSN: 2828-3325 Jurnal Ilmiah Pariwisata dan Bisnis Vol 02 No 7, 2023: 1679-1684 https://dx.doi.org/10.22334/paris.v2i7 Ucapan Terimakasih Pertama-tama penulis ingin memanjatkan puji syukur kedapa Tuhan Yang Maha Esa, karena atas berkat-Nya penulis dapat menyelesaikan tugas akhir tepat pada waktunya. Penulis menyadari bahwa tugas akhir ini tidak akan dapat terselesaikan tanpa adanya dukungan, bimbingan, dan nasehat dari orang tua, teman, dosen pembimbing, serta orang-orang tercinta. 5. DAFTAR PUSTAKA Apriyanto, A., D. Fardiaz, N.L. Puspitasari, S. Yasni dan S. Budiyanto. 1989. Petunjuk Praktikum Analisis Pangan. IPB Press, Bogor. Ega, A. (2017). Pengaruh Perbedaan Metode Pengeringan Bengkuang (Pachyrhizus erosus) Terhadap Sifat Fisikokimia dan Organoleptik Keripik yang Dihasilkan (Doctoral dissertation, Universitas Andalas). stiasih, T dan K. Ahmadi. 2009. Teknologi Pengolahan Pangan. PT Bumi Aksara. Jaka https://adoc.pub/prostdcg-senicrior-nobcortol-teknolog-inovatrf-pascoponen- un.html (Diakses pada tanggal 30 Mei 2022) https://ummaspul.e-journal.id/pengabdian/article/view/2205 (Diakses pada tanggal 30 Mei 2022) http://scholar.unand.ac.id/23114/2/I.%20PENDAHULUAN%20aaa%20aplouad.pd f (Diakses pada tanggal 31 Mei 2022) http://zmtirta.blogspot.com/2017/04/keripik-bengkuang-manfaat-resep-dan.html (Diakses pada tanggal 31 Mei 2022) https://www.scribd.com/presentation/426778591/Keripik-bengkoang (Diakses pada tanggal 1 Juni 2022) https://docplayer.info/49555900-Usaha-keripik-bengkoang-guna-meningkatkan- nilai-jual-buah-bengkoang-sebagai-salah-satu-ciri-khas-kecamatan- prembun-kabupaten-kebumen.html (Diakses pada tanggal 1 Juni 2022) Kamsiati, E. (2010). Peluang pengembangan teknologi pengolahan keripik buah dengan menggunakan penggoreng vakum. Jurnal Litbang Pertanian, 29(2), 73-77. Maulana, W. (2019). Modifiksi mesin perajang kentang bergelombang portable dengan motor listrik 200 watt (Doctoral dissertation, Universitas Bangka Belitung). Maulana, W. (2019). Modifiksi mesin perajang kentang bergelombang portable dengan motor listrik 200 watt (Doctoral dissertation, Universitas Bangka Belitung). Sulistywati, A. 1999. Membuat Keripik Buah dan Sayur. Puspaswara. Jakarta. Sulistywati, A. 1999. Membuat Keripik Buah dan Sayur. Puspaswara. Jakarta. 1684 https://paris.ipb-intl.ac.id/ Except where otherwise noted, content on this site is licensed under a Creative Commons Attribution 4.0 International license. (CC BY 4.0)
https://openalex.org/W2093954773	https://pure.rug.nl/ws/files/16069379/journal.pone.0112806.pdf	English	null	Individual Differences in Learning a Novel Discrete Motor Task	PloS one	2,014	cc-by	10,967	University of Groningen University of Groningen University of Groningen Individual Differences in Learning a Novel Discrete Motor Task enia, Laura; Schoemaker, Marina M.; Mouton, Leonora J.; Bongers, Raoul M. DOI: 10.1371/journal.pone.0112806 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 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Golenia, L., Schoemaker, M. M., Mouton, L. J., & Bongers, R. M. (2014). Individual Differences in Learning a Novel Discrete Motor Task. PLoS ONE, 9(11), Article e112806. https://doi.org/10.1371/journal.pone.0112806 Citation for published version (APA): Golenia, L., Schoemaker, M. M., Mouton, L. J., & Bongers, R. M. (2014). Individual Differences in Learning a Novel Discrete Motor Task. PLoS ONE, 9(11), Article e112806. https://doi.org/10.1371/journal.pone.0112806 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). Abstract 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. pyright: 2014 Golenia et al. This is an open-access article distributed under the terms of the Creative Commons Attribution L restricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: These authors have no support or funding to report. Funding: These authors have no support or funding to report. Funding: These authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * Email: r.m.bongers@umcg.nl * Email: r.m.bongers@umcg.nl Copyright 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/taverne- amendment. 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. 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. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to 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. 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: 24-10-2024 Individual Differences in Learning a Novel Discrete Motor Task Laura Golenia, Marina M. Schoemaker, Leonora J. Mouton, Raoul M. Bongers* University of Groningen, University Medical Center Groningen, Center for Human Movement Sciences, Groningen, The Netherlands Abstract Many motor learning studies focus on average performance while it is known from everyday life experience that humans differ in their way of learning new motor tasks. This study emphasises the importance of recognizing individual differences in motor learning. We studied individual tool grasping profiles of individuals who learned to pick up objects with a novel tool, a pair of pliers. The pair of pliers was attached to the thumb and the index finger so that the tip of the thumb and the tip of the index finger were displaced to the beaks of the pair of pliers. The grasp component was manipulated by varying the location of the hinge of the pair of pliers, which resulted in different relations between beak opening and closing and finger opening and closing. The Wider Beak group had the hinge at 7 cm, the Same Beak group had the hinge at 10 cm (i.e., in the middle), and the Smaller Beak group had the hinge at 13 cm from the digits. Each group consisted of ten right- handed participants who picked up an object with one of the pairs of pliers 200 times on two subsequent days. Hand opening, plateau phase, hand closing, grasping time and maximum aperture were analyzed. To characterize individual changes over practice time, a log function was fitted on these dependent variables and the ratio of improvement was determined. Results showed that at the beginning stage of tool use learning the characteristic grasping profile consisted of three phases; hand opening, plateau phase and hand closing. Over practicing individual participants differed in the number of phases that changed, the amount of change in a phase and/or the direction of change. Moreover, with different pliers different learning paths were found. The importance of recognizing individual differences in motor learning is discussed. Schoemaker MM, Mouton LJ, Bongers RM (2014) Individual Differences in Learning a Novel Discrete Motor Task. PLoS ONE 9(11): e112 one.0112806 oemaker MM, Mouton LJ, Bongers RM (2014) Individual Differences in Learning a Novel Discrete Motor Task. PLoS ONE 9(11): e112806 0112806 Editor: Andrea Antal, University Medical Center Goettingen, Germany Received April 16, 2014; Accepted October 17, 2014; Published November 11, 2014 Copyright: 2014 Golenia et al. November 2014 \| Volume 9 \| Issue 11 \| e112806 Introduction individual differences are already found during development, developmental literature can be taken as an inspiring model and as starting point for adult motor learning studies. The dynamical system framework has influenced the field of developmental studies substantially, hence, one can also look at individual differences in adult motor learning from this perspective. This framework proposes that movements are produced from the interactions among person, task and environment e.g. [13,14]. Properties of the sub-systems making up the person, the task, or the environment determine the result of these interactions. Zanone & Kelso [15], Kelso & Zanaone [16] and a more recent study by Kostubiec et al. [7] focused on adult motor learning of rhythmic tasks by examining learning of new relative phase relations between two fingers that are rhythmically moved. It was shown that individual differences reflect differences in the individuals’ intrinsic dynamics, thus learning of rhythmically motor tasks occurs on the background of pre-existing repertoires of the individual learner. The current study on individual differences of adult motor learning was inspired by both developmental studies and studies in adults from a dynamical system framework. However, in the current study a new aspect of individual differences is examined: individual differences in learning a novel discrete task instead of a novel rhythmic task. Because of this, the The key interest in motor learning studies was, and often still is, to find general laws by averaging performance across several participants, as for instance illustrated by power laws of learning [1,2]. However, showing generality in learning does not inform us about possible individual differences in motor learning between participants. Throughout the last decades, the few motor learning studies that did examine the nature of individual differences, have shown evidence of the importance of individual differences for both theoretical and practical aspects of motor learning e.g. [3–8]. To provide further evidence of individual differences in motor learning, the current study addresses individual differences in performance over time when learning a novel discrete motor task. The performance curve along which one individual evolves over time is what we define as the individual learning path. Individual learning paths are studied by examining grasping profiles over practicing to grasp an object with a novel tool, a pair of pliers. Individual differences have been emphasized in developmental studies across different tasks and movements e.g. [9–12]. Introduction Here, we study whether there are differences between individuals in the number of phases of tool grasping that change throughout practicing, in the directions of change and in the magnitude of change in these phases, aiming to reveal differences in individual learning paths. Some earlier studies [3–6,8] although not from a dynamical systems approach, did emphasize individual differences in motor learning. However, these studies did not examine how individual performance evolves over practicing because performance was only analysed at discrete moments in time; either at the beginning [6], at the end [3,5] or at the beginning and towards the end of learning [4,8]. For example, a study analysing the beginning stage of learning by King et al. [6] examined how individuals minimize a performance score, composed of spatial error and movement time, in a star tracing task. Results showed that different groups could be distinguished, one reducing spatial error, one reducing movement time and another one reducing both variables [6]. Cesqui et al. [5], using experts who were able to show consistent behaviour in an unconstrained one-handed ball catching task, showed that also at the end stage of learning different ways of catching a ball can be observed. Although these papers pointed to differences between individuals they did not address how the performance of one individual evolves over practicing, thus individual learning paths were not analysed. To get a better understanding of individual learning paths, we manipulated the grasp component by varying the location of the hinge of the pair of pliers. Varying the hinge location over the handles while keeping the length of the handles the same altered the aperture ratio between digits and beaks, which may have an impact on the grasping profile and therefore on the individual learning paths. Summarizing, the importance of emphasizing individual differences has been shown in developmental studies and in studies conducted from a dynamical system framework. To get a better understanding of individual differences in motor learning, the current study focused on individual differences in a novel discrete task. The aim of the current study was therefore to examine individual differences in how participants learn to use a novel pair of pliers when objects have to be picked up. Introduction Developmental studies regarding the development of goal-directed reaching showed that individual differences are present very early in development [11,12]. It was shown that infants differed in the timing of reach onset and the transition to stable periods when learning to reach during the first year [11,12]. Considering that The key interest in motor learning studies was, and often still is, to find general laws by averaging performance across several participants, as for instance illustrated by power laws of learning [1,2]. However, showing generality in learning does not inform us about possible individual differences in motor learning between participants. Throughout the last decades, the few motor learning studies that did examine the nature of individual differences, have shown evidence of the importance of individual differences for both theoretical and practical aspects of motor learning e.g. [3–8]. To provide further evidence of individual differences in motor learning, the current study addresses individual differences in performance over time when learning a novel discrete motor task. The performance curve along which one individual evolves over time is what we define as the individual learning path. Individual learning paths are studied by examining grasping profiles over practicing to grasp an object with a novel tool, a pair of pliers. Individual differences have been emphasized in developmental studies across different tasks and movements e.g. [9–12]. Developmental studies regarding the development of goal-directed reaching showed that individual differences are present very early in development [11,12]. It was shown that infants differed in the timing of reach onset and the transition to stable periods when learning to reach during the first year [11,12]. Considering that November 2014 \| Volume 9 \| Issue 11 \| e112806 1 PLOS ONE \| www.plosone.org Individual Differences in Motor Learning methodological techniques used within the dynamical system framework are not used in the current study. hands [4,24,25]. This suggests that the characteristic grasping profile of the beginning stage of tool use learning consists of three phases; hand opening, plateau phase, and hand closing. Interest- ingly, Bouwsema et al. [24] showed that prosthesis users who are more skilled in using their prostheses have a shorter duration of the plateau phase than prosthesis users who are less skilled. Moreover, Bouwsema et al. [25] revealed that the plateau phase shortened over learning to grasp an object with a prosthesis, suggesting that the grasp profile changes over learning. Participants Thirty right-handed participants were semi-random distributed over three groups of ten (in each group 5 males and 5 females; age 21.161.68 year). Each participant had no prior experience using the particular pairs of pliers that were used in the current study. The participants had no neurological diseases, recent injuries or musculoskeletal problems in the neck, shoulder, arm or hand regions, and had normal or corrected to normal visual sight. Those criteria were verified by self-reports of the participants. The participants received verbal and written descriptions of all procedures and signed an informed consent before the experiment started. The tool that was used in the current study is a pair of pliers that is usually not used in daily living, assuring novelty of the task. The pair of pliers was attached to the thumb and the index finger so that the tip of the thumb and the tip of the index finger were displaced to the beaks of the pair of pliers. This is a tool that comes very close to a functional displacement of the tip of the thumb and index finger to the tip of the tool. In order to pick up an object with this pair of pliers, participants had to shape the aperture of the beaks of the tool as they moved the tool towards the object to be grasped. Thus, grasping an object with this pair of pliers required the participants to learn to coordinate hand opening and hand closing, which together make up the grasping profile. The ethics committee of the Center for Human Movement Sciences, University Medical Center Groningen approved the study that was conducted according to the principles expressed in the Declaration of Helsinki. Introduction The two key questions addressed in the current study are 1) how the different phases of tool grasping (hand opening, plateau phase and hand closing) evolve per individual throughout practicing and 2) whether the use of different pliers is learned differently. As the present study aims at revealing individual learning paths in a discrete task, we chose a goal-directed action with a novel tool. This choice was based on the following reasons: First of all, when performing goal-directed actions with a novel tool the movements of the body need to be transformed to movements of the new end- effector, the tool. These transformations are often complex [17– 19] and have to be learned. Secondly, studies about motor learning of goal-directed actions with a tool [3,4] pointed at the existence of individual differences. That is, Bouwsema et al. [4] indicated that participants who learned to control hand opening of a prosthetic device differed in their learning capacity. Biryukova & Bril [3] showed that expert stone knappers (detaching stone flakes) differed in the amount of kinetic energy transmitted to the stone and in the kinematic patterns of the arm. Again, these two studies did not analyse learning paths. Importantly, both studies demonstrate the suitability of studying individual differences in motor learning by means of a task in which participants have to learn to perform goal directed movements with a novel tool. Individual Differences in Motor Learning registered with one Optotrak 3020 system sensor (Northern Digital, Waterloo, Canada), at a sampling frequency of 100 Hz. Six markers were used, two markers were attached to the tips of the pairs of pliers, two markers on the legs near the digits, and two markers on the digits themselves (index finger and thumb). For the current study, only the markers on the tips of the pair of pliers were used for analyses. end of the phases. Thus, hand opening was defined as the time between the start of the hand opening and the end of hand opening; hand closing was defined as the time between the start of hand closing and the end of hand closing. The period from the end of hand opening to the start of hand closing was defined as the plateau phase. Maximum aperture was computed as the maxi- mum in the grasp component. Changes over time in in the variables grasp time, hand opening, plateau phase, hand closing and maximum aperture were analyzed and characterized on an individual level by using a set of statistical markers. The so-called ratio of improvement (E/B) and the R2 of a logarithmic fit (R2) of the practice trials were employed. These two variables were computed for each dependent variable (see later) and separately for each individual participant. First, the ratio of improvement of the different dependent variables was calculated using the mean of the first 15 trials of session 1 as begin value, and the mean of the last 15 trials of session 2 as end value of the relevant variables (E/B). The ratio of improvement is therefore a statistical marker that can be considered as a percentage-changed measure as it indicates the amount of change over practicing. In order to determine the consistency of the change over practicing, a second statistical marker was calculated; the R2. To determine the value of R2, for each of the dependent variables the learning rate (Equation 1) was fitted to the series averaged over blocks of five trials. The equation used was based on Newell et al. [20]: The task was performed at a table, in which a large television screen (Panasonic, 62*111 cm) was horizontally mounted and on which the starting location and object location was indicated. These locations were 30 cm apart in the anterior-posterior direction. Design The study was performed in two sessions that were conducted on two subsequent days. In each session, participants picked up the object with the pair of pliers 100 times, thus 200 times in total. Individual Differences in Motor Learning The object that had to be picked up was a grey wooden cylinder (diameter 3 cm, height 3.5 cm) [28–30]. Learning rate : Vs n ð Þ~Vinfzase{Ysn ð1Þ Where Vinf represents the asymptotic target value, a relates to the initial performance value and c represents the slope of the function representing the learning rate. Its parameters were determined using the fminsearch function in Matlab. R2 was then calculated with linear regression in SPSS. Material and apparatus When grasping an object with the natural hand using a pincer grip, thus without a tool, opening of the digits up to a maximum is usually immediately followed by closing of the digits around the object [20,21]. Therefore, most often a single peak is found in the natural grasping profile [22]. In tool grasping on the other hand, a plateau in the aperture profile is very consistently seen [4,23–27]. Bongers [23] and Gentilucci et al. [26] both reported the presence of a plateau phase when grasping with pairs of pliers even though the pairs of pliers that were used were very different in how they were held and how they transformed the movements of the fingers to the movement of the new end-effectors. Also during prosthetic use a plateau phase was reported in the hand aperture; when using a body-powered prosthesis [27] and when using myo-electric Three different pairs of pliers were tested, all with a length of 20 cm. The pairs of pliers differed in the location of the hinge (Figure 1). The first group (Wider Beak group) executed the task with the Wider Beak pair of pliers with the hinge placed 7 cm away from the digits resulting in the beak opening wider than the opening of the digits. The second group (Same Beak group) used the Same Beak pair of pliers in which the hinge was placed in the middle, 10 cm away from the digits, resulting in the beak opening to be the same as the opening of the digits. The third group (Smaller Beak group) used the Smaller Beak pair of pliers with the hinge located 13 cm away from the digits causing the beak opening to be smaller than digit opening. 3D trajectories were November 2014 \| Volume 9 \| Issue 11 \| e112806 PLOS ONE \| www.plosone.org 2 Individual Differences in Motor Learning Procedure Participants were asked to sit comfortably in a chair in front of the table, in such a way that the start and object location were aligned with the shoulder, parallel to the sagittal plane. One leg of the pliers was attached to the thumb and one to the index finger, using elastic bands. In all trials, participants started the task with the beaks and digits closed. Participants initiated the movement following a ‘ready signal’ of the experimenter. They were instructed to reach with the pair of pliers to the object as rapidly and accurately as possible, lift it up approximately 10 cm, put it down and hold on to it until the TV screen would turn black (the TV screen turned black after 3 s). Then participants let go of the object and returned the beak to the starting location for the next trial to start. It was chosen to let the participants pick up the object because it is a quite regular procedure in prehension studies [26,31] as it mimics the manipulation of the to be grasped object. Learning rate : Vs n ð Þ~Vinfzase{Ysn ð1Þ ð1Þ Results In total, 6000 trials were measured in the current study. 228 unusable trials were removed from the dataset because markers were invisible so that one or more of the variables could not be determined, or when the task was executed incorrectly, for instance when the object was dropped. This left 5772 trials that were used for analysis. Out of the trials where the object has been dropped, 67.4% occurred in the first 15 trials, indicating that participants failed to perform the task. This shows that the task cannot be performed right away, but has to be learned. Individual Differences in Motor Learning times (i.e. E/B,1), and from individuals showing no changes in grasping times over practicing (i.e. E/B = 1). Then, the R2 was also included and compared to the learning paths and the ratio of improvement. Based on this comparison, criterion values for the R2 and the ratio of improvement were chosen by each researcher. After consensus of the three researchers about the criterion values; the criterion value of R2 was set at 0.4 meaning that a R2 larger than 0.4 indicated that changes had occurred during the 200 trials. A ratio of improvement smaller than 0.65 or larger than 1.35 was taken as boundaries to state that changes had occurred (i.e., E/B, 0.65 or E/B.1.35 indicate a change). Thus, the combination of a low value of R2 and a ratio of improvement near one indicated that no changes had occurred. The third step in the procedure was to determine whether a participant showed changes over practicing in each dependent variable by scoring a ‘change’ if both scores for each dependent variable met the criterion value and scoring a ‘no change’ if no or only one criterion value was met. times (i.e. E/B,1), and from individuals showing no changes in grasping times over practicing (i.e. E/B = 1). Then, the R2 was also included and compared to the learning paths and the ratio of improvement. Based on this comparison, criterion values for the R2 and the ratio of improvement were chosen by each researcher. After consensus of the three researchers about the criterion values; the criterion value of R2 was set at 0.4 meaning that a R2 larger than 0.4 indicated that changes had occurred during the 200 trials. A ratio of improvement smaller than 0.65 or larger than 1.35 was taken as boundaries to state that changes had occurred (i.e., E/B, 0.65 or E/B.1.35 indicate a change). Thus, the combination of a low value of R2 and a ratio of improvement near one indicated that no changes had occurred. The third step in the procedure was to determine whether a participant showed changes over practicing in each dependent variable by scoring a ‘change’ if both scores for each dependent variable met the criterion value and scoring a ‘no change’ if no or only one criterion value was met. Grasping patterns The grasping pattern of all participants was characterized by the hand opening to a certain aperture close to the maximum (hand opening), minimal changes of that aperture for a certain time (plateau phase), followed by the closure around the object (hand closing) (Figure 2). During the plateau phase the hand opening velocity stayed around zero what can clearly be seen in Figure 2. Three aspects of these grasping patterns stood out: First, the grasping pattern of all three pairs of pliers showed a pronounced plateau phase. Importantly, this plateau phase was observable in the aperture profile of the first trials in all participants. Second, the length of the three phases (hand opening, plateau, hand closing) changed over practice in most of the participants. And third, the maximum aperture of the grasp differed for the different pairs of pliers; as expected the maximum aperture was widest for the Wider Beak plier and smallest for the Smaller Beak plier for most of the participants (Figure 2). The mean maximum aperture for the Same Beak plier was 54.01 (611.59), for the Wider Beak plier 61.92 (619.69) and for the Smaller Beak plier 38.31 (65.68). Results of the one-way ANOVA showed that the maximum aperture of the three pairs of pliers was significantly different (F(2,27) = 13.52, p,0.001). Post-hoc tests showed differences between the Same Beak plier (p,0.01) and the Smaller Beak plier as well as between the Wider Beak plier and the Smaller Beak plier (p,0.01). The term ‘practicing’ was used for repeating the task over the days and the term ‘learning’ was used for changes in behavior over time. Therefore, it can be said that changes when repeating the task over the days, reflected practicing, and a ‘change’ in both the ratio of improvement and the R2 was an indication of learning, as behavior changed over time. Data analysis A three step procedure was applied to examine the individual data. First, for each individual participant both the ratio of improvement and the R2 were calculated for all five dependent variables, i.e. maximum aperture, grasp time, hand opening, plateau phase, and hand closing. Second, criterion values for the R2 and the ratio of improvement were chosen to determine which participants showed a change in either of the dependent variables. The criterion values were determined independently by three different researchers. The three researchers independently perused the learning paths of individual participants visually and compared them first with the corresponding ratio of improvement. The focus was set at distinguishing individuals with a ratio of improvement that indicated prolongation of the grasping times over practicing (i.e. E/B.1), from individuals with a decrease in the grasping The trajectories of the tips of the pair of pliers were analyzed in Matlab (MathWorks; Natick, Massachusetts) using customized programs. Hand aperture was defined as the three-dimensional distance between the two markers on the beaks. Aperture velocity was computed with a three point difference algorithm. The total grasp time as well as the times used for the different movement phases (hand opening, plateau phase, and hand closing) were distinguished. To determine the duration of these phases, a backward and forward search was performed from the maximum (for hand opening) and minimum (for hand closing) in the grasp velocity profile until a threshold of 3 cm/s and 23 cm/s, respectively. The points closest to and above threshold for opening and below threshold for closing were taken as the beginning and Figure 1. The three pairs of pliers. Note that for each pair of pliers depicted the opening at the digit side (i.e., right side) is kept the same in each picture. doi:10.1371/journal.pone.0112806.g001 Figure 1. The three pairs of pliers. Note that for each pair of pliers depicted the opening at the digit side (i.e., right side) is kept the same in each picture. doi:10.1371/journal.pone.0112806.g001 p doi:10.1371/journal.pone.0112806.g001 November 2014 \| Volume 9 \| Issue 11 \| e112806 November 2014 \| Volume 9 \| Issue 11 \| e112806 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 3 Individual Differences in Motor Learning Individual learning path participants merely revealed changes in hand opening and/or closing time. Thus, participants differed in the number of phases that changed over learning. Figure 3 shows the data distribution of the ratio of improvement and the R2 for all participants for the variables hand opening, plateau phase and hand closing (data of Table 1). As can be seen for all variables the ratio of improvement and the R2 vary gradual over participants; there are no strong natural cuts in the data distributions. This indicates that there are a large variety of individual learning paths and that it is hard to distinguish clear-cut learning strategies. Therefore, to distinguish learning path categories the combination of the plots of the individual change over practicing, together with the values for the ratio of improvement and the R2 are used to determine the criterion values as described in the methods. These criterion values were chosen and shown in the figure with the red lines. For the R2 the red line indicates that the participants above the line were showing changes over practicing and for the ratio of improvement they indicate that the participants above the high line and below the low line were showing changes. Note that eventually both criteria must be complied for that variable of the participant to be marked as a change. The different learning paths that we found in this way are described in detail in the next paragraph. Regarding the magnitude of change, Figure 4 and 5 show that participants differed in the magnitude of change over practicing in the variables hand opening, plateau phase, and hand closing. This is evidenced by the values of R2 and the ratios of improvement (Table 1). In understanding and interpreting the numbers presented in Table 1, remember that the closer the value of R2 to one and the further away the ratio of improvement to one, the higher the magnitude of change. To depict one example, participant 11 who used the Same Beak plier showed a low magnitude of change in hand opening and closing but showed a high magnitude of change in plateau phase. This is demonstrated by ratios of improvement near one (E/B = 0.98 and E/B = 0.87) and low R2’s (0.02 and 0.05) for hand opening and closing and a low ratio of improvement (E/B = 0.37) and a high R2 (0.71) for plateau phase (Table 1). Individual learning path Finally, the direction of change over learning differed between participants. The duration of the phases could either increase (E/ B.1) or decrease (E/B,1). The majority of the participants showed a decrease of the duration of the phases, especially in the plateau phase. An increase in the duration occurred more often in hand opening and closing. There were substantial individual differences in learning paths: in number of phase that changed, in magnitude of change per variable and in direction of change, which can be seen in Figure 4 and 5 as well as in Tables 1 and 2. Concerning the number of phases that were changed over practicing, results revealed that twenty-five out of the thirty participants showed a change in at least one of the phases (Table 2). The remaining five participants did not show changes in any of the phases, indicating that when learning to use a novel pair of pliers learners and non-learners could be identified. Out of the twenty-five learning participants, twenty-one revealed changes in the duration of the plateau phase, whereby thirteen of these participants only showed changes in plateau phase but no changes in other variables, and six participants showed both changes in plateau phase as well as in hand opening and/or hand closing. Four out of the twenty-five In sum, individuals differed in the number of phases that changed, in magnitude of change per phase, and in the direction of change showing that there are strong individual differences in the learning paths. Quantitative analysis All statistical analyses were executed using SPSS software (IBM, Armonk, New York). To determine whether there is a difference between pairs of pliers in maximum aperture, a between-subject one-way ANOVA was conducted. A repeated measures multivar- iate ANOVA was performed with the R2 as dependent variable, and grasping phases (hand opening, plateau phase, hand closing) as within-subject variable and pairs of pliers as between-subject variable (Wider Beak, Same Beak, Smaller Beak). Post-hoc tests were performed with Bonferonni correction. The same analysis was performed for the ratio of improvement. The level of significance was set at a#0.05. Figure 2. Grasping patterns of three different individuals each using a different pairs of pliers. The top row shows grasping patterns from beginning of the first practice sessions and the bottom row from the end of the second practice session. The aperture profile is the dark line and the aperture velocity is the light line. doi:10.1371/journal.pone.0112806.g002 Figure 2. Grasping patterns of three different individuals each using a different pairs of pliers. The top row shows grasping patterns from beginning of the first practice sessions and the bottom row from the end of the second practice session. The aperture profile is the dark line and the aperture velocity is the light line. doi:10.1371/journal.pone.0112806.g002 November 2014 \| Volume 9 \| Issue 11 \| e112806 PLOS ONE \| www.plosone.org 4 Individual Differences in Motor Learning Figure 3. Data distribution of the R2 and the ratio of improvement of all participants. The R2 and the ratio of improvement are plotted for all participants for the variables hand opening, plateau phase and hand closing. The Wider Beak group is indicated by blue points, the Same Beak group by red points and the Smaller Beak group by green points. The red lines indicate the criterion values determined for the corresponding variable. doi:10.1371/journal.pone.0112806.g003 Figure 3. Data distribution of the R2 and the ratio of improvement of all participants. The R2 and the ratio of improvement are plotted for all participants for the variables hand opening, plateau phase and hand closing. The Wider Beak group is indicated by blue points, the Same Beak group by red points and the Smaller Beak group by green points. The red lines indicate the criterion values determined for the corresponding variable. doi:10.1371/journal.pone.0112806.g003 doi:10.1371/journal.pone.0112806.g003 doi:10.1371/journal.pone.0112806.g003 Aperture About 93% of the participants did not adjust maximum aperture over practicing which indicates that differences within the grasping phases are not due to the size of the aperture to which the hand opened (Figure 4 and 5). November 2014 \| Volume 9 \| Issue 11 \| e112806 PLOS ONE \| www.plosone.org 5 Individual Differences in Motor Learning Table 1. R2 and ratio of improvement (E/B) for all participants and dependent variables maximum aperture, grasp time, hand opening, plateau phase and hand closing. Plier Partic. Discussion The qualitative analysis of the individual data revealed that the different pairs of pliers showed different learning paths. The differences between the pliers became particularly clear in the number of phases that changed over learning. With two pliers, the Same Beak and the Smaller Beak, most of the participants changed the duration of the plateau phase over learning, whereas for the Wider Beak plier more often the duration of hand opening and hand closing changed. In the Same Beak group all participants who did show changes over learning revealed changes in the plateau phase over practicing (Table 1). Two out of the seven participants who showed changes in plateau phase also showed changes in hand opening or hand closing. All participants in the Smaller Beak group also revealed changes in plateau phase and three participants also showed changes in hand opening in addition to plateau phase. In the Wider Beak group only four participants changed plateau phase. Changes in hand opening and hand closing over learning occurred more often in the Wider Beak group (Table 1). This study demonstrated that individual differences should be taken into account when studying motor learning. We investigated changes in individual grasping profiles when practicing to pick up an object with a novel pair of pliers, a novel discrete motor task. The main findings of the current study can be summarized as follows: (a) individuals differed in their learning path, (b) the changes over learning to use a pair of pliers showed up most prominently in changes of the plateau phase, and (c) different pliers with different transformation rules showed different learning processes. Our approach of focusing on individual differences sets us apart from most of the motor learning studies that average across participants and thereby neglect individual differences resulting in possible inaccurate descriptions of practice related changes. We captured individual learning paths that may have been masked when employing averaging techniques in search for a general principle of learning. How recognizing individual differ- ences in the learning process can contribute to the understanding of motor learning will be discussed in the following. Results of the repeated measures ANOVA showed a main effect of plier for the dependent variable R2 (F (2,27) = 5.85, p,0.01). The mean R2 for hand opening for the Wider Beak plier was 0.41 (60.21), for the Same Beak plier 0.14 (60.16) and for the Smaller Beak plier 0.40 (60.31). Discussion For the Wider Beak plier the mean R2 for plateau phase was 0.36 (60.28), for the Same Beak plier 0.55 (60.29) and for the Smaller Beak plier 0.70 (60.11).The mean R2 for hand closing for the Wider Beak plier was 0.29 (60.18), for the Same Beak plier 0.23 (60.21) and for the Smaller Beak plier 0.34 (60.18). Post-hoc tests showed differences between the Same Beak plier and the Wider Beak plier (p,0.01). The R2 differed for the grasping phases (F (2,26) = 8.75, p = 0.01). The interaction between these effects was not significant. The ratio of improve- ment showed a main effect of grasping phase (F (2,26) = 9.19, p = 0.01) but no significant differences between pliers. The mean ratio of improvement for hand opening for the Wider Beak plier was 0.66 (60.23), for the Same Beak plier 1.00 (60.26) and for the Smaller Beak plier 0.79 (60.28). For the Wider Beak plier the mean ratio of improvement for plateau phase was 0.76 (60.31), for the Same Beak plier 0.50 (60.28) and for the Smaller Beak plier 0.44 (60.14).The mean ratio of improvement for hand closing for the Wider Beak plier was 0.69 (60.26), for the Same Beak plier 1.05 (60.27) and for the Smaller Beak plier 1.13 (61.07). The two main effects interacted (F (4,54) = 3.53, p,0.05), showing that the ratio of improvement was the same for the three phases for the Wider Beak plier but the ratio differed over the phases for the Same Beak and the Smaller Beak pliers. g g The key finding of the current study was that individuals revealed different learning paths. In particular, the tool grasping profile consisted of hand opening, plateau phase and hand closing and during learning, these phases changed in a different way per individual. Participants differed in the number of phases that changed, in the amount of change in each of the phases and in the direction of the change resulting in different individual learning paths. To our knowledge, individual learning paths have not yet been studied before this explicit in a discrete motor task and hence no methods were available to characterize the changes over learning of individual participants. As mentioned in the introduc- tion, the dynamical system framework e.g. [13,14] and several developmental studies e.g. [9–12] have emphasized individual differences. Aperture Assessment of changes in duration of hand opening, plateau phase and/or hand closing over learning. Changes in hand opening Changes in plateau phase Changes in hand closing Total number participants Number participants Wider plier Number participants Same plier Number participants Smaller plier 2 + 2 13 2 6 5 + ++ 2 6 2 1 3 2 + + 2 0 1 1 + 2 + 3 3 0 0 2 2 + 1 1 0 0 2 2 2 2 5 2 2 1 Notes: A ‘change’ (indicated with +) or ‘no change’ (indicated with 2) in the duration of hand opening, hand closing and plateau phase were scored based on the criteria of R2 and the ratio of improvement; both criteria must be complied. doi:10.1371/journal.pone.0112806.t002 Changes in hand opening Changes in plateau phase Changes in hand closing Total number participants Number participants Wider plier Number participants Same plier Number participants Smaller plier 2 + 2 13 2 6 5 + ++ 2 6 2 1 3 2 + + 2 0 1 1 + 2 + 3 3 0 0 2 2 + 1 1 0 0 2 2 2 2 5 2 2 1 Notes: A ‘change’ (indicated with +) or ‘no change’ (indicated with 2) in the duration of hand opening, hand closing and plateau phase were scored based on the criteria of R2 and the ratio of improvement; both criteria must be complied. doi:10.1371/journal.pone.0112806.t002 Notes: A ‘change’ (indicated with +) or ‘no change’ (indicated with 2) in the duration of hand opening, hand closing and plateau phase were scored based on the criteria of R2 and the ratio of improvement; both criteria must be complied. doi:10.1371/journal.pone.0112806.t002 Aperture Aperture Grasp Open Plateau Close E/B R2 E/B R2 E/B R2 E/B R2 E/B R2 Wider 1 0.97 0.03 0.54 0.72 0.25 0.73 0.69 0.27 0.44 0.41 2 0.61 0.27 0.75 0.40 0.63 0.45 1.04 0.12 0.37 0.49 3 0.93 0.24 0.62 0.61 0.34 0.73 0.86 0.14 0.37 0.47 4 0.75 0.59 1.04 0.00 0.82 0.35 1.26 0.40 0.58 0.45 5 0.98 0.05 0.92 0.14 0.79 0.43 1.04 0.01 0.89 0.20 6 0.77 0.67 0.84 0.26 0.73 0.26 0.98 0.05 0.77 0.20 7 1.21 0.27 0.58 0.85 0.60 0.40 0.45 0.73 1.19 0.03 8 1.33 0.52 0.52 0.83 0.64 0.43 0.40 0.79 0.79 0.03 9 1.16 0.48 0.62 0.72 0.86 0.09 0.50 0.58 0.85 0.16 10 0.89 0.44 0.57 0.74 0.97 0.18 0.42 0.47 0.72 0.44 Same 11 0.90 0.43 0.60 0.76 0.98 0.02 0.37 0.71 0.87 0.05 12 1.38 0.77 0.72 0.06 0.85 0.02 0.43 0.43 1.27 0.46 13 1.19 0.40 0.54 0.89 1.03 0.18 0.09 0.96 0.92 0.19 14 1.13 0.38 0.59 0.78 1.49 0.25 0.29 0.77 1.25 0.07 15 1.16 0.39 0.53 0.82 0.83 0.09 0.28 0.82 1.17 0.06 16 0.97 0.02 0.89 0.30 1.47 0.53 0.65 0.46 0.75 0.27 17 0.76 0.69 0.64 0.82 0.85 0.04 0.55 0.46 0.61 0.28 18 0.88 0.28 0.95 0.31 0.93 0.04 0.94 0.05 1.02 0.04 19 1.33 0.71 0.69 0.42 0.78 0.09 0.49 0.66 1.56 0.72 20 0.88 0.04 0.91 0.01 0.80 0.12 0.94 0.15 1.12 0.16 Smaller 21 0.85 0.77 0.50 0.72 0.79 0.05 0.37 0.74 0.36 0.68 22 0.78 0.63 0.53 0.66 1.42 0.03 0.35 0.75 1.27 0.51 23 0.99 0.06 0.46 0.86 0.59 0.76 0.37 0.69 0.74 0.28 24 0.90 0.37 0.44 0.81 0.54 0.65 0.34 0.69 0.82 0.18 25 1.02 0.15 0.52 0.80 1.06 0.01 0.36 0.74 2.77 0.25 26 0.92 0.22 0.80 0.43 0.77 0.15 0.78 0.42 3.35 0.29 27 0.98 0.14 0.61 0.83 0.92 0.30 0.48 0.78 0.74 0.16 28 1.05 0.21 0.48 0.80 0.68 0.60 0.35 0.73 0.72 0.35 29 0.97 0.04 0.52 0.71 0.68 0.77 0.43 0.62 0.41 0.19 30 1.22 0.69 0.57 0.87 0.48 0.66 0.55 0.79 1.34 0.55 d i 10 1371/j l 0112806 t001 Individual Differences in M PLOS ONE \| www.plosone.org 6 November 2014 \| Volume 9 \| Issue 11 \| e112806 November 2014 \| Volume 9 \| Issue 11 \| e112806 6 Individual Differences in Motor Learning Table 2. Discussion Because a discrete task was examined in the current study and not a rhythmical task, as is usually done in studies conducted within a dynamical system framework, the methods often used within this framework could not be applied in the current study. Therefore, the development of new methodological techniques was required. In the current study new potential techniques were introduced to analyze individual differences in a discrete task. We proposed to use the R2 of a logarithmic fit and the so called ratio of improvement, forming a combined measure to characterize individual learning paths. Due to the novelty of the current approach the criteria used to determine whether a change in behavior is scored as a change, were based solely on the current dataset. Using this combination of markers in future studies, November 2014 \| Volume 9 \| Issue 11 \| e112806 PLOS ONE \| www.plosone.org 7 Individual Differences in Motor Learning Figure 4. Individual learning paths. Changes over practice trials in hand opening, plateau phase, hand closing, and in aperture for all participants (10 participants in total for each pair of pliers). Blocks of five trials are presented; Block 1: Trial 1–5, Block 2: Trial 51–55, Block 3: Trial 101–105, Block 4: Trial 151–155, Block 5: Trial 195–200. Note that these are not the same blocks of trials that were used in the analyses. doi:10.1371/journal.pone.0112806.g004 Figure 4. Individual learning paths. Changes over practice trials in hand opening, plateau phase, hand closing, and in aperture for all participants (10 participants in total for each pair of pliers). Blocks of five trials are presented; Block 1: Trial 1–5, Block 2: Trial 51–55, Block 3: Trial 101–105, Block 4: Trial 151–155, Block 5: Trial 195–200. Note that these are not the same blocks of trials that were used in the analyses. doi:10.1371/journal.pone.0112806.g004 They demonstrate that participants who could at the outset of the study perform only two relative phase patterns in a stable manner showed different learning routes than participants who could perform more than two stable patterns at the outset. As mentioned before, we did not use the methods from the dynamical systems framework when investigating individual differences. Discussion However, when comparing the current study to the studies coming from this framework, it becomes clear that there is a large difference in the They demonstrate that participants who could at the outset of the study perform only two relative phase patterns in a stable manner showed different learning routes than participants who could perform more than two stable patterns at the outset. As mentioned before, we did not use the methods from the dynamical systems framework when investigating individual differences. However, when comparing the current study to the studies coming from this framework, it becomes clear that there is a large difference in the examining different tasks, might further inform about the generality of the settings of the criteria we used. A limitation of the method used is that the criteria used were inspired by the current dataset and therefore might be difficult to generalize. Moreover, although we distinguished different categories the data varied gradually. Studies examining individual differences in a rhythmical task [7,15,16], revealed groups who learned in two different ways: November 2014 \| Volume 9 \| Issue 11 \| e112806 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 8 Individual Differences in Motor Learning Figure 5. Individual changes in learning paths over the first trials of practice day 1. Changes over practice trials in hand opening, plateau phase, hand closing, and in aperture for all participants (10 participants in total for each pair of pliers) shown for block 1 (trial 1–5) and for block 2 (trials 51–55). doi:10.1371/journal.pone.0112806.g005 Figure 5. Individual changes in learning paths over the first trials of practice day 1. Changes over practice trials in hand opening, plateau phase, hand closing, and in aperture for all participants (10 participants in total for each pair of pliers) shown for block 1 (trial 1–5) and for block 2 (trials 51–55). doi:10.1371/journal.pone.0112806.g005 will be to find a way to characterise individual differences in a structured way and to better understand why they occur. number of different learning groups: six different learning groups were found in the current study whereas Kostrubiec et al. [7] found only two. A possible explanation for this could be the difference between tasks. However, finding too many learning groups could bear a challenge as it becomes difficult to find general principles of motor learning. Discussion Considering our result of finding individual differences in a healthy adult population, future studies should examine individual differences in heterogeneous patient groups who are known to have difficulty with learning new motor skills, such as children with Developmental Coordination Disorder (DCD). Based on the results of the current study, it should be expected to find pronounced differences between patients, which is supported by the relatively high standard deviations in performance measures of tracing movements of DCD children compared to typically developed children and adults [44]. Understanding these differ- ences would again improve our understanding of motor learning processes. Moreover, in rehabilitation, interventions focusing on enhancing motor skills should be tailored to the motor learning capacity and style of individual patients. This will help to better customize rehabilitation to the needs of patients and to improve its effect. The second main finding of the current study is that the pronounced plateau phase in the grasping profile can be seen in all participants from the first trials onward. It seems that in tool grasping, the plateau phase is an integral component of the grasping profile. This is in line with literature also showing the existing of the plateau phase in the grasping profile of various tools [4,23–27]. Currently, the origins of the plateau phase in tool prehension are far from being understood. It might well be that the existence of the plateau phase could be required in tool grasping because of the absence of proprioception. Because of the absence of the proprioceptive system, the control of movement occurs on the basis of the visual system only, which processes feedback slower than the proprioceptive system. The grasping network is said to operate on a very fast timescale [45]. Thus, it could be that the plateau phase emerges because of the slower processing of the visual information. The two origins of individual differences in the foregoing, that is, personal history and capacity to pick up information for learning, can be related to the literature on learning. Obviously, differences in individual motor experiences lay a foundation for the individual differences revealed here. One of the sources for differences in motor experiences is that individual differences are present very early in development, such as the development of reaching movements during infancy [11,12]. The capacity to pick up information for learning might originate from common genetic variations [39,40]. Discussion The challenge in the future A promising approach to perception-action learning that is helpful in understanding individual differences is the direct learning approach advocated by Jacobs and Michaels [32–34]. This view on learning is developed within the conceptual framework of the ecological approach, which has longstanding PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org November 2014 \| Volume 9 \| Issue 11 \| e112806 9 Individual Differences in Motor Learning ties with the dynamical systems framework to action [35–38]. The direct learning approach aims to explain how people change from using less useful variables to more useful variables over learning a perception-action task. Therefore, the learning behaviour of an individual is portrayed in an information space. An information space has on each axis a different source of perception-action information an individual can use to perform a certain task. Each position in that space represents the informational variable, which is a combination of the informational sources, used by the individual in a specific moment in time. In this space, learning can be seen as a path representing the sequence of variables an individual exploits during learning the task. In this direct learning approach individual differences could originate from at least two sources: First, the personal history of an individual could cause individuals to start at a different location in the information space. Different starting positions would lead to individual differences in learning because the path from less useful to more useful variables is different when the starting point (i.e., the less useful variable used in the beginning of learning) is different. Second, an important assertion of the direct learning approach is the existence of detectable information that specifies the path to follow to arrive at a more useful variable, which is called information for learning [32]. It seems reasonable to assume that individuals could differ in the capacity to pick up this information, hence differences in this capacity could be a source of individual differences. In short, the theory of direct learning provides some interesting leads to understand the origins of individual differences in learning an action. In the next paragraph we will turn to how this approach might be connected to other domains in the literature to reveal origins of individual differences. how participants acquire a novel task and can result in a deeper understanding of motor learning processes. Discussion For instance, a common variation (Val66Met polymorphism) in the Brain-derived neurotrophic factor (BDNF), which is encoded by the BDNF gene, affects the anatomy of the hippocampus and prefrontal cortex. Thus, such genetic variation can induce changes in morphology of brain areas involved in learning and memory [41]. Moreover, this same variation in BDNF is thought to modulate possible synaptic changes in the motor cortex following a simple motor learning task [39]. Together this shows that understanding individual differences is required to achieve a full understanding of motor learning processes. Following this suggestion, the slower processing speed of visual information could also explain why the plateau phase still exists towards the end of learning in most of the participants: First participants learned to rely on visual information only, thus the plateau phase decreased. It seems that the goal of learning is to resemble natural grasping. However, it could be possible that natural grasping cannot be exactly resembled with a pair of pliers, because it is inhibited by the slower processing speed of the visual system that will always result in a plateau phase. This supports the idea that the plateau phase is an integral component of tool grasping. This is in line with findings in the literature as Bouwsema et al. [4,24] showed that even experienced prosthesis users showed a plateau phase in their grasping pattern. Another finding was that different pliers with different transformation rules revealed different learning paths, that is, different changes in hand opening, plateau phase and hand closing between the Wider, Same, and Smaller pliers were observed. It should be noted that three groups with different participants were used whereby each group used a different pair of pliers. It could be that differences we found between the pliers were caused by differences between participants in the groups. However, partic- ipants were randomly divided into the three groups decreasing the likeliness of this possibility. In order to understand individual differences, they have to be recognized and analysed. Individual differences are sometimes observed in the literature but their potential for understanding motor learning is often overlooked. For instance, Campola et al. [42] examined movement variability during a static pointing task performed with a tool showing that individuals differed in how variability was explored. In the same line, Cluff et al. [43] studied joint recruitment and coordination processes without focusing on individual differences when learning pole-balancing. November 2014 \| Volume 9 \| Issue 11 \| e112806 References 1. Newell A, Rosenbloom PS (1981) Mechanisms of skill acquisition and the law of practice. In: Anderson JR, editor. Cognitive Skills and their Acquisition. Hillsdale: Erlbaum. pp. 1–51. 20. Jeannerod M (1984) The timing of natural prehension movements. J Mot Behav 16: 235–254. 21. Mon-Williams M, Tresilian TR (2001) A simple rule of thumb for elegant prehension. Current Biology 11: 1058–1061. pp 2. Snoddy GS (1926) Learning and stability. J Appl Psychol 10: 1–36. 22. Bongers RM, Zaal FT, Jeannerod M (2012) Hand aperture patterns in prehension. Hum Mov Sci 31: 487–501. 3. Biryukova EV, Bril B (2008) Organization of goal-directed action at a high level of motor skill: The case of stone knapping in India. Motor Control 12:181–209. 23. Bongers RM (2010) Do changes in movements after tool use depend on body schema or motor learning? In: Kappers AML, van Erp JGF, Bergmann Tiest WM, van der Helm FCT, editors. Haptics: Generating and Perceiving Tangible Sensations. Berlin Heidelberg: Springer. pp. 271–276. 4. Bouwsema H, van der Sluis CK, Bongers RM (2010) Learning to control opening and closing of a myoelectric hand. Arch Phys Med Rehabil 91: 1442– 1446. 5. Cesqui B, d’Avella A, Portone A, Lacquaniti F (2012) Catching a ball at the right time and place: Individual factors matter. PLoS One 7:e31770. 24. Bouwsema H, Kyberd PJ, Hill W, van der Sluis CK, Bongers RM (2012) Determining skill level in myoelectric prosthesis use with multiple outcome measures. J Rehabil Res Dev 49: 1331–1348. p 6. King AC, Ranganathan R, Newell KM (2012) Individual differences in the exploration of a redundant space-time motor task. Neurosci Lett 529: 144–149. 25. Bouwsema H, van der Sluis CK, Bongers RM (2014) Changes in performance over time while learning to use a myoelectric prosthesis. J Neuroeng Rehabil 11– 16: 1–15. 7. Kostrubiec V, Zanone PG, Fuchs A, Kelso JA (2012) Beyond the blank slate: Routes to learning new coordination patterns depend on the intrinsic dynamics of the learner-experimental evidence and theoretical model. Front Hum Neurosci 6: 1–14. 26. Gentilucci M, Roy AC, Stefanini S (2004) Grasping an object naturally or with a tool: are these tasks guided by a common motor representation? Exp Brain Res 157: 496–506. 8. Vegter RJK, Lamoth CJ, de Groot S, Veeger DHEJ, van der Woude LHV (2014) Inter-Individual Differences in the Initial 80 Minutes of Motor Learning of Handrim Wheelchair Propulsion. PLoS One 9: e89729. 27. Acknowledgments We thank Steffie Spruijt, Eirlys Pijpers, and Wybren Terpstra for their help with collecting the data, we thank Hans Tholen for constructing the pairs of pliers, we thank Wim Kaan and Emyl Smid for their help in developing the equipment and keeping it running, and Philipp Metzner for his help making the figures. We appreciate the useful feedback while writing this paper of Inge Tuitert, Christian Greve, and Tim Valk. Discussion When observing their data in reference to individual differences, results showed differences in joint configuration variability between participants. Thus, being open for possible individual differences when analyzing data can ensure a more accurate description of When learning to use a pair of pliers, participants had to adapt to the transformation assigned by the construction of the plier. The transformation of the Same Beak plier presupposed a one-to-one digit-beak mapping, whereas the transformation of the Wider Beak plier and the Smaller Beak plier presupposed that the digits-beak mapping is not one-to-one. Studies on tool transformation distinguish between compatible and incompatible tools [17,18]. November 2014 \| Volume 9 \| Issue 11 \| e112806 PLOS ONE \| www.plosone.org 10 Individual Differences in Motor Learning role in the understanding of motor learning and that individual differences should be considered more often in motor learning studies as well as in studies aiming to improve rehabilitation for patient groups. Incompatible tool transformations are defined as transformations where the direction of the movement of the hand does not correspond to the direction of the movement of the tool, which is in contrast to compatible tool transformations where the direction of both the movement of the hand and tool corresponds. This definition can also be conveyed to the current study, whereby the Same Beak plier reflected a compatible transformation and the Wider and Smaller Beak plier reflected an incompatible transfor- mation. Beisert et al. [17] showed that tools incorporating a compatible transformation rule were handled faster and more accurately than tools with an incompatible transformation rule. This is in agreement with the current study as the mean duration of the grasping time for the Same Beak plier is shorter (268 ms) than the mean duration of grasping time of the Wider Beak plier (339 ms) and the Smaller Beak plier (355 ms). However, this does not help to explain the differences between the pliers, because learning paths of the Same Beak plier correspond more with the Smaller Beak plier than do the learning paths of the Smaller Beak plier with the ones of the Wider Beak plier. Supporting Information Table S1 Data of analysis variables of all individuals. (XLS) Author Contributions In conclusion, the results of the current study showed individual learning paths when learning a novel discrete motor task. The motor learning differences we found stress the need for more individualized assessment of motor learning. Based on these findings we propose that individual differences play an important Conceived and designed the experiments: LG MMS LJM RMB. Performed the experiments: LG MMS LJM RMB. Analyzed the data: LG MMS LJM RMB. Contributed reagents/materials/analysis tools: LG MMS LJM RMB. Wrote the paper: LG MMS LJM RMB. References Wing AM, Fraser C (1983) The contribution of the thumb to reaching movements. 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Proceedings of the Human Factors and Ergonomics Society Annual Meeting 51: 1353–1357. 12. Thelen E, Corbetta D, Kamm K, Spencer JP, Schneider K, et al. (1993) The transition to reaching: Matching intention and intrinsic dynamics. Child Dev 64: 1058–1098. g 32. Jacobs DM, Michaels CF (2007) Direct Learning. Ecological psychology 19: 321–349. 13. Kelso JAS (1995) Dynamic Patterns: The Self-Organization of Brain and Behavior. Cambridge: Massachusetts Institute of Technology. 341 p. elso JAS (1995) Dynamic Patterns: The Self-Organization of Bra 33. Jacobs DM, Vaz DV, Michaels CF (2012) The learning of visually guided action: an information-space analysis of pole balancing. J Exp Psychol Hum Percept Perform 38: 1215–27. 14. Newell K (1986) Constraints on the development of coordination. In: Wade M, Whiting HTA, editors. Motor development in children: Aspects of coordination and control. Dordrecht: Martinus Nijhoff. pp. 341–360. 34. Michaels CF, Romaniak-Gross CA (2012) Geometric specification of dynamics: learning to visually perceive kinetic quantities from static images. Perception 41: 93–109. 15. Zanone PG, Kelso JAS (1992) Evolution of Behavioral Attractors With Learning: Nonequilibrium Phase Transitions. J Exp Psychol Hum Percept Perform 18: 403–421. 35. Michaels CF, Beek PJ (1995) The state of ecological psychology. Ecological psychology 7: 259–278. 16. Zanone PG, Kelso JAS (1997) Coordination Dynamics of Learning and Transfer: Collective and Component Levels. J Exp Psychol Hum Percept Perform 25: 1454–1480. 36. Turvey; MT (1990) Coordintation. p gy J p y 42. Campolo D, Widjaja F, Xu H, Ang WT, Burdet E (2012) Analysis of Accuracy in Pointing with Redundant Hand- held Tools: A Geometric Approach to the Uncontrolled Manifold Method. PLoS One 9: e1002978. 41. Pezawas L, Verchinski BA, Mattay VS, Callicott JH, Kolachana BS, et al. (2004) The brain-derived neurotrophic factor val66met polymorphism and variation in human cortical morphology. J Neurophysiol 24: 10099–10102. 40. Cheeran BJ, Ritter C, Rothwell JC, Siebner HR (2009) Mapping genetic influences on the corticospinal motor system in humans. Neuroscience 164: 156– 163. 44. Snapp-Childs W, Mon-Williams M, Bingham GP (2012) A Sensorimotor Approach to the Training of Manual Actions in Children With Developmental Coordination Disorder. J Child Neurol 28: 204–212. 45. Davare M, Kraskov A, Rothwell JC, Lemon RN (2011) Interactions between areas of the cortical grasping network. Curr Opin Neurobiol 21: 565–570. 43. Cluff T, Manos A, Lee TD, Balasubramaniam R (2012) Multijoint error compensation mediates unstable object control. J Neurophysiol 108: 1167–1175. Individual Differences in Motor Learning References American Psychologist 45: 938–953. 37. Beek PJ, Peper CE, Stegeman DF (1995) Dynamical models of movement coordination. Hum Mov Sci 14: 573–608. 17. Beisert M, Massen C, Prinz W (2010) Embodied rules in tool use: a tool- switching study. J Exp Psychol Hum Percept Perform 36: 359–372. 38. Zaal FTJM, Bootsma RJ, van Wieringen, PCW (1998) Coordination in prehension – Information-based coupling of reaching and grasping. Exp Brain Res 4: 427–435. 18. Massen C, Sattler C (2010) Bimanual interference with compatible and incompatible tool transformations. Acta Psychol (Amst) 135: 201–208. 39. Cheeran B, Talelli P, Mori F, Koch G, Suppa A, et al. (2008) A common polymorphism in the brain-derived neurotrophic factor gene (BDNF) modulates human cortical plasticity and the response to rTMS. J Physiol 586: 5717–5725. 19. Su¨lzenbru¨ck S, Heuer H (2009) Learning the visuomotor transformation of virtual and real sliding levers: simple approximations of complex transforma- tions. Exp Brain Res 195: 153–165. 11 November 2014 \| Volume 9 \| Issue 11 \| e112806 PLOS ONE \| www.plosone.org November 2014 \| Volume 9 \| Issue 11 \| e112806 November 2014 \| Volume 9 \| Issue 11 \| e112806 Individual Differences in Motor Learning November 2014 \| Volume 9 \| Issue 11 \| e112806 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 12
https://openalex.org/W3122368928	https://www.jstage.jst.go.jp/article/jbse/16/1/16_20-00516/_pdf	English	null	Cerebellar foliation via non-uniform cell accumulation caused by fiber-guided migration of granular cells	Journal of biomechanical science and engineering	2,021	cc-by	6,108	Abstract The cerebellum has a unique morphology characterized by fine folds called folia. During cerebellar morphogenesis, folia formation (foliation) proceeds with granule cell (GC) proliferation in an external granular layer, and subsequent cell migration to an internal granular layer (IGL). GC migration is guided along Bergmann glial (BG) fibers, whose orientation depends on the deformation of cerebellar tissue during folia formation. The aim of this study is to investigate the contribution of the fiber-guided GC migration on folia formation from a mechanical viewpoint. Based on a continuum mechanics model of cerebellar tissue deformation and GC dynamics, we simulated foliation process caused by GC proliferation and migration. By changing migration speeds, we showed that the fiber-guided GC migration caused the non-uniform accumulation of GCs and folia lengthening. Furthermore, the simulation of impaired GC migration under pathological conditions, where GCs did not migrate along BG fibers, revealed that fiber-guided GC migration was necessary for folia lengthening. These simulation results successfully recapitulated the features of physiological and pathological foliation processes and validated the mechanisms that guidance of GC migration by BG fibers causes folia lengthening accompanied by non-uniform IGL. Our computational approach will help us understand biological and physical morphogenesis mechanisms, facilitated by interactions between cellular activities and tissue behaviors. Keywords: Cerebellar morphogenesis, Foliation, Finite element analysis, Continuum mechanics, Cell migration, Tissue growth Journal of Biomechanical Science and Engineering Cerebellar foliation via non-uniform cell accumulatio caused by fiber-guided migration of granular cells Hironori TAKEDA,, Yoshitaka KAMEO,,, Takahiro YAMAGUCHI,, Kazunori NAKAJIMA** and Taiji ADACHI,,** Institute for Frontier Life and Medical Sciences, Kyoto University 53 Shogoin-Kawahara-cho, Sakyo, Kyoto 606-8507, Japan E-mail: adachi@infront.kyoto-u.ac.jp Department of Micro Engineering, Graduate School of Engineering, Kyoto University 53 Shogoin-Kawahara-cho, Sakyo, Kyoto 606-8507, Japan Department of Mammalian Regulatory Network, Graduate School of Biostudies, Kyoto University 53 Shogoin-Kawahara-cho, Sakyo, Kyoto 606-8507, Japan **Department of Anatomy, Keio University School of Medicine 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan J-STAGE Advance Publication date : 16 January, 2021 Paper No.20-00516 [DOI: 10.1299/jbse.20-00516] Vol.16, No.1, 2021 Vol.16, No.1, 2021 1. Introduction The cerebellum has a unique morphology, comprising fine folds called folia. This morphology is associated with cerebellar function, e.g., motor control and cognition, based on evidence that a dysfunctional cerebellum has morphological defects (Sillitoe and Joyner, 2007; Leto et al., 2016). Therefore, to understand formation mechanisms underpinning the functional cerebellum, it is vital to investigate folia formation during cerebellar morphogenesis. During cerebellar development, cerebellar morphology is formed via foliation mechanisms including folia formation, elongation, and bifurcation. This foliation process is driven by cellular activities including cell proliferation and migration (Corrales et al., 2006; Legue et al., 2016; Leto et al., 2016; Govek et al., 2018; Lejeune et al., 2019). At initial foliation stages, granule cell (GC) precursors proliferate in the external granular layer (EGL) (Sudarov and Joyner, 2007; Legue et al., 2015; Lawton et al., 2019). These GCs extend parallel fibers along a mediolateral axis, and they then migrate inwards along Bergmann glia (BG) fibers (Edmondson and Hatten, 1987; Yacubova and Komuro, 2002a). After migration J-STAGE Advance Publication date : 16 January, 2021 Paper No.20-00516 [DOI: 10.1299/jbse.20-00516] © 2021 The Japan Society of Mechanical Engineers 1 Takeda, Kameo, Yamaguchi, Nakajima and Adachi, Journal of Biomechanical Science and Engineering, Vol.16, No.1 (2021) through the EGL, molecular layer (ML), and Purkinje cell layer (PCL), GCs halt, and accumulate to generate an internal granular layer (IGL). Consequently, the IGL becomes thicker around a ridge (lobule), when compared to a folium groove (fissure). Additionally, the height of lobule increases relatively as the fissure position is fixed, leading to the formation of characteristic cerebellar morphology (Sudarov and Joyner, 2007). Fiber-guided GC migration is believed to be crucial for foliation mechanisms, as impaired GC migration and disorganized BG fibers cause abnormal foliation, including the decrease in lobule height (Men et al., 2015; Ryan et al., 2017; Hughes et al., 2020). A previous study proposed a mechanism of folia lengthening (increase in lobule height), suggesting that fiber-guided migration caused IGL with non-uniform GC accumulation, and subsequent folia outgrowth (Sudarov and Joyner, 2007). This study suggests that BG fibers radiate inward in a fan shape around fissure depending on the tissue deformation. Since the GCs migrate along BG fibers, GCs spread to larger area in the IGL. Conversely, around lobule, BG fibers radiate outward and GCs concentrate to smaller area in the IGL. 1. Introduction Therefore, the oriented BG fibers depending on tissue deformation may cause non-uniform GC accumulation and associated tissue growth in the IGL, which cause folia lengthening. Based on this foliation mechanism, we investigated the contribution of GC migration along BG fibers toward folia lengthening, using computational simulations. For foliation simulation, a continuum mechanics model of tissue deformation and cell migration was developed with reference to a previous cerebral morphogenesis study (Rooij and Kuhl, 2018). Based on our mathematical model, we simulated foliation caused by GC proliferation in the EGL, and then GC migration from the EGL to the IGL. By altering GC migration speeds, we showed that GC migration caused folia lengthening. Furthermore, a simulation of impaired GC migration under pathological conditions showed that fiber orientation was necessary for folia lengthening. 2.2 Modeling GC proliferation and migration To model GC proliferation and migration, the balance equation for cell number density 𝑐 in the current state was described as: To model GC proliferation and migration, the balance equation for cell number density 𝑐 in the current state was described as: = −∂(𝑐𝒗) 𝜕𝒙 + 𝐷∂2𝑐 𝜕𝒙2 + 𝑓𝑐, (4) 𝜕𝑐 𝜕𝑡+ 𝑐1 𝐽 𝜕𝐽 𝜕𝑡= −∂(𝑐𝒗) 𝜕𝒙 + 𝐷∂2𝑐 𝜕𝒙2 + 𝑓𝑐, (4) where 𝐽 (> 0) is the Jacobian of the deformation gradient tensor 𝑭, the vector 𝒗 is cell migration velocity, 𝐷 is a diffusion coefficient, and 𝑓𝑐 is the production rate of cell number density at the current state. The production rate at the initial state 𝐹𝑐 is represented using 𝑓𝑐 as 𝐹𝑐= 𝐽𝑓𝑐. The second term on the left side of Eq. (4) shows the effect of tissue deformation on cell number density. The first term on the right side of Eq. (4) shows an advection term for GC migration, the second one shows a diffusion term, and the third one shows a production term for GC proliferation. where 𝐽 (> 0) is the Jacobian of the deformation gradient tensor 𝑭, the vector 𝒗 is cell migration velocity, 𝐷 is a diffusion coefficient, and 𝑓𝑐 is the production rate of cell number density at the current state. The production rate at the initial state 𝐹𝑐 is represented using 𝑓𝑐 as 𝐹𝑐= 𝐽𝑓𝑐. The second term on the left side of Eq. (4) shows the effect of tissue deformation on cell number density. The first term on the right side of Eq. (4) shows an advection term for GC migration, the second one shows a diffusion term, and the third one shows a production term for GC proliferation. Under physiological conditions, where GC migration is guided along BG fibers, GC migration velocity 𝒗 is described by a directional unit vector 𝒂 denoting fiber orientation as: (5) 𝒗= \|𝒗\|𝒂. By assuming current fiber orientation 𝒂 depends on tissue deformation, thus 𝒂 is described by the deformation gradient tensor 𝑭 as: By assuming current fiber orientation 𝒂 depends on tissue deformation, thus 𝒂 is described by the deformation gradient tensor 𝑭 as: 𝒂= 𝑭𝒂0 \|𝑭𝒂0\|, (6) 𝒂= 𝑭𝒂0 \|𝑭𝒂0\|, (6) where 𝒂0 is the initial fiber orientation. Takeda, Kameo, Yamaguchi, Nakajima and Adachi, Journal of Biomechanical Science and Engineering, Vol.16, No.1 (2021) The cerebellar tissue was modeled as a continuum body. We considered points in the body, whose position was denoted by 𝑿 at the initial state. In the current (deformed) state at time 𝑡, the position of the same point was moved to 𝒙 by a deformation map χ (𝒙= χ(𝑿, 𝑡)). Tissue deformation was represented by the deformation gradient tensor 𝑭 defined as 𝑭 = ∂χ(𝑿, 𝑡)/ ∂𝑿. To model volumetric growth, we assumed that the deformation gradient tensor 𝑭 was multiplicatively decomposed into a growth part 𝑭g and an elastic deformation part 𝑭e (Rodriguez et al., 1994) as: 𝑭= 𝑭e𝑭g. (1) In this study, we assumed isotropic tissue growth because the anisotropy of GC accumulation in IGL was not observed. The deformation gradient 𝑭g can be described by using growth stretch 𝜃 as: (2) 𝑭g = 𝜃𝑰, where 𝑰 was a second order unit tensor. To model tissue growth caused by GC accumulation, we assumed that the volumetric growth depended on cell number density. Thus, the growth stretch was described as a function of cell number density 𝑐 (≥0) in the current state as: where 𝑰 was a second order unit tensor. To model tissue growth caused by GC accumulation, we assumed that the volumetric growth depended on cell number density. Thus, the growth stretch was described as a function of cell number density 𝑐 (≥0) in the current state as: 𝜃(𝑐) = (1 + 𝑘𝑐)𝛼, + 𝑘𝑐)𝛼, (3) 𝜃(𝑐) = (1 + 𝑘𝑐)𝛼, (3) where 𝑘 and 𝛼 are constants to represent the relationship between tissue growth and cell number density (Rooij and Kuhl, 2018). where 𝑘 and 𝛼 are constants to represent the relationship between tissue growth and cell number density (Rooij and Kuhl, 2018). 2.1 Continuum mechanics modeling for cerebellar tissue deformation 2.1 Continuum mechanics modeling for cerebellar tissue deformation During cerebellar morphogenesis, foliation proceeds with GC dynamics including GC proliferation in the EGL and GC migration to the IGL along BG fibers (Fig. 1A). To investigate foliation mechanisms, we developed a mathematical model of cerebellar tissue deformation, GC proliferation, and migration based on continuum mechanics. Fig. 1 Modeling cerebellar morphogenesis. (A) Folia formation is accompanied by granule cell (GC) proliferation in the external granular layer (EGL), and subsequent migration into the internal granular layer (IGL) along Bergmann glial (BG) fibers. This folia formation example shows murine cerebellar morphogenesis from embryonic day E16.5–18.5. (B) Initial morphology of cerebellar tissue for finite element analysis consisting of EGL, molecular layer (ML), Purkinje cell layer (PCL) and IGL. The tissue had a length 𝐿𝑥= 400 μm and a thickness 𝐿𝑦= 50 μm. Fig. 1 Modeling cerebellar morphogenesis. (A) Folia formation is accompanied by granule cell (GC) proliferation in the external granular layer (EGL), and subsequent migration into the internal granular layer (IGL) along Bergmann glial (BG) fibers. This folia formation example shows murine cerebellar morphogenesis from embryonic day E16.5–18.5. (B) Initial morphology of cerebellar tissue for finite element analysis consisting of EGL, molecular layer (ML), Purkinje cell layer (PCL) and IGL. The tissue had a length 𝐿𝑥= 400 μm and a thickness 𝐿𝑦= 50 μm. [DOI: 10.1299/jbse.20-00516] © 2021 The Japan Society of Mechanical Engineers 22 Takeda, Kameo, Yamaguchi, Nakajima and Adachi, g j Journal of Biomechanical Science and Engineering, Vol.16, No.1 (2021) g j Journal of Biomechanical Science and Engineering, Vol.16, No.1 (2021) Journal of Biomechanical Science and Engineering, Vol.16, No.1 (2021) 3. Cerebellar morphogenesis; simulation results 3. Cerebellar morphogenesis; simulation results 2.2 Modeling GC proliferation and migration To model impaired GC migration under pathological conditions, where GCs do not migrate along BG fibers, we randomly set an angle 𝜑r (−𝜑max/2 ≤𝜑r ≤𝜑max/2) between the fiber direction 𝒂 and the migration direction 𝒗. where 𝒂0 is the initial fiber orientation. To model impaired GC migration under pathological conditions, where GCs do not migrate along BG fibers, we randomly set an angle 𝜑r (−𝜑max/2 ≤𝜑r ≤𝜑max/2) between the fiber direction 𝒂 and the migration direction 𝒗. Through abovementioned modeling, we constructed the mathematical model for GC migration guided by the fiber orientation 𝒂, which changes from the initial orientation 𝒂0 depending on tissue deformation 𝑭, as formulated in Eqs. (5) and (6). The cell number density of GC temporally changes through GC migration and production according to Eq. (4) and affects tissue growth and subsequent tissue deformation, as per Eq. (1-3). [DOI: 10.1299/jbse.20-00516] © 2021 The Japan Society of Mechanical Engineers 23 2.3 A simulation model for cerebellar foliation To investigate foliation mechanisms, we performed numerical simulations based on the above models, using a finite element method. Cerebellar tissue, consisting of EGL, ML, PCL, and IGL, was modeled in a two-dimensional space by assuming plane strain condition (Fig. 1B). Based on experimental observations (Sudarov and Joyner 2007), the tissue had a length 𝐿𝑥= 400 μm and a thickness 𝐿𝑦= 50 μm, at the initial state (𝑡= 0) and each layer had the same thickness. By presupposing the presence of folia at the embryonic day E16.5 (Sudarov and Joyner 2007), the initial tissue shape was slightly curved and followed a sine function having an amplitude of 5 μm. In numerical simulations, the out- of-plane displacement on the lateral surfaces was constrained with fixed top points. During cerebellar foliation, not all the GCs in the EGL proliferate. We used a cell number density c of the migrating GCs, which do not have a proliferative ability, and assumed that a fixed number of proliferative GCs existed in EGL. Therefore, to model GC production in the EGL, the production rate of cell number density 𝐹𝑐(= 𝐽𝑓𝑐) was set to 𝐹𝑐= 𝐹0 𝑐 (𝐹0 𝑐> 0), and 𝐹𝑐= 0 in the other layers. To model GC migration from the EGL to the IGL, the migration speed \|𝒗\| was set to \|𝒗\| = 𝑣0 (𝑣0 ≥0 ) in the EGL, ML and PCL, and \|𝒗\| = 0 in the IGL. The migration direction was determined depending on BG fiber orientation as described in Eq. (5). The directional unit vector of fiber orientation 𝒂0 at the initial state was set to 𝑎0𝑥= 0 and 𝑎0𝑦= −1, where 𝑎0𝑥 and 𝑎0𝑦 are x- and y-component of 𝒂0. These model parameters were defined as listed in Table 1. Table 1 Model parameters. Symbol Value Description 𝐿𝑥 400 μm Length of cerebellar tissue 𝐿𝑦 50 μm Thickness of cerebellar tissue 𝑘 1.0 Constitutive parameter in Eq. (3) 𝛼 0.5 Constitutive parameter in Eq. (3) 𝐷 5 μm2/h Diffusion coefficient in Eq. (4) 𝐹0 𝑐 0.1 /h Production rate of cell number density in the external granule layer (EGL) 𝑣0 0,5, 10 μm/h Cell migration speed in the external granule layer (EGL), molecular layer (ML) and Purkinje cell layer (PCL) 𝜑max 0,𝜋, 3/2𝜋 Range of random angle 𝜑r Table 1 Model parameters. Table 1 Model parameters. 3. Cerebellar morphogenesis; simulation results 3. 2.3 A simulation model for cerebellar foliation Cerebellar morphogenesis; simulation results 3.1 Under physiological conditions GC migration causes non-uniform cell accumulation in the IGL, and folia lengthening 3. Cerebellar morphogenesis; simulation results 3.1 Under physiological conditions GC migration causes non-uniform cell accumulation in the IGL, and folia lengthening To understand the contribution of GC migration along the BG fibers (oriented based on tissue deformation) to cerebellar morphogenesis, we performed numerical analyses of cerebellar tissue morphogenesis, by altering the cell migration velocity 𝑣0 as 10 μm/h with reference to the experimental measurement (Yacubova and Komuro, 2002b) and as 5 and 0 μm/h for comparison. Our simulation data showed that GC proliferation in the EGL and their subsequent migration from the EGL to the IGL, caused folia formation (Fig. 2 A-C). The distribution of GC cell number density was temporally changed (Fig. 2A-C; right section). When the migration speed 𝑣0 was 10 μm/h, GC increased in the IGL when compared to other layers, over time 𝑡= 20 h (Fig. 2A). Cell numbers density in the IGL had a non-uniform distribution, and the density were higher around the lobule than the fissure. As the migration speed decreased (𝑣0 = 5 μm/h), cell number density decreased in the IGL, but increased in other layers, including the EGL, ML, and PCL, over time 𝑡= 20 h (Fig. 2B). When GCs did not migrate (𝑣0 = 0 μm/h), cell number density became uniformly distributed in the EGL, over time 𝑡= 20 h (Fig. 2C). These data suggested that higher migration speeds caused a non-uniform GC distribution. To quantitatively analyze the non-uniformity of IGL thickness, temporal changes in difference 𝑑 in IGL thickness around lobule 𝑑l and fissure 𝑑f were plotted (Fig. 2D). Differences in the IGL thickness were larger when GCs migrate (𝑣0 = 10, 5 μm/h) than when GCs do not migrate (𝑣0 = 0 μm/h); this result suggested that GC accumulation mediated by GC migration contributed to the formation of an IGL of non-uniform thickness. To analyze the effects of GC migration © 2021 The Japan Society of Mechanical Engineers [DOI: 10.1299/jbse.20-00516] 24 Takeda, Kameo, Yamaguchi, Nakajima and Adachi, Journal of Biomechanical Science and Engineering, Vol.16, No.1 (2021) Takeda, Kameo, Yamaguchi, Nakajima and Adachi, g j Journal of Biomechanical Science and Engineering, Vol.16, No.1 (2021) on folia lengthening, temporal changes in the lobule height ℎ was plotted (Fig. 2E). We observed that lobule height increased as migration speed increased. These data suggested that fiber-guided GC migration caused the formation of IGL of non-uniform thickness and folia lengthening, which are characteristic morphogenesis features of the cerebellar tissue. on folia lengthening, temporal changes in the lobule height ℎ was plotted (Fig. 2E). 3. Cerebellar morphogenesis; simulation results 3.1 Under physiological conditions GC migration causes non-uniform cell accumulation in the IGL, and folia lengthening We observed that lobule height increased as migration speed increased. These data suggested that fiber-guided GC migration caused the formation of IGL of non-uniform thickness and folia lengthening, which are characteristic morphogenesis features of the cerebellar tissue. Fig. 2 The effects of fiber-guided migration on granule cell (GC) accumulation and folia length. GCs proliferate in external granular layers (EGLs) and migrate to internal granular layers (IGLs). (A–C) Morphological changes in the cerebellar tissue resulting from GC migration; (A) 𝑣0 = 10 μm/h, (B) 𝑣0 = 5 μm/h and (C) 𝑣0 = 0 μm/h (no migration). The left section of each image shows the cerebellar layer constitution, and the right section shows the distribution of cell number density caused by GC accumulation. (D) Temporal changes in IGL thickness. (E) Temporal changes in lobule height. Fig. 2 The effects of fiber-guided migration on granule cell (GC) accumulation and folia length. GCs proliferate in external granular layers (EGLs) and migrate to internal granular layers (IGLs). (A–C) Morphological changes in the cerebellar tissue resulting from GC migration; (A) 𝑣0 = 10 μm/h, (B) 𝑣0 = 5 μm/h and (C) 𝑣0 = 0 μm/h (no migration). The left section of each image shows the cerebellar layer constitution, and the right section shows the distribution of cell number density caused by GC accumulation. (D) Temporal changes in IGL thickness. (E) Temporal changes in lobule height. Journal of Biomechanical Science and Engineering, Vol.16, No.1 (2021) 3.2 Impaired GC migration causes defects in folia formation under pathological conditions 3.2 Impaired GC migration causes defects in folia formation under pathological conditions As proposed in a previous study, the orientation of BG fibers, depending on tissue deformation, may be key to the formation of IGL of non-uniform thickness and folia elongation (Sudarov and Joyner, 2007). To investigate the contribution of fiber-guided GC migration, we performed numerical simulations on impaired GC migration, under pathological conditions. Impaired GC migration was modeled, as the migration direction was disturbed by a random angle 𝜑, ranging from −𝜑max/2 to 𝜑max/2. We fixed the migration speed at 𝑣0 = 10 μm/h. The direction field in the EGL, ML and PCL at time 𝑡= 20 h was distributed as shown (Fig 3 A–C; grey arrows in the enlarged view). When the migration direction generated no disturbance (𝜑max = 0) corresponding to a physiological condition as previously simulated (Section 3.1), the migration direction spread radially toward the IGL around the fissure, and the migration direction converged toward the IGL around the lobule (Fig. 3A). When the random angle range 𝜑max was set to π and 3π/2 under pathological conditions, the migration direction was disoriented (Fig. 3B and C; grey arrows in the enlarged view). Cell number density decreased in the IGL, but increased in other layers as random angle range increased (Fig. 3A–C; lower section). To investigate the effects of fiber-guided migration on folia morphology, we plotted temporal changes in difference in IGL thickness between the fissure and lobule 𝑑 (= 𝑑l −𝑑f), and lobule height ℎ (Fig. 3D and E). These data indicated that the difference in IGL thickness and the lobule height were smaller as migration randomness increased. Thus, we suggested that an elongated lobule and non-uniform IGL thickness during cerebellar morphogenesis were caused by deformation-dependent GC migration. © 2021 The Japan Society of Mechanical Engineers [DOI: 10.1299/jbse.20-00516] 25 Takeda, Kameo, Yamaguchi, Nakajima and Adachi, Journal of Biomechanical Science and Engineering, Vol.16, No.1 (2021) Fig. 3 The effects of impaired granule cell (GC) migration under pathological conditions, where GC migration is not guided on Bergmann glial (BG) fibers, on folia formation. (A-C) The direction field of GC migration over time 𝑡= 20 h, as indicated by arrows in the external granule layer (EGL), molecular layer (ML) and Purkinje cell layer (PCL), depending on the random angle 𝜑max. 3.2 Impaired GC migration causes defects in folia formation under pathological conditions Left sections show the cerebellar layer constitution, and right sections show the distribution of cell number density caused by GC accumulation. (D) Temporal changes in IGL thickness. (E) Temporal changes in lobule height. Fig. 3 The effects of impaired granule cell (GC) migration under pathological conditions, where GC migration is not guided on Bergmann glial (BG) fibers, on folia formation. (A-C) The direction field of GC migration over time 𝑡= 20 h, as indicated by arrows in the external granule layer (EGL), molecular layer (ML) and Purkinje cell layer (PCL), depending on the random angle 𝜑max. Left sections show the cerebellar layer constitution, and right sections show the distribution of cell number density caused by GC accumulation. (D) Temporal changes in IGL thickness. (E) Temporal changes in lobule height. 4. Discussion During cerebellar morphogenesis, the characteristic morphology of the cerebellar tissue is formed via GC migration along BG fibers (Corrales et al., 2006; Legue et al., 2016; Leto et al., 2016; Govek et al., 2018; Lejeune et al., 2019). To investigate the GC mediated foliation, we performed numerical simulations of the cerebellar tissue, driven by GC proliferation and migration. Our simulation results under physiological conditions showed that GC migration from the EGL to the IGL caused elongated folia and a non-uniform IGL which was thicker at the lobule, than the fissure (Fig. 2). Moreover, impaired GC migration under pathological conditions, modeled as random migration directions, resulted in reduced folia lengthening and formation of IGL with uniform thickness (Fig. 3). These results suggested that GC migration along BG fibers was implicated in the formation of characteristic cerebellar morphology, including non- uniform GC accumulation in the IGL, and lengthening folia. This study validated foliation mechanisms via computer simulation, i.e., folia lengthening accompanied by non- uniform IGL appeared to be caused by GC proliferation and migration along BG fibers (Sudarov and Joyner, 2007). The direction of GC migration corresponding to BG fiber orientation (Fig. 3A) agreed with a previous study (Govek et al., 2018). With impaired and random GC migration, we observed that abnormal foliation, including reduced folia lengthening and formation of IGL with uniform thickness, was caused (Fig. 3B and C). Importantly, these results agree with experimental data; impaired GC migration leads to widely-distributed GCs and a thin IGL (Govek et al., 2018). Therefore, our numerical simulation data successfully recapitulated the features of physiological/pathological foliation processes previously observed in experimental studies (Sudarov and Joyner, 2007; Govek et al., 2018). We simulated and identified the physical mechanisms that underpin deformation-dependent cell migration and cause non-uniform layer formation during foliation. Such layer formation has been previously explained as the result of biochemically-regulated neuronal migration (D'Arcangelo and Curran, 1998; Sekine et al., 2012; Caffrey et al., 2014; Matsunaga et al., 2017). In addition, the identification of mechanical perspectives is also important in understanding layer formation mechanisms (Lejeune et al., 2016; Engstrom et al., 2018; Lawton et al., 2019). It was previously shown that tissue elastic deformations, such as stretch and compression, during wrinkling caused differences in thickness of multi-layered tissue between lobule and fissure (Tallinen et al., 2014; Holland et al., 2018). 4. Discussion In addition to this mechanical © 2021 The Japan Society of Mechanical Engineers [DOI: 10.1299/jbse.20-00516] 26 Takeda, Kameo, Yamaguchi, Nakajima and Adachi, Journal of Biomechanical Science and Engineering, Vol.16, No.1 (2021) Takeda, Kameo, Yamaguchi, Nakajima and Adachi, Journal of Biomechanical Science and Engineering, Vol.16, No.1 (2021) Takeda, Kameo, Yamaguchi, Nakajima and Adachi, mechanism, we also proposed a mechanism based on plastic deformation caused by changes in cell number via fiber- guided migration. In physiological cerebellar tissue, GC number around lobule is clearly more than around fissure (Sudarov and Joyner, 2007). Because fiber-guided migration is commonly observed during brain morphogenesis such as neuronal migration along radial glial fibers in cerebral development (Borrell and Gotz, 2014; Del Toro et al., 2017; Rahimi-Balaei et al., 2018), our physical mechanism will be important for our increased understanding of layer formation during this process. In our simulations, we assumed the thickness of each layer at the initial state to be equal (Fig. 1B). The thickness of each layer is considered to affect the number of produced GCs and extent of GC migration. In addition, we assumed the initial folia height as a specific value, which can influence the magnitude of deformation. However, because these assumptions affected only the quantitative results, the setting of the initial tissue state does not affect the qualitative results for the proposed foliation mechanism, where the folia elongate via non-uniform GC accumulation caused by fiber- guided GC migration. For the simulation of a specific condition, a further analysis is required. To apply this model to a three-dimensional analysis for the entire cerebellum, the effects of a cerebellar tissue curvature on foliation must be investigated in the future. The foliation mechanism investigated in this research presupposed the existence of the initial fissure, even though initial foliation was important for the determination of cerebellar morphology. To comprehensively understand the foliation, it is important to investigate the formation of an initial fissure by considering other mechanisms, such as fluid- like tissue behavior resulting from cell migration in the EGL (Engstrom et al., 2018; Lawton et al., 2019). Moreover, folia bifurcation in parallel with folia lengthening is important for the study of cerebellar morphogenesis. To investigate folia bifurcation, other mechanical factors that cause bifurcation must be considered, such as lobule-lobule contact. 5. Conclusion In this study, we constructed the computational model for cerebellar foliation caused by fiber-guided cell migration. Through finite element analysis, we successfully recapitulated the features of foliation processes and validated foliation mechanisms, i.e., folia lengthening accompanied by non-uniform IGL appeared to be caused by GC proliferation and migration along BG fibers. Our computational approach has provided insights on the biological and physical mechanisms that characterize cerebellar morphogenesis, and may facilitate a greater understanding of interactions between cellular activities and tissue behaviors. 4. Discussion In addition to mechanisms implicated in the formation of characteristic tissue morphology, further developments may reveal mechanisms for establishment of the physiological function by considering the tissue morphology associates with its function (Sillitoe and Joyner, 2007; White and Sillitoe, 2013; Leto et al., 2016). 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https://openalex.org/W3025194065	https://europepmc.org/articles/pmc7227657?pdf=render	English	null	Long-term outcomes of repaired and unrepaired truncus arteriosus: 20-year, single-center experience in Thailand	PeerJ	2,020	cc-by	10,120	Long-term outcomes of repaired and unrepaired truncus arteriosus: 20-year, single-center experience in Thailand Ekkachai Dangrungroj1, Chodchanok Vijarnsorn1, Prakul Chanthong1, Paweena Chungsomprasong1, Supaluck Kanjanauthai1, Kritvikrom Durongpisitkul1, Jarupim Soongswang1, Kriangkrai Tantiwongkosri2, Thaworn Subtaweesin2 and Somchai Sriyoschati2 Ekkachai Dangrungroj1, Chodchanok Vijarnsorn1, Prakul Chanthong1, Paweena Chungsomprasong1, Supaluck Kanjanauthai1, Kritvikrom Durongpisitkul1, Jarupim Soongswang1, Kriangkrai Tantiwongkosri2, Thaworn Subtaweesin2 and Somchai Sriyoschati 1 Department of Pediatrics, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand 2 1 Department of Pediatrics, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand 2 Department of Surgery, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand How to cite this article Dangrungroj E, Vijarnsorn C, Chanthong P, Chungsomprasong P, Kanjanauthai S, Durongpisitkul K, Soongswang J, Tantiwongkosri K, Subtaweesin T, Sriyoschati S. 2020. Long-term outcomes of repaired and unrepaired truncus arteriosus: 20-year, single-center experience in Thailand. PeerJ 8:e9148 http://doi.org/10.7717/peerj.9148 ABSTRACT Background. Truncus arteriosus (TA) is a complex congenital heart disease that carries morbidities in the first year of life. Previous authors have reported an operative mortality of 50%. In this report, we aim to report on the survival of patients with TA in our medical center in the recent era. Methods. A retrospective review of all patients diagnosed with TA in Siriraj Hospital, Thailand from August 1995 to March 2018 was performed. Patients with single ventricle, hemiTA were excluded. The characteristics and outcomes of repaired and unrepaired TA patients with a known recent functional status in 2018 were reviewed. Operative mortality risks were analyzed using a multivariate model. Results. A total of 74 patients (median age at referral: 70 days) were included in the cohort. One-third of the patients had associated anomalies including DiGeorge syndrome (13.5%). Anatomical repair was not performed in 22 patients (29.7%). The median age at time of repair for the 52 patients was 133 days (range: 22 days to 16.7 years). Complex TA was 10%. Early mortality occurred in 16 patients (30.8%). Five patients (9.6%) had late deaths at 0.3–1.2 years. Significant mortality risk was weight at time of operation <4 kg (HR 3.05, 95% CI [1.05–8.74], p-value 0.041). Of the 31 operation survivors, 17 required re-intervention within 0.4–11.4 years. Eight patients had reoperation at 8.7 years (range: 2.7–14.6 years) post-repair. Freedom from reoperation was 93%, 70.4%, and 31%, at 5, 10, and 15 years, respectively. All late survivors were in functional class I–II. Of the 22 unrepaired TA patients, 11 patients (50%) died (median age: 13.6 years; range: 14 days–32.8 years). Survival of unrepaired TA patients was 68.2%, 68.2%, and 56.8, at 5, 10, and 15 years of age, respectively. At the end of study, 11 survivors of TA with palliative treatment had a recent mean oxygen saturation value of 84.1 ± 4.8% and a mean weight for height of 81.4 ± 12.7%, which were significantly lower than those of 31 late-survivors who had undergone anatomical repair. Submitted 16 September 2019 Accepted 17 April 2020 Published 12 May 2020 Corresponding author Chodchanok Vijarnsorn, cvijarnsorn@yahoo.com Academic editor Cheng Zhan Additional Information and Declarations can be found on page 15 DOI 10.7717/peerj.9148 Copyright 2020 Dangrungroj et al. INTRODUCTION Truncus arteriosus (TA) is a rare, complex congenital heart disease characterized by a single great artery supplying both systemic and pulmonary circulations, as the so called common arterial trunk (Collett & Edwards, 1949). Based on Collett and Edwards’ classification (Collett & Edwards, 1949; Jacobs, 2000), TA was classified into four anatomical types originating from the pulmonary artery. However, type IV currently corresponds to pulmonary atresia with ventricular septal defect, which is not precise TA nomenclature. Clinically, TA leads to cyanosis because of the mixing of deoxygenated and oxygenated blood at the common arterial trunk. Heart failure is inevitable when the pulmonary vascular resistance physiologically declines in infancy. Truncal valve regurgitation and aortic arch anomaly often aggravate heart failure symptoms and mortality (Jacobs, 2000). If patients were left untreated, pulmonary vascular adaptive mechanisms would lead to rapid pulmonary arterial hypertension and subsequently to Eisenmenger syndrome (ES) (Marcelletti, McGoon & Mair, 1976). Anatomical repair of TA was reported in 1967 by Rastelli, Titus & McGoon (1967) and surgical techniques have progressed steadily since then (Mavroudis, Jonas & Bove, 2015). The timing of the operation tends to be an early repair in neonates or during infancy within the first three months of age to reduce the risk of pulmonary hypertension (Brown et al., 2001; Hanley et al., 1993). In any case, the surgical procedure is widely known to carry a significant risk of mortality and the operation requires meticulous postoperative management. According to the Society of Thoracic Surgeons Congenital Heart Surgery database (STS-CHSD) from 2000–2009, TA repair with truncal valve surgery had a significantly higher rate of mortality than without truncal valve surgery (30% vs. 11%) (Russell et al., 2012). Furthermore, in a recent multicenter cohort study in the US, 20% of the children who underwent simple TA repair were reported to experience major adverse cardiac events postoperatively (Mastropietro et al., 2019). Currently, complex TA surgery is categorized as a level-4 procedure according to the Society of Thoracic Surgeons— European Association for Cardio-Thoracic Surgery (Russell et al., 2012; Mastropietro et al., 2019). Siriraj Hospital is one of referral cardiac centers that performs surgical corrections and provides postoperative care for children with TA in Thailand. In a report of early cases prior to 2004, Loahaprasitiporn and colleagues found an operative mortality of 50% following corrective surgery, within 6 weeks to 6 months of age (Laohaprasitiporn et al., 2008). ABSTRACT Distributed under Creative Commons CC-BY 4.0 Submitted 16 September 2019 Accepted 17 April 2020 Published 12 May 2020 Corresponding author Chodchanok Vijarnsorn, cvijarnsorn@yahoo.com Academic editor Cheng Zhan Additional Information and Declarations can be found on page 15 DOI 10.7717/peerj.9148 Copyright Conclusion. Contemporary survival rates of patients with TA following operation in the center has been gradually improved over time. Most of the operative mortality occurs in the early postoperative period. Compared to patients with TA who had palliative treatment, operative survivors have a better functional status even though they carry a risk for re-intervention. Distributed under Creative Commons CC-BY 4.0 Distributed under Creative Commons CC-BY 4.0 OPEN ACCESS Subjects Cardiology, Pediatrics, Surgery and Surgical Specialties Keywords Truncus arteriosus, Survival, Mortality risk, Congenital heart defect Subjects Cardiology, Pediatrics, Surgery and Surgical Specialties Keywords Truncus arteriosus, Survival, Mortality risk, Congenital heart defect Subjects Cardiology, Pediatrics, Surgery and Surgical Specialties Dangrungroj et al. (2020), PeerJ, DOI 10.7717/peerj.9148 MATERIALS AND METHODS This study was a single-centered, observational study using a hospital database from a large referral cardiac center in Thailand. Following approval from the Siriraj Institutional Review Board Faculty of Medicine, Siriraj Hospital, Mahidol University (COA no. Si 379/2017), all consecutive patients who had a confirmed diagnosis of TA type I–III (Collett and Edward) (Collett & Edwards, 1949) by echocardiography between August 1, 1995 and March 31, 2018 in the center were retrospectively reviewed. Patients with single ventricle, hemiTA, or TA type IV, or patients who had undergone an operation from another hospital were excluded. The requirement for informed consent from patients was waived with the approval of the Siriraj Institutional Review Board Faculty of Medicine, Siriraj Hospital, Mahidol University. Demographic data was collected for age at referral, diagnosis, gender, weight at referral, oxygen saturation, associated anomalies, presence of DiGeorge syndrome, initial presentation, and cardiac findings including type of TA (I, II, III), truncal valve leaflets, degree of truncal valve regurgitation, presence of aortic arch interruption, pulmonary artery stenosis, arch sidedness and coronary abnormality. Patient management was recorded, including surgical interventions, postoperative complications, and clinical outcomes including weight, height, functional class, oxygen saturation, and mortality following diagnosis and at the most recent follow-up at the end of 2018. Patients who were lost to a follow-up or their latest functional status was unknown in 2018 were excluded from the study cohort. Early mortality following total correction for TA was defined as 30-day mortality including patients who died after 30 days without being discharged from the hospital when admitted for the operation. Patient information was anonymized and de-identified prior to the analysis. INTRODUCTION Medically conservative treatments were provided to patients who did not meet operability criteria, such as late referral with ES. Currently, some survivors in both repaired and unrepaired groups reach adulthood. In the present study, we aim to evaluate: (1) the survival rates of patients who were diagnosed with TA, and either repaired or unrepaired TA, at Siriraj Hospital; and (2) operative mortality risks for the past two decades. Dangrungroj et al. (2020), PeerJ, DOI 10.7717/peerj.9148 2/18 Dangrungroj et al. (2020), PeerJ, DOI 10.7717/peerj.9148 Demographics A total of 74 patients with TA (45% were male) was included in the study cohort. Median age of referral and diagnosis at the center was 70 days of age (range: 0–25.9 years). The patients’ demographics are summarized in Table 1. Almost one-third of the patient population had an associated anomaly such as VACTREL association, anorectal malformation, tracheoesophageal fistula, micropthalmia, hypospadia, including DiGeorge syndrome (13.5%). The most common type of TA was type I. Six patients had pulmonary artery stenosis (8.1%), five patients had moderate or severe truncal valve regurgitation (6.7%), four patients had partial anomalous pulmonary venous return (5.4%), and two patients had interrupted aortic arch (2.7%). Following the identification of operability and obtaining consent from the patients’ parents, 52 patients underwent TA anatomical repair and 22 patients did not. Two patients in the unrepaired TA group underwent palliative pulmonary artery banding (PAB); one was unable to perform further anatomical repair for TA due to underlying Jacobsen syndrome with severe thrombocytopenia while the other died after repair interrupted the aortic arch concurrent with PAB. From observations, the initial oxygen saturation in the repaired TA group was significantly higher than that of the patients who received palliative treatment. The flow of the study is shown in Fig. 1. Dangrungroj et al. (2020), PeerJ, DOI 10.7717/peerj.9148 STATISTICAL ANALYSIS The patients’ baseline characteristics and outcomes were summarized using descriptive statistics. Normally distributed data was presented as mean ± SD, while the median (with range) was used where the distribution of data was not normal. Categorical data was represented as a number and a percentage (%).The comparison of continuous variables between groups was assessed using an unpaired T test for normally distributed data and a Wilcoxon rank-sum test for non-normally distributed data. Differences between the categorical data were assessed with a Chi-square or Fisher’s exact test. Patients were classified into two groups; those who underwent anatomical repair for TA and those who had palliative treatment. Cumulative survival, from date of birth and date of operation to the endpoint, was calculated using the Kaplan-Meire analysis with log-rank test. The association between baseline characteristics and postoperative mortality was evaluated with multivariate analysis. A p-value < 0.05 was considered to be statistically significant. Statistical analyses were performed with SPSS 20.0 for Windows (SPSS Inc., Chicago, IL, USA). Dangrungroj et al. (2020), PeerJ, DOI 10.7717/peerj.9148 3/18 Surgical correction and mortality Table 2 shows patient demographics, operative data, and early postoperative course in 52 repaired TA patients. Anatomical repair of TA has been performed at the medical center since 1999. Of the 52 patients, 19 underwent total repair in 1999–2006, representing various age groups at the time of operation: neonate (n = 2), aged 1–6 months old (n = 31), aged 6 months–1 year old (n = 13), age more than 1 year old (n = 6). Three patients underwent PAB prior to a total correction. The right ventricular to pulmonary artery valve conduit diameter varied from 12–25 mm, depending on the patient’s size, with z-score of 2.3 ± 0.9. The types of conduit are shown in Table 2. One patient in the cohort underwent direct anastomosis from right ventricle to pulmonary artery. Repair of complex TA, which is defined as TA with significant pathology of truncal valve and aortic arch (Russell et al., 2012; Mastropietro et al., 2019), accounted for 5 of 52 (9.6%) of the cohort). Among four patients who underwent concurrent truncal valve repair, three died postoperatively. The patient who survived (13.9 years postoperatively) had progressive aortic valve (native truncal valve) and conduit regurgitation, and the patient is currently on a list for redo surgery. One patient who underwent concomitant aortic arch repair for interrupted aortic arch died early post-operation due to pulmonary hypertensive crisis. A total of 16 early deaths (30.8%) occurred with a median postoperative time of 3 days (range: 0–40 days). Most of the patients with early mortality died from low cardiac output syndrome (LCOS), related to pulmonary hypertensive crisis or myocardial ischemia, followed by sepsis and multi-organ dysfunction (Table 3). All early survivors (n = 36) were complete in a follow up with a median duration of 5.7 years (range: 0.46–19.8 years). Late mortality was reported in five patients (9.6%) at a median duration of seven months after operation (range: 5.5 months–1.2 years), related to infection and possible persistent pulmonary hypertension postoperatively (Table 3). Dangrungroj et al. (2020), PeerJ, DOI 10.7717/peerj.9148 4/18 Table 1 Patients’ characteristics (n = 74). Surgical correction and mortality Variable All patients Repaired TA Unrepaired TA p-value (n = 74) (n = 52) (n = 22) Male gender 34 (45.9%) 25 (48.1%) 9 (40.9%) 0.57 Age at suspicion of TA at primary hospital (years) 0.05 (0–25.91) 0.05 (0–2.01) 0.07 (0–25.91) 0.79 Age at referral and diagnosis of TA at the center (years) 0.19 (0–25.91) 0.16 (0–2.01) 1.85 (0–25.91) 0.07 Body weight at referral (kg) 3.67 (1.50–48.00) 3.38 (1.50–9.40) 8.05 (2.30–48.00) 0.05 Oxygen saturation, % 88.88 ± 5.86 90.33 ± 4.68 85.45 ± 6.96 0.005* Cardiothoracic ratio on chest radiography 0.62 ± 0.05 0.62 ± 0.04 0.63 ± 0.06 0.42 Prenatal diagnosis 4 (5.4%) 3 (5.7%) 1 (4.5%) 1.00 Associated anomalies 24 (32.4%) 18 (34.6%) 6 (27.3%) 0.53 - DiGeorge syndrome 10 (13.5%) 8 (15.4%) 2 (9.1%) 0.47 TA type (Collett and Edwards) - Type I 54 (72.9%) 37 (71.1%) 17 (77.3%) 0.58 - Type II 17 (23.0%) 13 (25%) 4 (18.2%) - Type III 3 (4.1%) 2 (3.9%) 1 (4.5%) Truncal valve leaflets - Bicuspid 14 (19.0%) 9 (17.3%) 5 (22.7%) 0.84 - Tricuspid 40 (54.0%) 31 (59.6%) 9 (40.9%) - Quardricuspid 20 (27.0%) 12 (23.1%) 8 (36.4%) Presence of moderate and severe truncal valve regurgitation 5 (6.7%) 4 (7.7%) 1 (4.5%) 1.00 Presence of pulmonary artery stenosis 6 (8.1%) 5 (9.6%) 1 (4.5%) 0.66 Presence of interrupted aortic arch 2 (2.7%) 1 (1.9%) 1 (4.5%) 0.51 Presence of right side aortic arch 22 (29.7%) 17 (32.7%) 5 (22.7%) 0.35 Presence of partial anomalous pulmonary venous return 4 (5.4%) 4 (7.7%) 0 0.31 Left ventricular ejection fraction (%) 66.99 ± 8.90 68.53 ± 8.09 62.80 ± 9.86 0.015* Notes. Data represented by median (range), mean ± SD and number (% within column). Statistically significant at p-value < 0.05 by Chi-square or Fishers exact test and independent T test (for normally distributed data) or Wilcoxon rank-sum test (for non- normally distributed data). TA, truncus arteriosus. Notes. Data represented by median (range), mean ± SD and number (% within column). Statistically significant at p-value < 0.05 by Chi-square or Fishers exact test and independent T test (for normally distributed data) or Wilcoxon rank-sum test (for non- ll di ib d d ) The postoperative survival of patients with repaired TA (n = 52) was 63.5% at 1-year and steady at 59.5% after 2 years following the operation. Dangrungroj et al. (2020), PeerJ, DOI 10.7717/peerj.9148 Reoperation and reintervention Thirty-one late survivors following total correction were reported surviving until the end of 2018 and were having regular follow-ups at the medical center. The median postoperative follow-up was 6.4 years (range: 1.0–19.8 years). Seventeen patients (54.8%) required either catheter intervention or reoperation for conduit change at a median time of 3.1 years post-operation (range: 0.4–11.4 years). The range in number of catheter interventions was 0–4 times per patient. Freedom from re-intervention was reported to be 93.3%, 48.3%, 43.9%, and 35% at 1, 5, 10, and 15 years post-repair, respectively. Reoperation for conduit replacement had been performed in 8 patients (25.8%) at the median time of 8.7 years (range: 2.7–14.6 years) after their corrective surgery. Freedom from reoperation in survivors with repaired TA was 100%, 93%, 70.4%, and 31% at 1, 5, 10, and 15 years postoperation, respectively. Surgical correction and mortality Consequently, survival rates of early survivors (n = 36) at 1, 5, 10, and 15 years following repair were 91.7%, 85.9%, 85.9%, and 85.9%, respectively. A univariate analysis of 17 variables revealed 2 potential risk factors of mortality following repaired TA: weight at operation <4 kg and preoperative major infection within 1 month (Table 4). The independent risk of postoperative mortality, which was identified by multivariate analysis was only weight at operation <4 kg (HR 2.71, 95% CI [1.05–6.95]; p-value 0.039) (Table 4). With regards to early death postoperatively, weight at operation <4 kg was found to be an associated factor by univariate and multivariate analyses (HR 3.05, 95% CI [1.05–8.74], p-value 0.041). In patients with late mortality, preoperative major infection within one month and pulmonary hypertensive crisis in the operating room requiring inhaled nitric oxide were shown to be mortality risks by the univariate analysis. Nevertheless, only pulmonary hypertensive crisis in the operating room Dangrungroj et al. (2020), PeerJ, DOI 10.7717/peerj.9148 5/18 Figure 1 Flow of study. Full-size DOI: 10.7717/peerj.9148/fig-1 requiring inhaled nitric oxide was found to be an independent risk factor for late death by the multivariate analysis (hazard ratio 6.59, 95% CI [1.02–42.7], p-value 0.048). Figure 1 Flow of study. Flow of study. requiring inhaled nitric oxide was found to be an independent risk factor for late death by the multivariate analysis (hazard ratio 6.59, 95% CI [1.02–42.7], p-value 0.048). requiring inhaled nitric oxide was found to be an independent risk factor for late death by the multivariate analysis (hazard ratio 6.59, 95% CI [1.02–42.7], p-value 0.048). Dangrungroj et al. (2020), PeerJ, DOI 10.7717/peerj.9148 Pulmonary arterial hypertension (PAH) therapy Of the 52 patients who underwent surgical correction, 47 patients (90.3%) received postoperative pulmonary vasodilators including inhaled nitric oxide, iloprost, or oral forms such as sildenafil, bosentan or beraprost sodium. In five patients who had not been given pulmonary vasodilators, four underwent total correction prior to 2000, when inhaled nitric oxide was unavailable in the theatre and intensive care unit. Sixteen early deaths (30.8%) occurred with a median postoperative time of three days (range: 0–40 days), related to pulmonary hypertensive crisis or myocardial ischemia, followed by sepsis and multi-organ dysfunction. Twenty-eight patients were prescribed oral pulmonary vasodilators when they were discharged home after their operation. Monotherapy was given: beraprost in 3 patients and sildenafil in 25 patients. The median time of post-operative pulmonary arterial hypertension therapy was 6.9 months (range 1–109 months). Pre-treatment targeted therapy was used in two patients who underwent total correction at age 14 and 16 years, because of late referrals and the decision of parents. They were placed on sildenafil (25–50 mg oral three times/day) prior to the cardiac catheterization in the referral center. Interestingly, both patients had baseline oxygen saturation in room air of 90–94%. Cardiac catheterization preoperatively showed elevated pulmonary artery pressure (PAP) in both patients. The first patient had a mean PAP (mPAP) of 80 mmHg, Qp:Qs of 1.36, and pulmonary vascular resistance (PVR) index of 13.6 WU m2 in room air and mPAP 72 mmHg; the PVR index declined to 0.9 WU m2 after inhaled nitric oxide 20 PPM testing. The second patient had a mPAP of 63 mmHg, Qp:Qs of 6, PVR of 3.4 WU, and PVR index of 3.9 WU m2 in room air. Following surgical repair, these two patients continued with the pulmonary vasodilator (oral sildenafil, for 48.6 and 57.9 months following operation, respectively). Repeat cardiac catheterizations at two years post repair were performed, with a decreased mean PAP to 30 mmHg in the first patient and 28 mmHg in the second patient. Of the 22 patients who received palliative treatment, 11 were mortality cases; 7 patients died in infancy (less than 1.5 years of age) due to heart failure. These 7 patients had no indication for receiving PAH therapy. Four of the patients who died (13–32.8 years of age) had late referrals and were diagnosed with ES. Of these four patients, two received oral pulmonary vasodilator (one beraprost and one sildenafil). TA with palliative treatment Twenty-two patients in the cohort had not undergone anatomical repair for TA. Cardiac catheterization, which was performed in 6 patients showed mean pulmonary arterial pressure of 70.8 ± 14.0 mmHg, baseline pulmonary vascular resistance index of 20.1 ± 14.11 WU m2 and post-pulmonary vasodilator testing pulmonary vascular resistance index of 6.9 ± 4.9 WU m2. Some patients who were deemed to be receiving a conservative treatment were referred back to the primary hospital. All 22 patients in this study had been in recent contact and had a known clinical status in 2018. Since their most recent visit (median age: 13.6 years; range: 14 days–32.8 years), 11 patients (50%) had died. Seven patients had died in infancy (less than 1.5 years of age), and the primary cause of death was heart failure, precipitated by infections such as sepsis or pneumonia. Four patients who died (13–32.8 years of age) had late referral and diagnoses of ES. Of the four patients, two died Dangrungroj et al. (2020), PeerJ, DOI 10.7717/peerj.9148 6/18 from pneumonia and two patients had sudden death, which was likely related to adverse cardiac events. The survival rates of the TA with palliative treatment were 72.7%, 68.2%, 68.2%, and 56.8, at 1, 5, 10, and 15 years of age, respectively. In comparing the survival ages of 52 patients with TA repair, 63.5% were surviving at 1-year and 59.6% were steady at 5-years of age, with no statistical differences. The survival of 36 early-survivors following TA repair was significantly higher than that of 22 patients with palliative treatment (Log rank p 0.03). At the end of study cohort in 2018, 11 survivors of TA with palliative treatment had a recent mean oxygen saturation value of 84.1 ± 4.8% and a mean weight for height of 81.4 ± 12.7%, which were significantly lower than those of 31 late-survivors who had undergone anatomical repair. Dangrungroj et al. (2020), PeerJ, DOI 10.7717/peerj.9148 Pulmonary arterial hypertension (PAH) therapy Of the 11 surviving patients, 6 received oral pulmonary vasodilator (3 beraprost and 3 sildenafil). Dangrungroj et al. (2020), PeerJ, DOI 10.7717/peerj.9148 7/18 Table 2 Demographics, operative data and early postoperative course of patients who underwent truncus arteriosus repair (n = 52). (continued on next page) Dangrungroj et al. (2020), PeerJ, DOI 10.7717/peerj.9148 Pulmonary arterial hypertension (PAH) therapy - Aortic homograft - Pulmonary homograft Pulmonary arterial hypertension (PAH) therapy Variable All TA repair (n = 52) Mortality (n = 21) Survivors in 2018 (n = 31) p-value Male gender 25 (48.1%) 9 (42.9%) 16 (51.6%) 0.53 Prenatal diagnosis 3 (5.7%) 2 (9.5%) 1 (3.2%) 0.55 Age at surgery (days) 133 (22–6111) 88 (22–319) 189 (48–6111) 0.11 Weight at total repair (kg) 4.2 (2.2–38.0) 3.6 (2.2–6.5) 5.1 (2.9–38.0) 0.06 Associated anomalies 18 (34.6%) 11 (52.4%) 7 (22.5%) 0.027* - DiGeorge syndrome 8 (15.4%) 4 (19.0%) 4 (12.9%) 0.54 TA type I (Collett and Edward) 37 (71.1%) 13 (61.9%) 24 (77.4%) 0.23 Presence of moderate and severe truncal valve regurgitation 4 (7.7%) 3 (14.3%) 1 (3.2%) 0.29 Presence of pulmonary artery stenosis 5 (9.6%) 3 (14.3%) 2 (6.5%) 0.38 Presence of interrupted aortic arch 1 (1.9%) 1 (4.7%) 0 0.40 Presence of right side aortic arch 17 (32.7%) 7 (33.3%) 10 (32.6%) 0.93 Presence of partial anomalous pulmonary venous return 4 (7.7%) 0 4 (12.9%) 0.13 Presence of coronary abnormalities 8 (15.4%) 4 (19.0%) 4 (12.9%) 0.54 Pulmonary artery banding prior to repair 3 (5.8%) 1 (4.8%) 2 (6.5%) 1.00 Preoperative major infection within 1 month 10 (19.2%) 7 (33.3%) 3 (9.7%) 0.03* Preoperative being on mechanical ventilator 2 (3.8%) 1 (4.8%) 1 (3.2%) 1.00 Operative data Surgical era 1999-2006 15 (28.8%) 7 (33.3%) 8 (25.8%) 0.55 Cardiopulmonary bypass time (min) 169.6 ± 43.8 181.3 ± 51.1 161.7 ± 36.9 0.07 Aortic clamp time (min) 107.2 ± 29.6 105.8 ± 26.6 108.1 ± 31.9 0.79 Truncal valve repaired 4 (7.7%) 3 (14.3%) 1 (3.2%) 0.14 Aortic arch repaired 1 (1.9%) 1 (4.8%) 0 0.22 Conduit size (mm) 14.2 ± 3.1 12.9 ± 2.9 15.0 ± 2.9 0.01* Conduit size z-score 2.3 ± 0.9 2.3 ± 0.8 2.3 ± 1.0 0.89 Type of conduit 0.18 - Aortic homograft 5 (9.6%) 0 5 (16.1%) - Pulmonary homograft 10 (19.2%) 3 (14.3%) 7 (22.6%) - Handcock/Carpentier–Edwards valved conduit 12 (23.1%) 5 (23.8%) 7 (22.6%) - Contegra bovine jugular valved conduit 24 (46.2%) 12 (57.1%) 12 (38.7%) -Direct anastomosis with monocusp 1 (1.9%) 1 (4.8%) 0 Intraoperative usage of inhaled nitric oxide 9 (17.3%) 6 (28.5%) 3 (9.6% 0.07 Early postoperative course Postoperative ECMO 4 (7.7%) 4 (19.0%) 0 0.02* Postoperative usage of inhaled nitric oxide 34 (65.4%) 16 (76.2%) 18 (58.1%) 0.17 Postoperative acute kidney injury requiring renal replacement therapy 14 (26.9%) 11 (52.4%) 3 (9.7%) 0.001* Postoperative pneumonia 24 (46.2%) 8 (38.1%) 16 (51.6%) 0.34 Postoperative septicemia 10 (19.2%) 6 (28.6%) 4 (12.9%) 0.16 Postoperative fatal arrhythmia 4 (7.7%) 4 (19.0%0 0 0.02* (continued on next page) data and early postoperative course of patients who underwent truncus arteriosus repair (n = 52). DISCUSSION In this 20-year, single-center database, 52 of 74 patients with a diagnosis of TA had undergone total repair at a median age of 133 days (range: 22 days–16.7 years). Repair of complex TA, which required truncal valve repair or aortic arch interruption repair was performed in 10% of the cases. Early and late mortality was 30.8% and 9.6%, respectively. Mean survival rate of early repaired TA survivors (n = 36) was 91.7% at 1-year and 85.9% at 2-years postoperatively. The independent risk factor for overall mortality was weight at operation <4 kg (hazard ratio: 3.05, 95% CI [1.05–8.74], p-value 0.041). At the median postoperative time of 6.4 years (range: 1.0–19.8 years), more than half of the late-survivors required either catheter intervention or reoperation for conduit change. Freedom from reoperation in repaired TA survivors was 100%, 93%, 70.4%, and 31%, at 1, 5, 10, and 15 years, respectively. All survivors were in the WHO functional class I-II. Of the 22 patients who had palliative treatment (median age at the most recent visit: 13.6 years; range: 14 days–32.8 years), 11 patients (50%) died, with survival rates of 72.7%, 68.2%, 68.2%, and 56.8, at 1, 5, 10, and 15 years of age. This cohort was first to be used in a report of long-term outcomes of patients with TA in Thailand, following the report of Loahaprasittiporn and colleagues who reported early outcomes using the 1995-2004 database from our medical center (Laohaprasitiporn et al., 2008). Survival here is shown with aggregated times and numbers of patients. Although the surgical mortality in this study is still high, it is less than the 50% reported previously for our medical center (Laohaprasitiporn et al., 2008). Furthermore, this cohort includes outcomes for unrepaired TA patients. These findings may aid in counseling patients with regards to treatment and prognosis. The timing of surgical repair of TA has an impact on outcome as early mortality risk is associated with elevated pulmonary vascular resistance leading to postoperative pulmonary hypertensive crisis (Brown et al., 2001; Hanley et al., 1993; Urban et al., 1998). Hanley and colleagues indicated that when the age at repair was above 100 days, it was an independent predictor of perioperative death. Early postoperative course (continued on next page) Dangrungroj et al. (2020), PeerJ, DOI 10.7717/peerj.9148 8/18 Table 2 (continued) Variable All TA repair (n = 52) Mortality (n = 21) Survivors in 2018 (n = 31) p-value Total intensive care unit stay 8.5 (1–167) 5 (1–134) 11 (1–167) 0.59 Total hospital length of stay 23 (1–206) 12 (1–151) 24 (9–206) 0.99 Notes. Data represented by median (range), mean ± SD and number (% within column). Statistically significant at p-value < 0.05 by Chi-square or Fishers exact test and independent T test (for normally distributed data) or Wilcoxon rank-sum test (for non- normally distributed data) TA, truncus arteriosus. ECMO, extracorporeal membrane oxygenator. Data represented by median (range), mean ± SD and number (% within column). Statistically significant at p-value < 0.05 by Chi-square or Fishers exact test and independent T test (for normally distributed data) or Wilcoxon rank-sum test (for non- normally distributed data) TA, truncus arteriosus. ECMO, extracorporeal membrane oxygenator. Dangrungroj et al. (2020), PeerJ, DOI 10.7717/peerj.9148 DISCUSSION Moreover, pulmonary artery pressure was significantly lower in patients undergoing the operation during the neonatal period (Hanley et al., 1993), which agrees with the study of Urban who found fewer pulmonary hypertensive episodes in patients with repaired TA under 90 days of age (7%), in contrast to above 90 days of age (40%) (Urban et al., 1998). Elective repair during the first three months of age is historically advocated in many centers (Brizard et al., 1997; Lacour-Gayet et al., 1996), though the benefits from repairing TA in the neonatal period is widely accepted (Brown et al., 2001; Hanley et al., 1993; Urban et al., 1998; Naimo et al., 2016). In this decade, the Dangrungroj et al. (2020), PeerJ, DOI 10.7717/peerj.9148 9/18 Table 3 Characteristics and causes of death following operation (n = 21). Dangrungroj et al. (2020), PeerJ, DOI 10.7717/peerj.9148 DISCUSSION ID Operation year Gender Associated anomalies Age at repair (days) Interval post operation (days) Repaired TA type Cause of death 64 2003 Male None 133 1 Type I Early postoperative PH crisis 50 2004 Female None 47 1 Type II Early postoperative LCOS, my- ocardial failure, AKI, possible PH crisis, myocardial ischemia 62 2004 Female Microcephaly 92 2 Type I Early postoperative PH crisis, hemopericardium 85 2004 Female Congenital iris cyst 49 14 Type I Early postoperative PH crisis, sep- sis, pneumonia, atrial tachycardia, idioventricular rhythm 22 2005 Female None 47 0 Type III with prox- imal pulmonary artery stenosis Early postoperative PH crisis 86 2007 Male None 135 4 Type III Early postoperative PH crisis, AKI 60 2008 Male DiGeorge syn- drome 319 380 Type I Late mortality due to Persistent PAH post-operation, infection, pneumonia 46 2009 Female None 86 1 Type I Early postoperative myocardial failure possible myocardial is- chemia, JET, AV block 61 2011 Male None 67 0 Type I, repaired truncal valve Early postoperative PH crisis, LCOS, VT, VF 23 2012 Female Ex preterm, congenital hypothyroid 184 8 Type I PH crisis, myocardial failure, pro- longed CBP on ECMO 24 2013 Male Ex preterm, hypospadias 77 459 Type I Late mortality, persistent PAH post-operation, conduit failure, redo conduit change and died due to pneumonia post reoperation 2 months) 19 2013 Female Fetal alcohol syndrome 90 219 Type II Late mortality: septic shock 78 2013 Male None 134 5 Type I, post pul- monary artery banding Early postoperative PH crisis 93 2014 Male Complete bi- lateral cleft lips and cleft palate 60 25 Type II Early postoperative PH crisis, LCOS on ECMO, severe intratho- racic infection, bowel ischemia, septic shock 26 2014 Female Tracheoesophageal fistula 22 168 Type I Late mortality: sepsis, pneumonia 25 2015 Male None 115 40 Type I In-hospital mortality, postopera- tive septic shock, pneumonia 83 2015 Female None 251 0 Type II Early postoperative PH crisis, VT, VF, arrest on ECMO, cardiac tam- ponade (continued on next page) Table 3 Characteristics and causes of death following operation (n = 21). (continued on next page) Dangrungroj et al. Dangrungroj et al. (2020), PeerJ, DOI 10.7717/peerj.9148 DISCUSSION (2020), PeerJ, DOI 10.7717/peerj.9148 10/18 Table 3 (continued) ID Operation year Gender Associated anomalies Age at repair (days) Interval post operation (days) Repaired TA type Cause of death 31 2015 Female None 93 1 Type II + repaired IAA, multiple small muscular VSD left opened Early postoperative PH crisis 20 2015 Male DiGeorge syndrome, hypospadia 36 214 Type I Late mortality: sepsis, pneumonia 21 2016 Female DiGeorge syndrome 88 7 Type II Early postoperative PH crisis 92 2018 Female VACTREL association, rib anomaly, hemivertebra 27 4 Type I + repaired truncal valve Early postoperative PH crisis, sep- sis, pneumonia Notes. TA, truncus arteriosus; PH crisis, pulmonary hypertensive crisis; LCOS, low cardiac output syndrome; AKI, acute kidney injury; ECMO, extracorporeal membrane oxy- genator; PAH, pulmonary arterial hypertension; VT, ventricular tachycardia; VF, ventricular fibrillation; IAA, interrupted aortic arch; VSD, ventricular septal defect. 20 2015 Male DiGeorge syndrome, hypospadia 36 214 Type I Late mortality: sepsis, pneumonia 21 2016 Female DiGeorge syndrome 88 7 Type II Early postoperative PH crisis 92 2018 Female VACTREL association, rib anomaly, hemivertebra 27 4 Type I + repaired truncal valve Early postoperative PH crisis, sep- sis, pneumonia Notes. TA, truncus arteriosus; PH crisis, pulmonary hypertensive crisis; LCOS, low cardiac output syndrome; AKI, acute kidney injury; ECMO, extracorporeal membrane oxy- genator; PAH, pulmonary arterial hypertension; VT, ventricular tachycardia; VF, ventricular fibrillation; IAA, interrupted aortic arch; VSD, ventricular septal defect. Table 4 Predictors of overall postoperative mortality (n = 52). Variables Crude HR (95%CI) p-value Adjusted HR (95%CI) p-value Male gender 0.69 (0.29–1.66) 0.42 Presence of associated anomaly 2.06 (0.87–4.66) 0.09 1.52 (0.59–3.87) 0.38 DiGeorge syndrome 1.16 (0.39–3.45) 0.78 Prenatal diagnosis 1.65 (0.38–7.14) 0.49 Weight at operation <4 kg. 3.18 (1.31–7.71) 0.01* 2.71 (1.05–6.95) 0.039* TA type II and III 0.59 (0.25–1.43) 0.25 Presence of moderate and severe truncal valve regurgitation 2.26 (0.66–7.78) 0.19 Presence of pulmonary artery stenosis 0.63 (0.18–2.17) 0.47 Presence of interrupted aortic arch 4.66 (0.59–36.41) 0.14 Presence of partial anomalous pulmonary venous return 0.04 (0-24.57) 0.33 Presence of coronary abnormalities 1.61 (0.54–4.81) 0.38 Pulmonary artery banding prior to repair 0.71 (0.09–5.3) 0.79 Preoperative major infection within 1 month 2.42 (1.0–6.04) 0.05* 1.23 (0.42–3.61) 0.69 Preoperative being on mechanical ventilator 1.1 (0.15–8.61) 0.88 Operation in 1997–2006 1.39 (0.56–3.45) 0.47 CBP time >150 min 0.45 (0.15–1.32) 0.14 Intraoperative usage of inhaled nitric oxide 2.24 (0.87–5.71) 0.09 1.71 (0.62–4.71) 0.29 Notes. Univariate and multivariate analysis by Cox regression. DISCUSSION Statistically significant at p-value < 0.05. HR, hazard ratio; TA, truncus arteriosus; CBP, cardiopulmonary bypass. Notes. TA, truncus arteriosus; PH crisis, pulmonary hypertensive crisis; LCOS, low cardiac output syndrome; AKI, acute kidney injury; ECMO, extracorporeal membrane oxy- genator; PAH, pulmonary arterial hypertension; VT, ventricular tachycardia; VF, ventricular fibrillation; IAA, interrupted aortic arch; VSD, ventricular septal defect. Notes. TA, truncus arteriosus; PH crisis, pulmonary hypertensive crisis; LCOS, low cardiac output syndrome; AKI, acute kidney injury; ECMO, extracorporeal membrane oxy- genator; PAH, pulmonary arterial hypertension; VT, ventricular tachycardia; VF, ventricular fibrillation; IAA, interrupted aortic arch; VSD, ventricular septal defect. Notes. TA, truncus arteriosus; PH crisis, pulmonary hypertensive crisis; LCOS, low cardiac output syndrome; AKI, acute kidney injury; ECMO, extracorporeal membrane oxy- genator; PAH, pulmonary arterial hypertension; VT, ventricular tachycardia; VF, ventricular fibrillation; IAA, interrupted aortic arch; VSD, ventricular septal defect. Table 4 Predictors of overall postoperative mortality (n = 52). Variables Crude HR (95%CI) p-value Adjusted HR (95%CI) p-value Male gender 0.69 (0.29–1.66) 0.42 Presence of associated anomaly 2.06 (0.87–4.66) 0.09 1.52 (0.59–3.87) 0.38 DiGeorge syndrome 1.16 (0.39–3.45) 0.78 Prenatal diagnosis 1.65 (0.38–7.14) 0.49 Weight at operation <4 kg. 3.18 (1.31–7.71) 0.01 2.71 (1.05–6.95) 0.039* TA type II and III 0.59 (0.25–1.43) 0.25 Presence of moderate and severe truncal valve regurgitation 2.26 (0.66–7.78) 0.19 Presence of pulmonary artery stenosis 0.63 (0.18–2.17) 0.47 Presence of interrupted aortic arch 4.66 (0.59–36.41) 0.14 Presence of partial anomalous pulmonary venous return 0.04 (0-24.57) 0.33 Presence of coronary abnormalities 1.61 (0.54–4.81) 0.38 Pulmonary artery banding prior to repair 0.71 (0.09–5.3) 0.79 Preoperative major infection within 1 month 2.42 (1.0–6.04) 0.05* 1.23 (0.42–3.61) 0.69 Preoperative being on mechanical ventilator 1.1 (0.15–8.61) 0.88 Operation in 1997–2006 1.39 (0.56–3.45) 0.47 CBP time >150 min 0.45 (0.15–1.32) 0.14 Intraoperative usage of inhaled nitric oxide 2.24 (0.87–5.71) 0.09 1.71 (0.62–4.71) 0.29 Notes. Univariate and multivariate analysis by Cox regression. *Statistically significant at p-value < 0.05. HR, hazard ratio; TA, truncus arteriosus; CBP, cardiopulmonary bypass. median age at the time of anatomical correction of TA has decreased significantly in many medical centers and has favorably affected surgical outcomes (Naimo et al., 2016; Ivanov et al., 2019). In developing countries; however, delaying the referral often leads to delaying the operation, which remains a health care issue. DISCUSSION These findings suggest that repair of TA in patients who are older than one year of age is feasible, though physicians need to deliberately select the cases (Arslan et al., 2014; Chen et al., 2016). diagnosis has gradually developed, though in our study cohort, only four patients had prenatal diagnoses. The median age of referral to our center tended to decrease from 126 days (0–13.9 years of age) in 1995-2006 to 63.5 days (0–25.9 years of age) after 2006 with increased operable opportunity. The age of the patient at time of operation; however, was mostly during infancy (median: 133 days). At our medical center, in 2014, neonatal repairs were initiated and performed in two patients. Unfortunately, one died in hospital and the other had late death at 168 days postoperatively due to pneumonia. In our study, six patients who survived beyond their first year of life had undergone anatomical repair for TA. Pre-operative cardiac catheterization showed that the mean pulmonary artery pressure was 57.2 ± 17.6 mmHg and the pulmonary vascular resistance index in room air, and after a pulmonary vasodilator were 6.2 ± 4.9 and 1.6 ± 1.2 WU m2, respectively. All of these patients survived postoperatively and remained in follow-up until the end of study with a median duration of 5.6 years (range: 4.1–12.7 years). These findings suggest that repair of TA in patients who are older than one year of age is feasible, though physicians need to deliberately select the cases (Arslan et al., 2014; Chen et al., 2016). diagnosis has gradually developed, though in our study cohort, only four patients had prenatal diagnoses. The median age of referral to our center tended to decrease from 126 days (0–13.9 years of age) in 1995-2006 to 63.5 days (0–25.9 years of age) after 2006 with increased operable opportunity. The age of the patient at time of operation; however, was mostly during infancy (median: 133 days). At our medical center, in 2014, neonatal repairs diagnosis has gradually developed, though in our study cohort, only four patients had prenatal diagnoses. The median age of referral to our center tended to decrease from 126 days (0–13.9 years of age) in 1995-2006 to 63.5 days (0–25.9 years of age) after 2006 with increased operable opportunity. The age of the patient at time of operation; however, was mostly during infancy (median: 133 days). DISCUSSION At a few centers, prenatal Dangrungroj et al (2020) PeerJ DOI 10 7717/peerj 9148 11/18 CBP time >150 min median age at the time of anatomical correction of TA has decreased significantly in many medical centers and has favorably affected surgical outcomes (Naimo et al., 2016; Ivanov et al., 2019). In developing countries; however, delaying the referral often leads to delaying the operation, which remains a health care issue. At a few centers, prenatal median age at the time of anatomical correction of TA has decreased significantly in many medical centers and has favorably affected surgical outcomes (Naimo et al., 2016; Ivanov et al., 2019). In developing countries; however, delaying the referral often leads to delaying the operation, which remains a health care issue. At a few centers, prenatal median age at the time of anatomical correction of TA has decreased significantly in many medical centers and has favorably affected surgical outcomes (Naimo et al., 2016; Ivanov et al., 2019). In developing countries; however, delaying the referral often leads to delaying the operation, which remains a health care issue. At a few centers, prenatal Dangrungroj et al. (2020), PeerJ, DOI 10.7717/peerj.9148 diagnosis has gradually developed, though in our study cohort, only four patients had prenatal diagnoses. The median age of referral to our center tended to decrease from 126 days (0–13.9 years of age) in 1995-2006 to 63.5 days (0–25.9 years of age) after 2006 with increased operable opportunity. The age of the patient at time of operation; however, was mostly during infancy (median: 133 days). At our medical center, in 2014, neonatal repairs were initiated and performed in two patients. Unfortunately, one died in hospital and the other had late death at 168 days postoperatively due to pneumonia. In our study, six patients who survived beyond their first year of life had undergone anatomical repair for TA. Pre-operative cardiac catheterization showed that the mean pulmonary artery pressure was 57.2 ± 17.6 mmHg and the pulmonary vascular resistance index in room air, and after a pulmonary vasodilator were 6.2 ± 4.9 and 1.6 ± 1.2 WU m2, respectively. All of these patients survived postoperatively and remained in follow-up until the end of study with a median duration of 5.6 years (range: 4.1–12.7 years). Dangrungroj et al. (2020), PeerJ, DOI 10.7717/peerj.9148 DISCUSSION At our medical center, in 2014, neonatal repairs A number of single-center and multicenter studies have recently reported acceptable operative outcomes for patients with TA. In-hospital mortality varied from 5 to 17.5% (Brown et al., 2001; Mastropietro et al., 2019; Naimo et al., 2016; Ivanov et al., 2019; Asagai et al., 2016; Morgan et al., 2019), and the presence of associated cardiac anomalies, such as interrupted aortic arch, significant truncal valve regurgitation, coronary abnormalities, and pulmonary arteries were reported to increase the operative mortality (Brown et al., 2001; Hanley et al., 1993; Russell et al., 2012; Naimo et al., 2016). In a large cohort of patients with STS-CHSD, complex TA (defined as TA with significant aortic arch anomaly, interruption or coarctation, and truncal valve regurgitation requiring concomitant repair) accounted for 10% of the TA population, carrying an operative mortality of 30%, which is much higher than that of simple TA repair (6.9–11%) (Russell et al., 2012; Mastropietro et al., 2019). In accordance with a recent report from a Toronto group, the 10-year survival of patients with complex TA (defined as TA with significant aortic arch anomaly, interruption or coarctation, truncal valve regurgitation requiring concomitant repair, and branch pulmonary artery stenosis/ hypoplasia) who had undergone an operation since 2000 was 68%, compared to 95% for patients who had simple TA (Morgan et al., 2019). The overall operative mortality for these patients decreased from 36% to 7% since 2000, which coincided with the average reduction in cardiopulmonary bypass time. Low birth weight, complex TA, and year of diagnosis prior to 2000 were associated with decreased survival for patients up to one year of age in the study by Morgan et al. (2019). In a large study from a Melbourne group, 11.7% early mortality and 11.1% late death were reported following anatomical repair (Naimo et al., 2016). Weight at repair <2.5 kg, prior surgical intervention, coronary abnormalities, and use of postoperative extracorporeal membrane oxygenator were found to be risk factors for early death while DiGeorge syndrome was a risk factor for late death (Naimo et al., 2016). Comparing to our initial report from our medical center (Laohaprasitiporn et al., 2008), early mortality following TA repair was reduced from 50% to 30.8%. This decrease was greater than that recently reported for experienced centers (Russell et al., 2012; Mastropietro et al., Dangrungroj et al. DISCUSSION (2020), PeerJ, DOI 10.7717/peerj.9148 12/18 2019; Naimo et al., 2016; Ivanov et al., 2019; Chen et al., 2016; Morgan et al., 2019), but comparable to the 2016 publication from Asagai and colleagues who reviewed 52 patients with repaired TA between 1974 and 2002 (Asagai et al., 2016). The plausible explanation may be because both Asagai’s (Asagai et al., 2016) and ours report had mostly included patients who were beyond the neonatal period. Pulmonary hypertensive crisis and LCOS, followed by precipitated infection were the main causes of early death. The independent risk factor for overall mortality was weight at operation <4 kg (HR 3.05, 95% CI [1.05–8.74], p-value 0.041). Complex TA, coronary abnormality, and extracardiac associated anomalies were not found to be strong predictors of death in our study. The number of truncal valve and interrupted arch repairs in our study was only five, which may have been too small a sample size to demonstrate a statistic difference in the multivariate analysis. The reports of late death (9.6%) were consistent with several other previous studies (Naimo et al., 2016; Ivanov et al., 2019; Asagai et al., 2016; Rajasinghe et al., 1997). The optimal methods for right ventricular outflow tract reconstruction for TA repair have been long debated (Brown et al., 2001; Lacour-Gayet et al., 1996; Poynter et al., 2013; Hickey et al., 2008). Homograft conduit provides good biocompatibility, stable hemodynamic, and a competent valve to deal with the elevated pulmonary pressure in TA; however, its poor growth potential, especially in TA repair that is usually performed in infancy, leads to an unsatisfactory freedom from reoperation (Poynter et al., 2013). Heterograft valve conduit has been developed and may be used based on material limitations and the desired size of the homograft. Drawbacks have been reported such as possible unfavorable biocompatibility resulting in pseudomembrane formation, aneurysm, and thrombosis and its poor longevity (Hickey et al., 2008). The direct anastomosis technique without extracardiac conduit was proposed by Barbero-Marcial & Tanamati (1999), and it may overcome the limited growth of conduit and decrease the need for reintervention (Raisky et al., 2009), though progressive pulmonary regurgitation, postoperative pulmonary branch stenosis, and compression to the left anterior descending artery, especially in patients with coronary abnormalities, remain as significant issues (Lacour-Gayet et al., 1996; Ivanov et al., 2019). In our medical center, homografts were used in 15 patients (28%) and heterografts were used in 36 patients (69%). Dangrungroj et al. (2020), PeerJ, DOI 10.7717/peerj.9148 DISCUSSION Of the 36 heterografts, 24 patients who underwent the operation after 2010 were repaired with a Contegra bovine valve conduit. Conduit size selection in the center was within z-score of 2.3 ± 0.9, which was within the acceptable range mentioned in previous publications (Poynter et al., 2013; Hickey et al., 2008). One patient in this cohort who underwent TA repair with direct anastomosis at 49 days of age died early postoperatively due to pulmonary hypertensive crisis, sepsis, and myocardial failure with atrial tachycardia followed by idioventricular rhythm. In some cases, following TA repair, re-intervention may be performed to conduit and branch the pulmonary arteries (Mavroudis, Jonas & Bove, 2015; Naimo et al., 2016; Ivanov et al., 2019; Morgan et al., 2019; Rajasinghe et al., 1997). Catheter intervention such as balloon dilatation or stent implantation may be initially used at our center in the management to alleviate stenotic lesions as a strategy for delaying reoperation. At the median postoperative duration (6.4 years; range: 1.0–19.8 years), the freedom from re-intervention in the study was 48.3% and 43.9%, at 5 and 10 years, respectively, which is consistent with previous reports (Russell Dangrungroj et al. (2020), PeerJ, DOI 10.7717/peerj.9148 13/18 et al., 2012; Naimo et al., 2016; Ivanov et al., 2019; Morgan et al., 2019). Reoperation for conduit replacement was performed in 8 patients (25.8%) at a median time of 8.7 years (range: 2.7–14.6 years) post total correction. Freedom from reoperation in survivors with repaired TA at 10 years was 70.4%. With regards to truncal valve reoperation, a 14-year-old patient who had undergone concomitant truncal valve repair with primary TA repair, was on a list for reoperation to change the conduit and redo the truncal valve repair at 13 years from the first operation. Among those with a less than moderate truncal valve, 1 patient had infective endocarditis with vegetation at the truncal valve, requiring late truncal valve repair and conduit change at 5.2 years post-operation, and 1 patient had progressive to moderate truncal regurgitation with stable hemodynamic. Of 22 patients who had TA with palliative treatment, 12 patients were definitely late diagnosed and then referred, which led to a progression of the disease to severe pulmonary arterial hypertension or Eisenmenger syndrome, where total repair should not be performed. Dangrungroj et al. (2020), PeerJ, DOI 10.7717/peerj.9148 DISCUSSION Four patients were referred in infancy, and were on the list for TA repair, but they died while waiting for surgery due to infection and multi-organ failure at their local hospital. Three patients were diagnosed and then referred as neonates, but they died from neonatal sepsis/NEC/pneumonia in the center. The parents of two patients denied surgery for their children. One patient had Jacobsen syndrome with severe thrombocytopenia, for their children. One patient had Jacobsen syndrome with severe thrombocytopenia, which has a high risk for major surgery. The parents agreed to have palliative pulmonary artery banding to alleviate heart failure symptoms. The economic burden may play a role in late diagnoses and referrals and in the decision to deny surgery in the earlier era. The fate of patients with palliative treatment for TA was also a focus of this study. Mortality can be classified into 2 groups: the first group (n = 7) died within 1.5 years of age due to intractable heart failure and infection. The second group (n = 4) died at 10-years and older due to ES and progressive truncal valve regurgitation, precipitated by infection. In previous publications, almost 80% of the patients with unrepaired TA died before 1 year of age and few survived until adulthood (Marcelletti, McGoon & Mair, 1976; Niwa et al., 1999; Williams et al., 1999). Niwa and colleagues reviewed 10 adults with ES secondary to unrepaired TA and found the mean age of survival was 41.5 ± 5.1 years, which was a shorter life-span compared to ES secondary to ventricular septal defect (Niwa et al., 1999). In our series, the survival rates of 22 patients with unrepaired TA were 72.7%, 68.2%, 68.2%, and 56.8 at 1, 5, 10, and 15 years of age. Half of these patients were deceased at an estimated 15.2 ± 3.8 years of age (median ± SE). All survivors that were encountered progressed to the ES stage that led to hypoxemia and limited somatic growth. In comparing the ages of survival for patients with repaired and unrepaired TA, no statistical difference was found since most of the operative mortalities included late death occurring before 2 years of age. The survival curve of 36 early survivors following TA repair was superior to that of 22 patients with palliative treatment (Log rank p = 0.03). Therefore, complete repair of TA likely improves the survival of patients with TA. CONCLUSION Contemporary survival rates of patients with TA following operation in the center has been gradually improved over time. Most of the operative mortality occurs in the early postoperative period. Compared to patients with TA who had palliative treatment, operative survivors have a better functional status even though they carry a risk for re-intervention. Weight at operation under 4 kg is identified as a significant early and all-mortality risk factor. The survival rate of patients with repaired TA, who can be discharged from hospital after their operation is better than patients with unrepaired TA. We encourage primary physicians to detect this lesion as early as possible for the optimal repair and management. DISCUSSION To improve public health outcomes for patients with this lesion, early detection and referral can help manage and repair the lesion in patients who are neonates or at least younger than three months of age. The optimal surgical and postoperative care in neonates and infants in the center is the main focus. which has a high risk for major surgery. The parents agreed to have palliative pulmonary artery banding to alleviate heart failure symptoms. The economic burden may play a role in late diagnoses and referrals and in the decision to deny surgery in the earlier era. The fate of patients with palliative treatment for TA was also a focus of this study. Mortality can be classified into 2 groups: the first group (n = 7) died within 1.5 years of age due to intractable heart failure and infection. The second group (n = 4) died at 10-years and older due to ES and progressive truncal valve regurgitation, precipitated by infection. In previous publications, almost 80% of the patients with unrepaired TA died before 1 year of age and few survived until adulthood (Marcelletti, McGoon & Mair, 1976; Niwa et al., Dangrungroj et al. (2020), PeerJ, DOI 10.7717/peerj.9148 14/18 LIMITATIONS Selective bias is inevitable in retrospective studies. Consequently, we included patients with diagnoses of TA type I, II, or III who had undergone TA repair or received palliative treatment at the medical center. All patients were in follow-up or their functional status was known at the end of study (2018). The small number of patients in the study affected the multivariate analysis. In addition, the age of referral and diagnosis of TA at the center is mainly known for patients who were older than neonate, because of the limited resources available in the developing country. Management and surgical strategies also tend to be inconsistent, as they often depend on the preferences of individual surgeons and pediatric cardiologists. Competing Interests Competing Interests The authors declare there are no competing interests. The authors declare there are no competing interests. ACKNOWLEDGEMENTS The authors thank the faculty and staff of the Cardiovascular Thoracic Surgery, Faculty of Medicine Siriaj Hospital for their support and care of patients with TA. We acknowledge Prof. Duangmanee Loahaprasitiporn, Deputy Dean of Quality Development, Faculty of Medicine Siriaj Hospital, who established the original research and published the preliminary outcomes of patients with TA in Siriraj Hospital since 2008. We also thank Miss Julaporn Poolium who greatly assisted in the statistical analysis. Dangrungroj et al. (2020), PeerJ, DOI 10.7717/peerj.9148 ADDITIONAL INFORMATION AND DECLARATIONS Funding The authors received no funding for this work. The authors received no funding for this work. The authors received no funding for this work. Human Ethics The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers): The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers): The Siriraj Institutional Review Board Faculty of Medicine, Siriraj Hospital, Mahidol University has approved this study (COA no. Si 379/2017). The Siriraj Institutional Review Board Faculty of Medicine, Siriraj Hospital, Mahidol University has approved this study (COA no. Si 379/2017). Supplemental Information Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.9148#supplemental-information. Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.9148#supplemental-information. peerj.9148#supplemental-information. Data Availability The following information was supplied regarding data availability: The following information was supplied regarding data availability: The raw data set is available as a Supplemental File. The raw data set is available as a Supplemental File. Author Contributions • Ekkachai Dangrungroj conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, and approved the final draft. • Ekkachai Dangrungroj conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, and approved the final draft. Dangrungroj et al. (2020), PeerJ, DOI 10.7717/peerj.9148 • Chodchanok Vijarnsorn conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft. • Prakul Chanthong, Paweena Chungsomprasong, Supaluck Kanjanauthai, Kritvikrom Durongpisitkul, Jarupim Soongswang, Kriangkrai Tantiwongkosri, Thaworn Subtaweesin and Somchai Sriyoschati performed the experiments, authored or reviewed drafts of the paper, and approved the final draft. Dangrungroj et al. 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https://openalex.org/W4243997832	https://www.researchsquare.com/article/rs-3303/latest.pdf	English	null	Influence of complete uncinate process removal on 2-year assessment of radiologic outcomes: subsidence and sagittal balance in patients receiving one-level anterior cervical discectomy and fusion	Research Square (Research Square)	2,020	cc-by	6,036	Association of complete uncinate process removal on 2-year assessment of radiologic outcomes: subsidence and sagittal balance in patients receiving one-level anterior cervical discectomy and fusion Sung Hyun Noh National Health Insurance Corporation Ilsan Hospital Jeong Yoon Park Gangnam Severance Hospital Sung Uk Kuh Gangnam Severance Hospital Dong Kyu Chin Gangnam Severance Hospital Keun Su Kim Gangnam Severance Hospital Yong Eun Cho Gangnam Severance Hospital Kyung Hyun Kim (  gramlin11@naver.com ) Gangnam Severance Hospital Sung Hyun Noh National Health Insurance Corporation Ilsan Hospital Research article Posted Date: June 20th, 2020 DOI: https://doi.org/10.21203/rs.2.12547/v5 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on July 6th, 2020. See the published version at https://doi.org/10.1186/s12891-020-03443-7. Page 1/19 Abstract Background: Many patients with cervical radiculopathy experience stenosis of the neural foramens due to cumulative osteophyte or uncovertebral joint hypertrophy. For cervical foraminal stenosis, complete uncinate process resection (UPR) is often conducted concurrently with anterior discectomy and fusion (ACDF). The aim of this study was to assess the clinical and radiological outcomes of ACDF with complete UPR versus ACDF without UPR. Methods: In total, 105 patients who performed one-level ACDF with a cage-and-plate construct between 2011 and 2015 were retrospectively reviewed. Among them, 37 patients had ACDF with complete UPR, and 68 patients had ACDF without UPR. Radiologic outcomes of disc height, C2–C7 lordosis, T1 slope, C2–C7 sagittal vertical axis (SVA), center of the sella turcica–C7 SVA (St-SVA), spino-cranial angle (SCA), and fusion rate were evaluated on plain X-ray at pre-operation, immediately post-operation, and at 2-year follow-up. For statistically matched pairs analysis, ACDF with UPR group (24 patients) and ACDF without UPR (24 patients) were compared. Results: All of the clinical parameters improved at the 2-year follow up (P<0.0001). Improvement in visual analogue scale (VAS) scores for arm pain was significantly improved in the ACDF with complete UPR group immediately post-operation. All cervical sagittal parameters, including cervical lordosis, segmental angle, disc height, C2-C7 SVA, St-SVA, T1 slope, and SCA, except for preoperative St-SVA, SCA, and disc height of 2 years follow-up, were similar between the ACDF with complete UPR and ACDF without UPR groups. Differences in disc height, C2-C7 SVA, and SCA at 2-year follow up after preoperative examination, however, were statistically significant (p<0.05). Subsidence occurred in 9 patients (ACDF with complete UPR: 8 cases [33%] versus ACDF without UPR: 1 cases [4%]; p < 0.05). Conclusions: Cervical sagittal alignment after ACDF with complete UPR is not significantly different from that achieved with ACDF without UPR. However, subsidence appears to occur more often after ACDF with complete UPR than after ACDF without UPR, although with little to no clinical impact. More precise and careful selection of patients is needed when deciding on additional complete UPR. Patient recruitment and inclusion criteria Between January 2011 and December 2015, 578 patients who underwent ACDF for cervical spondylotic disease at our institution were collected. Among them, we excluded 473 patients whose follow-up period was less than 2 years or the surgery level was two levels or more. In this retrospective study, 105 consecutive patients with single-level cervical spondylotic disease who underwent primary ACDF with a cage-and-plate construct between January 2011 and December 2015 at the author’s institution were included (Fig. 1). This study was approved by the Institutional Review Board of our hospital. The uncinate process was randomly removed totally according to the technical preference of the single surgeon (Fig. 2). Thus, we defined ACDF with UPR as complete unilateral or bilateral removal of the uncinate process, while ACDF without UPR was defined as the conventional removal of only the anterior and posterior parts of the uncinate process or no removal of the uncinate process. This was confirmed with postoperative computed tomography scans. The patients were divided into two groups: 37 patients underwent ACDF with complete UPR and 68 patients were treated with ACDF without UPR. For statistically matched pairs analysis, ACDF with UPR group (24 patients) and ACDF without UPR (24 patients) were compared. The inclusion criteria included the following: 1) patients with symptoms of degenerative cervical disease; 2) patients who received primary ACDF with UPR at only one level; and 3) a follow-up period greater than 24 months. The exclusion criteria were as follows: 1) patients who had previous cervical spine surgery due to ossification of posterior longitudinal ligaments, fractures, tumors, etc.; 2) patients who underwent ACDF for more than two levels; and 3) a follow-up period less than 24 months. Background Anterior cervical discectomy and fusion (ACDF) aiming to improve the stability of the vertebra by decompression of neural elements and fusion is regarded as the gold-standard procedure for symptomatic cervical spondylosis in patients in whom non-operative care has failed [1]. Clinical and radiologic results after ACDF appear to be good [2]. Many patients with cervical radiculopathy also experience stenosis of the neural foramens because of cumulative osteophyte or uncovertebral joint hypertrophy. Although most anterior cervical discectomy and fusion procedures include cervical uncosectomy or uncoforaminotomy to decompress nerve roots in patients with cervical radiculopathy, Lee DH et al. reported that complete uncinate process resection (UPR) during ACDF improves pain in a patient's arm more rapidly than conventional ACDF without UPR and provides similar fusion rates [3, 5]. Page 2/19 Page 2/19 Meanwhile, SH Lee et al. reported that complete UPR over 38% during ACDF increases the risk of subsidence during follow up [4]. Meanwhile, SH Lee et al. reported that complete UPR over 38% during ACDF increases the risk of subsidence during follow up [4]. At present, there is little evidence of whether this surgical technique provides good clinical and radiologic outcomes after complete unilateral or bilateral UPR, especially in regards to subsidence and cervical sagittal alignment. Accordingly, this study was undertaken to evaluate the association of complete UPR on subsidence and regional cervical sagittal balance by comparing the clinical and radiologic outcomes after ACDF with complete UPR versus ACDF without UPR. Surgical procedure Page 3/19 The patients were positioned under general anesthesia in the supine position. The surgical technique was chosen using a standard Smith–Robinson technique. After confirmation and exposure of the proper vertebral levels according to the compressive materials, a discectomy was performed, and a high-speed burr was applied to remove the anterior and posterior bony spurs and the endplate cartilage. The endplate cartilage was eliminated with a curette carefully to preserve the bony endplate as much as possible to prevent cage subsidence. Discs, endplate cartilaginous, and other compressive materials were subducted to achieve appropriate dural and neural decompression. Using an osteotome, a high-speed electric drill, and a Kerrison punch, the nerve roots were decompressed by completely removing the uncinate process. Page 3/19 If the patient had unilateral symptoms and if radiologic results were consistent, we performed removal of the uncinate process unilaterally. We used a plate (Atlantis; Medtronic, Minneapolis, MN, USA) and allograft cage (Cornerstone®-SR; Medtronic, Minneapolis, MN, USA) with local autologous bone. We did not use autologous iliac bone or growth factors, such as demineralized bone matrix and recombinant bone morphogenetic proteins (rhBMP), as graft material. The proper size for the allobone cage was decided by both preoperative evaluation and intraoperative formatting using a trial cage. The cage was placed into the disc space as described above. Fixed type screw was utilized to fix the anterior cervical spine plate. If there was no complication during operation, all patients were able to sit upright and walk with a neck collar on the first day after surgery. The patients wore a cervical collar for 1 month after surgery. Clinical and radiographic results were obtained by an independent observer for 5 days post- operatively. In the outpatient clinic, patients were continuously followed up post-operation. Clinical outcome assessment Intraoperative blood loss, operative time, days of hospitalization, and clinical outcomes were evaluated using the neck disability index (NDI), neck visual analog scale (VAS), and arm-VAS preoperatively, immediately after surgery, and at 2-year follow up. During the last follow up, the patient was assessed according to Odom’s criteria, from poor to excellent [6]. Radiological evaluation Preoperative radiologic examination evaluated plain radiographs, computed tomography scans, and magnetic resonance imaging. Plain radiological examinations of the cervical spine were also conducted immediately after surgery and at 2-year follow up for all patients. Cervical alignment was evaluated using the Cobb angle of C2–C7, working the process described by Borden [7]: this angle was made by the lines along the inferior endplate of C2 to the inferior endplate of C7 in the neutral position. Subsidence was decided by measuring the distance from the upper endplate of the upper vertebral body to the lower endplate of the lower vertebral body at the level of the operation. The segmental angle was calculated using the Cobb angle of the adjacent vertebrae in the intervertebral disc involved. The total intervertebral height was decided as the length from the upper endplate of the cephalad vertebrae to the inferior endplate of the caudal vertebrae of the fused segment, which was quantified as the mean value of the height of the anterior and posterior borders [8]. Subsidence was described as a decline in the height of the operative segment greater than 3 mm between immediate images after the operation and those acquired at the last follow up (Fig. 3A). Spino-cranial angle (SCA) was defined as the angle between the C7 line and the line joining the center of the sella turcica and the center of the inferior endplate of the C7 body. The center of the sella turcica – C7 sagittal vertical axis (St-SVA) was defined as the distance between a plumb line hung from the center of the sella turcica and the center of the C7 body (Fig. 3B). The C2–C7 sagittal vertical axis (SVA) was decided as the length from the postero-superior corner of C7 and the vertical line from the center of the C2 body. The T1 slope was defined as the angle between the upper endplate of T1 and the horizontal line (Fig. 3C). Because keeping horizontal gaze is the most important function of the cervical vertebrae, patients maintained a horizontal gaze position during radiologic Page 4/19 Page 4/19 examination. Occipital slope (O-s) is a postural variable reflecting the position of the skull, and it can reflect the degree of horizontal gaze. O-s represents the angle between the McGregor line and horizontal line (Fig. 3D). We decided the maximum difference in the O-s values at each examination as 2 degrees. Radiological evaluation Radiological fusion was decided to have occurred when there was ≤ 2° movement on flexion–extension and/or ≤ 2 mm of movement of the interspinous distance on flexion–extension across the fusion segment [9]. Statistical analysis The findings are presented as mean values ± standard deviations (SD) or counts, as indicated. The independent t-test and chi-squared test results were used to compare both groups. By checking the normality of continuous data through Kolmogorov-Smirnov test, if the normality assumption is satisfied, the data are expressed as mean ± SD, and an independent two sample t-test is performed, and if the normality assumption is not satisfied, median (Q1-Q3), and Mann-Whitney U test was performed. The binary multiple logistic regression test was used to determine the influencing radiologic factors of subsidence as dependent variable. Gender, age, BMD, BMI, smoking, DM, operation level, resection side, and whether to remove uncinate as independent variables were adjusted and radiologic parameters were analyzed by binary multiple logistic regression. All P values <0.05 were considered to indicate statistical significance. All statistical analyses were performed using SPSS (version 23.0, SPSS, Chicago, IL, USA). 1. Patient demographics (Table 1) In total, 105 patients underwent ACDF at the authors’ institution. Detailed demographics of 48 out of 105 patients were shown in Table 1. In the matched pair analysis, there was no statistically significant factor in the demographic between the two groups. The total of 105 patients’ ages ranged from 46 to 77 years (average age, 57.9±11.83 years old). The patients were followed for an average of 37.7±10.5 months. The operation level was primarily the C5/6 level (60 cases, 57%), followed by the C4/5 level (23 cases, 22%). 2. Comparison of intraoperative blood loss, operative time, days of hospitalization, and clinical parameters(Table 2) Intraoperative blood loss, operative time, days of hospitalization, Arm-VAS, Neck-VAS, NDI, and Odom’s criteria of the two groups are shown in Table 2. All of the clinical parameters improved at 2-year follow up (P<0.0001). Regarding Odom’s criteria, most of the surgical results were excellent and good in both groups. Also, there was no complication in either group. There was no statistically significant clinical outcome between the ACDF with UPR and ACDF without UPR groups except for postoperative Arm-VAS. 4. Binary multiple logistic regression of the five measurements as significant parameters on subsidence (Table 4) Radiologic factors that may potentially associate with subsidence were analyzed using binary multiple logistic regression test. The results are shown in Table 4. As an association factor of subsidence, preoperative SCA values were significant (P<0.05). In opposition to our hypothesis, complete UPR was not a significant factor affecting subsidence. 3. Comparison of radiologic parameters (Table 3) 3. Comparison of radiologic parameters (Table 3) Page 5/19 Cervical lordosis, segmental angle, disc height, C2-C7 SVA, St-SVA, T1 slope, SCA, incidence of subsidence, and fusion rate of the two groups are shown in Table 3. All cervical sagittal parameters, including cervical lordosis, segmental angle, disc height, C2-C7 SVA, St-SVA, T1 slope, and SCA, except for preoperative St-SVA, SCA, and disc height of 2 years follow-up, were similar between the ACDF with complete UPR and ACDF without UPR groups. Differences in disc height, C2-C7 SVA, and SCA at 2-year follow up after preoperative examination, however, were statistically significant (p<0.05). Subsidence occurred in 9 patients (ACDF with complete UPR: 8 cases [33%] versus ACDF without UPR: 1 cases [4%]; p < 0.05). Radiological images for representative patients in each group are displayed in Figures 4 and 5. There was no statistical significance because there were only a few cases of removal of uncinate on both sides. However, subsidence occurred more frequently in cases of removal of both sides than in cases of removing only one side. 4. Binary multiple logistic regression of the five measurements as significant parameters on subsidence (Table 4) Discussion ACDF is the treatment of choice for symptomatic cervical spondylosis in patients when conservative treatments, such as medication or physiotherapy, have failed [10]. Patients with arm pain with neural foramen stenosis due to osteophytes or hypertrophy of the uncovertebral joint should be treated with ACDF, as well as UPR. ACDF with complete UPR is known to improve pain in the arm better and faster [11]. However, inadequate removal of the uncinate process has been reported to contribute to poor outcomes in cervical spondylosis cases [12]. In our study, the ACDF with UPR group had better arm pain in the immediate post-operation period than the ACDF without UPR group. As the uncinate process is an important structure to maintaining the stability of adjacent vertebral bodies in the spinal axis, we investigated whether sagittal alignment or subsidence is affected by removing the uncinate process. Subsidence occurs as a natural process during the course of an interbody fusion procedure and is described as settlement of a body with a higher elasticity modulus (e.g., graft, cage, spacer) into a body with lower elasticity modulus (e.g., vertebral body), leading to a change in spine structure [13]. However, upon excessive subsidence, interbody spaces are narrowed and kyphosis of the spine occurs. This introduces instability of the screw-plate and screw-bone (e.g., pull-out, change of angulation, breakage of the instrumentation) [13]. To the best of our knowledge, end-plate preparation, type of cage and size, multilevel fusion, recombinant human bone morphogenetic protein-2 (rhBMP-2), process of instrumentation, and bone quality are significant factors of subsidence [14]. In our study, when the ACDF with complete UPR and ACDF without UPR were compared under the same conditions, Page 6/19 Page 6/19 subsidence was significantly higher when complete UPR was performed after 3 years on average. Considering these reasons, it would seem that end-plate preparations would be performed more in the process of UPR in the ACDF with UPR group. However, between the ACDF with UPR and ACDF without UPR groups, clinical results except postoperative Arm-VAS were not significantly different. This is because the foramen is widened due to the UPR, such that, even if subsidence occurs, radiculopathy due to pressing of the root does not occur. Overall, in the case of one-level ACDF, it is difficult to find a significant adverse effect of subsidence. Discussion However, caution against subsidence is needed, and a large- scale and long-term follow-up study of multiple-level ACDF with UPR is necessary. Sagittal balance has been suggested for cervical spine treatment. T1 slope determines the sagittal balance of the cervical spine, and this parameter is related with C2–C7 angle [15]. Previous studies have reported that C2-C7 lordosis is closely related to the other cervical and thoracic parameters (cervical lordosis, thoracic kyphosis) [16]. Cervical sagittal imbalance influences the health-related quality of life (HRQOL) of patients [17]. St-SVA and C2–C7 SVA are closely associated with the clinical results of neck pain and HRQOL [18]. The A study by Tang et al. suggested that increasing cervical SVA is a cause for clinical concern of cervical malalignment as reflected by poor HRQOL scores [19]. In our study, C2-C7 lordosis, segmental angle, disc height, C2-C7 SVA, St-SVA, T1 slope, and SCA were not different between ACDF with UPR and ACDF without UPR group, although the differences significant in disc height, C2-C7 SVA, and SVA at last follow-up and preoperatively were statistically between the two surgery groups (p<0.05). Accordingly, there were no differences in clinical outcomes between the two groups. A study by Tang et al. suggested that increasing cervical SVA is a cause for clinical concern of cervical malalignment as reflected by poor HRQOL scores [19]. In our study, C2-C7 lordosis, segmental angle, disc height, C2-C7 SVA, St-SVA, T1 slope, and SCA were not different between ACDF with UPR and ACDF without UPR group, although the differences significant in disc height, C2-C7 SVA, and SVA at last follow-up and preoperatively were statistically between the two surgery groups (p<0.05). Accordingly, there were no differences in clinical outcomes between the two groups. Global cervical spine lordosis was not influenced by single-level ACDF [20]. This is the natural mechanism of the human body, which keeps the head on a neutral axis in the optimal horizontal plane for the visiovestibular system and re-establishes sagittal balance [20]. In our study, single-level ACDF with UPR did not affect sagittal balance, although parameters of C2-C7 lordosis, segmental angle, disc height, C2- C7 SVA, St-SVA, and SVA were worse. Thus, long-term follow up and a large scale study of multiple-level ACDF with UPR or ACDF in kyphotic cervical spine are necessary. Technically, UPR usually proceeds from the inside to the outside. Discussion This technique needs to be performed carefully because of the possibility of injury to the nerve roots and vertebral arteries. It is recommended to use a punch rather than a drill when removing the lateral portion of the uncinated process. Availability of data and materials The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. Acknowledgements Not ApplicableNot ApplicableNot Applicable Conclusions Cervical sagittal alignment after ACDF with complete UPR is not significantly different from that achieved with ACDF without UPR. However, subsidence appears to occur more often after ACDF with complete UPR than after ACDF without UPR, although with little to no clinical impact. More precise and careful selection of patients is needed when deciding on additional complete UPR. Authors’ contributions All authors made substantive intellectual contributions to this study to qualify as authors. NSH and KKH contributed to study design, acquisition of data, analysis of data, and interpretation of results. PJY, KSU, CDK, KKS, CYE contributed to discuss the article. All authors read and approved the final manuscript. Limitations of this study Our study had a few limitations. The matched pair number of patients who underwent removal of the uncinate process was small. Also, cases with a bilaterally UPR were rare. And, because our study did not have a randomized controlled design, we could not completely control the possibility of selection bias. Additionally, because our study size was small, we were limited in our ability to make comparisons between the groups for several factors known to affect prognosis. Failure to indicate the extent to which the uncinate process was removed as an objective indicator was also a limitation. However, the results of this study suggest that when performing ACDF with complete UPR, the risk of subsidence should be Page 7/19 Page 7/19 considered. Prospective studies will be conducted using well-guided evidence-based protocols with adequate controls. considered. Prospective studies will be conducted using well-guided evidence-based protocols with adequate controls. Funding No funding Consent for publication Not applicable Competing interests The authors declare that they have no financial or other conflicts of interest in relation to this research and its publication. Abbreviations ACDF: Anterior cervical discectomy and fusion Page 8/19 RhBMP-2: Recombinant human bone morphogenetic protein-2 RhBMP-2: Recombinant human bone morphogenetic protein-2 HRQOL: Health-related quality of life HRQOL: Health-related quality of life References 1. CLOWARD RB. The anterior approach for removal of ruptured cervical disks. Journal of neurosurgery. 1958;15(6):602-617. 1. CLOWARD RB. The anterior approach for removal of ruptured cervical disks. Journal of neurosurgery. 1958;15(6):602-617. 1. CLOWARD RB. The anterior approach for removal of ruptured cervical disks. Journal of neurosurgery. 1958;15(6):602-617. 2. Wang JC, McDonough PW, Endow KK, Delamarter RB. Increased fusion rates with cervical plating for two-level anterior cervical discectomy and fusion. 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Does additional uncinate resection increase pseudarthrosis following anterior cervical discectomy and fusion? SPINE. 2018;43(2):97-104. 6. Odom GL, Finney W, Woodhall B. Cervical disk lesions. Journal of the American Medical Association. 1958;166(1):23-28. 6. Odom GL, Finney W, Woodhall B. Cervical disk lesions. Journal of the American Medical Association. 1958;166(1):23-28. Page 9/19 7. BORDEN AG, RECHTMAN AM, GERSHON-COHEN J. The normal cervical lordosis. Radiology. 1960;74(5):806-809. 8. Chen Y, Wang X, Lu X, et al. Comparison of titanium and polyetheretherketone (PEEK) cages in the surgical treatment of multilevel cervical spondylotic myelopathy: A prospective, randomized, control study with over 7-year follow-up. Eur Spine J. 2013;22(7):1539-1546. 9. Hwang S, Hwang Y, Lieu A, et al. Outcome analyses of interbody titanium cage fusion used in the anterior discectomy for cervical degenerative disc disease. Journal of spinal disorders & techniques. 2005;18(4):326-331. 10. 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The process of subsidence after cervical stabilizations by cage alone, cage with plate and plate-cage. A biomechanical comparative study. Neurologia i neurochirurgia polska. 2007;41(5):411. 14. Lim TH, Kwon H, Jeon CH, et al. Effect of endplate conditions and bone mineral density on the compressive strength of the graft-endplate interface in anterior cervical spine fusion. Spine. 2001;26(8):951-956. 14. Lim TH, Kwon H, Jeon CH, et al. Effect of endplate conditions and bone mineral density on the compressive strength of the graft-endplate interface in anterior cervical spine fusion. Spine. 2001;26(8):951-956. 15. Jun H, Kim J, Ahn J, et al. T1 slope and degenerative cervical spondylolisthesis. Spine. 2015;40(4):E226. 15. Jun H, Kim J, Ahn J, et al. T1 slope and degenerative cervical spondylolisthesis. Spine. 2015;40(4):E226. 16. Kato M, Namikawa T, Matsumura A, Konishi S, Nakamura H. Effect of cervical sagittal balance on laminoplasty in patients with cervical myelopathy. Global Spine Journal. 2017;7(2):154-161. 16. Kato M, Namikawa T, Matsumura A, Konishi S, Nakamura H. Effect of cervical sagittal balance on laminoplasty in patients with cervical myelopathy. Global Spine Journal. 2017;7(2):154-161. 17. Sakai K, Yoshii T, Hirai T, Arai Y, Shinomiya K, Okawa A. Impact of the surgical treatment for degenerative cervical myelopathy on the preoperative cervical sagittal balance: A review of prospective comparative cohort between anterior decompression with fusion and laminoplasty. Eur Spine J. 2017;26(1):104-112. 18. Wu S, Li Y, Zhang Y, et al. Porous Titanium‐6 Aluminum‐4 vanadium cage has better osseointegration and less micromotion than a Poly‐Ether‐Ether‐Ketone cage in sheep vertebral fusion. Artificial Organs. 2013;37(12):E201. 18. Wu S, Li Y, Zhang Y, et al. Porous Titanium‐6 Aluminum‐4 vanadium cage has better osseointegration and less micromotion than a Poly‐Ether‐Ether‐Ketone cage in sheep vertebral fusion. Artificial Organs. 2013;37(12):E201. 19. Tang JA, Scheer JK, Smith JS, et al. The impact of standing regional cervical sagittal alignment on outcomes in posterior cervical fusion surgery. Neurosurgery. 2012;71(3):662-669. 19. Tang JA, Scheer JK, Smith JS, et al. References The impact of standing regional cervical sagittal alignment on outcomes in posterior cervical fusion surgery. Neurosurgery. 2012;71(3):662-669. 20. Donk RD, Arnts H, Verhagen WI, Groenewoud H, Verbeek AL, Bartels, R. H. M. A. Cervical sagittal alignment after different anterior discectomy procedures for single-level cervical degenerative disc disease: Randomized controlled trial. Acta Neurochir. 2017;159(12):2359-2365. 20. Donk RD, Arnts H, Verhagen WI, Groenewoud H, Verbeek AL, Bartels, R. H. M. A. Cervical sagittal alignment after different anterior discectomy procedures for single-level cervical degenerative disc disease: Randomized controlled trial. Acta Neurochir. 2017;159(12):2359-2365. Page 10/19 Page 10/19 BMD; bone mineral density, BMI; body mass index, DM, diabetes mellitus; Tables Table 1. Patient demographics Table 1. Patient demographics ACDF without UPR ACDF with complete UPR p-value ACDF without UPR (n = 24) ACDF with complete UPR (n = 24) p-value Sex Female 15 16 Male 9 8 0.763 Mean age (years) 47.9 ± 9.78 49.1 ± 9.67 0.669 BMD (g/cm2) T-score -0.66 ± 1.21 -0.78 ± 0.77 0.681 BMI (kg/m2) 23.5 ± 2.47 23.5 ± 2.02 0.984 DM 5 6 0.731 Smoking 9 6 0.351 Operation level C2/3 C3/4 0 6 0 5 C4/5 C5/6 C6/7 15 3 0 15 4 0 0.999 Resection side Unilateral 20 Bilateral 4 mineral density, BMI; body mass index, DM, diabetes mellitus; ior cervical discectomy and fusion te process removal BMD; bone mineral density, BMI; body mass index, DM, diabetes mellitus; BMD; bone mineral density, BMI; body mass index, DM, diabetes mellitus; ACDF; anterior cervical discectomy and fusion UPR; uncinate process removal p < 0.05 comparing ACDF without UPR and ACDF with complete UPR p < 0.05 comparing ACDF without UPR and ACDF with complete UPR p < 0.05 comparing ACDF without UPR and ACDF with complete UPR p < 0.05 comparing ACDF without UPR and ACDF with complete UPR Page 11/19 Page 11/19 Table 2. Comparison of intraoperative blood loss, operative time, days of hospitalization, and clinical parameters Table 2. Comparison of intraoperative blood loss, operative time, days of hospitalization, and clinical parameters ACDF without UPR (n = 24) Median (Q1-Q3),(min-max) N(%) ACDF with complete UPR (n = 24) Median (Q1-Q3),(min-max) N(%) p- value Intraoperative blood loss (ml) 60.00(52.50-80.00),(50.00- 100.00) 77.50(57.50-90.00),(50.00-140.00) 0.175 Operation time (min) 100.00(90.00-120.00),(90.00- 150.00) 120.00(100.00-130.00),(90.00- 155.00) 0.086 Duration of hospitalization (day) 6.00(6.00-7.00),(5.00-9.00) 6.00(6.00-7.00),(5.00-9.00) 0.866 Arm VAS Preoperation Postoperation 2 years follow-up 9.00(8.00-9.00), (7.00-9.00) 4.00(3.00-5.00), (2.00-6.00) 2.00(1.00-2.00), (1.00-3.00) 8.50(8.00-9.00), (7.00-9.00) 3.00(2.00-3.50), (2.00-5.00) 2.00(1.00-2.00),(1.00-3.00) 0.116 0.003* 0.711 Neck VAS Preoperation Postoperation 2 years follow-up 9.00(8.00-9.00), (7.00-9.00) 2.00(1.00-5.00), (1.00-5.00) 1.00(1.00-2.00), (1.00-3.00) 9.00(8.00-9.00), (7.00-9.00) 2.00(2.00-3.50), (2.00-5.00) 1.00(1.00-1.00), (1.00-2.00) 0.817 0.657 0.281 NDI Preoperation Postoperation 2 years follow-up 38.00(37.00-41.50), (35.00- 44.00) 24.00(21.00-25.00), (15.00- 29.00) 14.00(13.50-16.50), (11.00- 19.00) 40.50(37.50-42.00), (35.00-44.00) 22.00(19.00-25.00),(15.00-27.00) 13.50(11.00-15.00),(11.00-17.00) 0.464 0.514 0.069 Odom’s criteria Excellent Good Fair Poor 9 15 0 0 9 14 1 0 0.999 0.999 VAS; Visual analog scale, NDI; Neck Disability Index Page 12/19 p < 0.05 comparing ACDF without UPR and ACDF with UPR p < 0.05 comparing ACDF without UPR and ACDF with UPR Table 3. Comparison of radiologic parameters Table 3. SVA; sagittal vertical axis, St-SVA; sellar turcica–sagittal vertical axis, SCA spinocranial angle Tables Comparison of radiologic parameters Page 13/19 ACDF without UPR (n = 24) Mean ± SD, N(%) ACDF with complete UPR (n = 24) Mean ± SD, N(%) p-value C2–C7 lordosis (°) Preoperation Postoperation 2 years follow-up 2 years follow-up - Preoperation 15.50(9.45-17.60), (3.90-20.80) 15.80(10.25-17.90), (7.70-27.90) 15.95(13.45-24.15), (10.20- 28.50) 3.80(0.70-8.50),(-5.70-17.00) 14.10(5.45-19.55), (3.90-26.40) 17.25(8.50-19.10), (3.00-27.30) 14.65(11.00-29.70), (1.10-45.50) 5.20(-3.60-15.30),(-12.20-30.00) 0.781 0.772 0.877 0.984 Segmental angle (°) Preoperation Postoperation 2 years follow-up 2 years follow-up - Preoperation 5.45(4.60-5.95), (1.80-7.10) 5.85(2.90-7.45), (0.50-14.40) 5.90(5.10-7.25), (1.20-9.60) 0.70(-0.75-2.90), (-4.80-5.30) 4.95(4.00-5.50), (1.30-7.10) 5.70(3.45-7.55), (1.00-14.40) 5.20(3.75-6.10), (0.90-10.00) 0.50(-1.60-2.30), (-4.80-6.90) 0.215 0.918 0.207 0.643 Disc height (mm) Preoperation Postoperation 2 years follow-up 2 years follow-up – Preoperation 5.60(5.15-6.18), (4.23-7.90) 7.16(6.46-7.90), (5.84-8.91) 6.22(5.41-6.58), (4.82-13.12) 0.08(-0.52-2.02), (-1.42-6.95) 5.96(5.58-6.26), (5.18-6.97) 7.53(7.27-7.84), (6.52-8.91) 5.19(5.15-5.55), (5.01-5.82) -0.44(-1.15--0.19), (-1.78-0.45) 0.173 0.117 <0.001* 0.007* C2–C7 SVA (mm) Preoperation Postoperation 2 years follow-up 2 years follow-up - Preoperation 20.05(15.39-26.31), (12.46- 30.53) 19.33(13.92-26.13), (9.77-29.04) 15.78(12.36-21.51), (10.62- 30.84) -3.98(-5.80--2.84), (-9.62-7.01) 17.06(15.15-24.72), (6.97-28.53) 18.58(13.25-29.88), (6.72-39.25) 17.28(11.18-29.57), (4.42-41.93) -0.26(-4.15-5.88), (-5.32-15.96) 0.261 0.877 0.703 0.005* St-SVA (mm) Preoperation Postoperation 2 years follow-up 30.77(24.05-35.06), (15.71- 42.98) 27.65(17.49-28.63), (10.59- 52 27) 25.68(20.72-29.36), (13.25-55.43) 28.94(17.12-30.42), (4.40-61.23) 28.56(11.53-41.36), (4.74-77.58) 0.018* 0.414 0.496 ACDF without UPR (n = 24) Mean ± SD, N(%) ACDF with complete UPR (n = 24) Mean ± SD, N(%) p-value ACDF without UPR Page 14/19 T1 slope (°) Preoperation Postoperation Last follow-up Last follow-up - Preoperation 25.15(20.25-27.90), (12.00- 31.60) 24.85(17.10-28.10), (13.60- 32.80) 23.50(17.60-27.00), (12.00- 33.20) -1.30(-3.00-1.25), (-14.90-2.90) 24.10(22.00-25.90), (11.90-44.00) 25.40(20.50-27.55), (14.80-32.50) 25.80(20.75-28.00), (15.40-49.20) 0.50(-0.70-2.75), (-15.40-7.10) 0.687 0.599 0.327 0.066 SCA (°) Preoperation Postoperation 2 years follow-up 2 years follow-up - Preoperation 104.65(101.20-108.65), (89.90- 115.90) 104.75(100.90-108.45), (94.60- 117.00) 105.80(100.60-111.60), (92.80- 115.50) 3.65(-4.50-8.35), (-13.30-10.70) 111.05(107.85-114.70), (101.20- 120.00) 105.90(103.65-111.45), (95.50- 113.60) 105.80(99.10-107.30), (87.30- 121.40) -8.15(-15.15-2.70), (-20.10-9.80) <0.001* 0.397 0.634 0.004* Subsidence 1 (4%) 8 (33%) 0.023* Fusion 22 (92%) 22 (92%) 0.999 SVA; sagittal vertical axis, St-SVA; sellar turcica–sagittal vertical axis, Page 15/19 Table 4. Binary multiple analysis of the five measurements as significant parameters on Factor Odds Ratio 95% CI p-value Preoperative C2–C7 SVA 1.034 0.896 – 1.192 0.651 Preoperative St-SVA 0.946 0.863 – 1.037 0.238 Preoperative SCA 1.237 1.074 – 1.425 0.003* Preoperative CL 0.950 0.817 – 1.104 0.503 Preoperative T1-slope 0.998 0.870 – 1.145 0.978 SVA; sagittal vertical axis, St-SVA; sellar turcica–sagittal vertical axis, Table 4. Binary multiple analysis of the five measurements as significant parameters on subsidence Table 4. SVA; sagittal vertical axis, St-SVA; sellar turcica–sagittal vertical axis, Tables Binary multiple analysis of the five measurements as significant pa SVA; sagittal vertical axis, St-SVA; sellar turcica–sagittal vertical axis, SVA; sagittal vertical axis, St-SVA; sellar turcica–sagittal vertical axis, SCA; spinocranial angle, CL; cervical lordosis, CI: confidence interval SCA; spinocranial angle, CL; cervical lordosis, CI: confidence interval * Statistically significant Figures Figures Page 16/19 Figures gure 1 ow chart of the patients in our study. g igure 1 Figure 1 Page 16/19 Figure 2 A: Cervical spine oblique radiographs at C4-5 (black arrow). B: Cervical spine CT (axial view) shows right foraminal stenosis (black arrow). C: ACDF with UPR was performed, and the right foramen was widened on post-operative CT (black arrow). D: The nerve root was decompressed by completely removing the uncinate process (black arrow) Figure 2 Figure 2 A: Cervical spine oblique radiographs at C4-5 (black arrow). B: Cervical spine CT (axial view) shows right foraminal stenosis (black arrow). C: ACDF with UPR was performed, and the right foramen was widened on post-operative CT (black arrow). D: The nerve root was decompressed by completely removing the uncinate process (black arrow). A: Cervical spine oblique radiographs at C4-5 (black arrow). B: Cervical spine CT (axial view) shows right foraminal stenosis (black arrow). C: ACDF with UPR was performed, and the right foramen was widened on post-operative CT (black arrow). D: The nerve root was decompressed by completely removing the uncinate process (black arrow). p ( ) Figure 3 A: Subsidence measurements were performed from the anterior, middle, and posterior portions of the vertebral bodies of interest. Subsidence was described as a greater than 3 mm decrease in height of the operative segment between images produced immediately after the operation and those acquired at 2 years follow up. B: The SCA was defined as the angle between the C7 line and the line joining the center of the sellar turcica and the center of the inferior endplate of the C7 body. The center of the St-SVA was defined as the distance between a plumb line from the center of the sellar turcica and the center of the C7 body. C: The C2–C7 SVA was decided as the length from the posterosuperior corner of C7 and the vertical line from the center of the C2 body. The T1 slope was defined as the angle between the upper endplate of T1 and the horizontal line. D: O-s is the angle between the McGregor line and the horizontal line. Figure 3 Figure 3 A: Subsidence measurements were performed from the anterior, middle, and posterior portions of the vertebral bodies of interest. Subsidence was described as a greater than 3 mm decrease in height of the operative segment between images produced immediately after the operation and those acquired at 2 years follow up. B: The SCA was defined as the angle between the C7 line and the line joining the center of the sellar turcica and the center of the inferior endplate of the C7 body. The center of the St-SVA was defined as the distance between a plumb line from the center of the sellar turcica and the center of the C7 body. C: The C2–C7 SVA was decided as the length from the posterosuperior corner of C7 and the vertical line from the center of the C2 body. The T1 slope was defined as the angle between the upper endplate of T1 and the horizontal line. D: O-s is the angle between the McGregor line and the horizontal line. Page 17/19 Figure 4 A case from the ACDF with complete UPR group. The patient underwent an ACDF operation of C5/6 with complete UPR. In this patient, C2–C7 SVA and St-SVA increased with time, but SCA decreased with time. Figure 4 A case from the ACDF with complete UPR group. The patient underwent an ACDF operation of C5/6 with complete UPR. In this patient, C2–C7 SVA and St-SVA increased with time, but SCA decreased with time. A case from the ACDF with complete UPR group. The patient underwent an ACDF operation of C5/6 with complete UPR. In this patient, C2–C7 SVA and St-SVA increased with time, but SCA decreased with time. A case from the ACDF with complete UPR group. The patient underwent an ACDF operation of C5/6 with complete UPR. In this patient, C2–C7 SVA and St-SVA increased with time, but SCA decreased with time. Page 18/19 Figure 5 A case from the ACDF without UPR group. The patient underwent an ACDF operation of C4/5 without UPR. In this patient, C2–C7 SVA and St-SVA decreased with time, but SCA increased with time. Figure 5 A case from the ACDF without UPR group. The patient underwent an ACDF operation of C4/5 without UPR. In this patient, C2–C7 SVA and St-SVA decreased with time, but SCA increased with time. Page 19/19
W4226451279.txt	https://www.researchsquare.com/article/rs-805973/latest.pdf	en		Application of deep learning image reconstruction algorithm to improve image quality in CT angiography of children with Takayasu arteritis	Journal of X-ray science and technology	2,021	cc-by	3,931	Application of Deep Learning Image Reconstruction (DLIR) Algorithm to Improve Image Quality in CT Angiography of Children with Takayasu Arteritis Jihang Sun Beijing Children’s Hospital Haoyan Li Beijing Children’s Hospital Jianying Li General Electric (United States) Haiyun Li Capital Medical University Michelle Li Stanford University Zuofu Zhou (  464481492@qq.com ) Fujian Provincial Maternity and Children's Hospital Yun Peng Beijing Children’s Hospital Research Article Keywords: Computed Tomography Angiography, Takayasu Arteritis, Pediatrics, Deep Learning Posted Date: September 21st, 2021 DOI: https://doi.org/10.21203/rs.3.rs-805973/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Journal of X-Ray Science and Technology on November 18th, 2021. See the published version at https://doi.org/10.3233/XST-211033. Page 1/12 Abstract purpose: To evaluate the image quality improvement in CTA of children with Takayasu arteritis (TAK) using a Deep learning image reconstruction (DLIR) in comparison to other reconstruction algorithms. Methods: 32 patients (9.14±4.51 years old) with TAK underwent neck, chest and abdominal CTA with 100kVp were enrolled. Images were reconstructed at 0.625mm slice thickness using Filtered BackProjection (FBP), 50% adaptive statistical iterative reconstruction-V (ASIR-V), 100%ASIR-V and DLIR with high setting (DLIR-H). The CT number and standard deviation (SD) of the descending aorta and back muscle were measured and contrast-to-noise ratio (CNR) for aorta was calculated. The vessel visualization, overall image noise and diagnostic confidence were evaluated using a 5-point scale (5, excellent; 3, acceptable) by 2 observers. Results: There was no significant difference in CT number across all reconstructions. The image noise values (in HU) were 31.36±6.01, 24.96±4.69, 18.46±3.91 and 15.58±3.65, and CNR values for aorta were 11.93±2.12, 15.66±2.37, 22.54±3.34 and 24.02±4.55 with FBP, 50%ASIR-V, 100%ASIR-V and DLIR-H, respectively. The 100%ASIR-V and DLIR-H images had similar noise and CNR (all P>0.05), and both had lower noise and higher CNR than FBP and 50%ASIR-V images (all P<0.05). The subjective evaluation suggested that all images were diagnostic for large arteries, but only 50%ASIR-V and DLIR-H met the diagnostic requirement for small arteries (3.03±0.18 and 3.53±0.51). Conclusions: DLIR-H improves the CTA image quality and diagnostic confidence for TAK patients compared with 50%ASIR-V, and best balances image noise and spatial resolution compared with 100%ASIR-V. 1. Background CT angiography (CTA) is a common examination, which can quickly and noninvasively identify the main artery malformation (1–7) and has been widely used for children. It is also an important evaluation method for Takayasu arteritis (TAK) (8–9). Children with TAK need to use immunosuppressants, the inflammatory indexes of children with TAK usually tend to be normal immediately after treatment, which cannot correctly reflect the severity and activity of the disease (8, 10). Therefore, CTA has become an important method to evaluate the status of TAK and sometime is even more sensitive than laboratory test results, especially for patients after using immunosuppressants (8–11). However, children are radiationsensitive and CT scans should be performed following the as low as reasonably achievable (ALARA) principle (12). How to maintain or even improve the image quality when the radiation dose decreases is worthy of continuous study. Iterative reconstruction (IR) algorithms have shown the ability to reduce image noise and have contributed to the radiation dose reduction in CTA for the last decade (13–15). However, constrained by the modelling complexity, there is a need to balance spatial resolution and image noise in IR algorithms, and IR with high weights are often limited in clinical applications to avoid blurring artifacts or blotchy appearance in IR images, which affects the efficiency of dose reduction (16– Page 2/12 17). Recently, a deep learning image reconstruction (DLIR) algorithm (GE Healthcare) has been developed based on the artificial intelligence and deep neural networks. The current version of DLIR consists of three different strength settings: low, medium, and high to yield different noise reduction capability. Studies have shown that DLIR at different strengths can further reduce image noise while avoiding the blurring artifacts, so as to improve the image quality compared with IR algorithms (18–21). In our study, we extended the study to pediatric patients with TAK reconstructed with DLIR at the high setting (DLIR-H) to investigate whether DLIR-H could improve image quality under normal scan conditions in comparison with the traditional Filter back projection (FBP) and the state-of-the-art adaptive statistical iterative reconstruction (ASIR-V) algorithm. 2. Methods 2.1. Patients The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). This was a retrospective study approved by the ethics committee of our hospital, and informed consents from patients were waived. The CTA was performed continuously from Nov 1, 2019 to Jun 30, 2020. 2.2. CT scan and image reconstruction All examinations were performed on a 256 rows multi-detector CT scanner (Revolution CT, GE Healthcare, USA), using a tube voltage of 100kVp, helical pitch value of 1.375:1 with 40mm detector width and 0.5s rotation speed. The automatic tube current modulation technique was used with tube current in the range of 50-500mA, noise index was 11. All scans were performed when children were in a quiet state. For those children who could not cooperate, the 10% chloral hydrate at a dose of 0.4ml/kg based on body weight was given orally and scans would not start until patients fell asleep. The scanning range included neck, chest, and abdomen, with the upper edge from the skull base and the lower edge to the anterior superior iliac spine. For the contrast-enhanced CT protocol, a peripheral venous cannula was pre-placed in the superficial vein of dorsum of the hand. An iodinated contrast agent (320 mg I/ml iodixanol; GE Healthcare, USA) was administered using a single-head power injector. The contrast medium dosage was calculated according to the body weight of each child: 1.6 ml/kg for children lower than 16.0 kg, 1.4 ml/kg for 16.1-35.0kg and 1.2 ml/kg for higher than 35.0kg (the maximum usage limit was 70ml). Flow rate was adjusted according to a fixed injection time of 15s and contrast enhanced scan started at 17s after the start of contrast injection. The raw data was reconstructed to 0.625mm thin slice images using 3 reconstruction algorithms into 4 groups: FBP group, 50%ASIR-V group, 100%ASIR-V group, and DLIR-H group. All reconstructions used a standard kernel. 2.3. Image quality evaluation Page 3/12 All images were transferred to an advantage workstation (AW4.7, GE Healthcare, USA). Two observers with 15 and 8 years of experience in reviewing CTA images subjectively evaluated image quality using a 5-point scoring system. The reviewers were aware of the clinical task of diagnosing TA but were blinded to patient information and reconstruction algorithms. The readers evaluated all 4 differently reconstructed images of the same patient before moving to different patients, but the order in which the different reconstructions appear on the AW changed randomly. The observers could freely adjust the window width and position for observation and used multiplanar reconstructions (MPR) and threedimensional images for the evaluation. If the scores given by the two observers were not the same, a third senior doctor with 20 years of experience in reviewing children chest CT images would evaluate the images and gave the final score. Subjective scores included the overall image noise, visualization of large arteries (artery diameter greater than the vertebral artery or proper hepatic artery), visualization of small arteries (artery diameter less than or equal to the vertebral artery or proper hepatic artery) and diagnostic confidence. Scores with at least 3 points were accepted for diagnosis with 5 points being the best. The specific evaluation criteria are listed in Table 1. Table 1 specific criteria for subjective evaluation 1 point 2 points 3 points 4 points 5 points Overall image noise severe high moderate little rare Vessel display ability nondetect detectable measurable clear clear with sharp edges Diagnostic confidence none little moderate high definite After the subjective evaluation, the two observers conducted objective measurement on the AW workstation together, selected the largest section of the liver portal, set a circular region of interest (ROI) on the descending aorta (Ao) on the liver portal section with a diameter half of that of Ao and on the back muscle (Mu) at the same imaging level to measure their CT attenuation value and standard deviation (SD) value. The contrast-to-noise ratio (CNR) for the descending aorta was calculated using the following formula: CNR = (CT (Ao) - CT (Mu)) / ((SD (Ao) + SD (Mu))/2) 2.4. Statistical analysis All the data were expressed using the form of mean ± standard deviation. The differences of the 4 image groups were analyzed, continuous variables following the normal distribution were analyzed by using the repeated measures analysis of variance with Bonferroni correction. The ordinal scales or variables that failed to follow normal distribution were analyzed by using Friedman's test. P < 0.05 was considered to have statistically significant difference. Page 4/12 3. Results A total of 32 patients (9 males and 23 females) with an average age of 9.14 ± 4.51 years (1–17 years) were included in the study. The volume CT dose index (CTDIvol) for the group was 4.53 ± 2.62mGy and the dose-length-product (DLP) was 258.39 ± 189.93 mGy.cm. The results of the subjective evaluation and objective measurement are listed in Table 2 and Table 3, respectively. For the subjective score, the overall image noise of the 50%ASIR-V, 100%ASIR-V and DLIR-H images could meet the diagnostic requirements, with the 100%ASIR-V and DLIR-H images having the highest scores, and there was no statistical difference between them; for the visualization of large arteries, all images could met the diagnostic requirements (Fig. 1), and 100%ASIR-V and DLIR-H images were the best, and there was no statistical difference between them; for the visualization of small arteries, the 100%ASIR-V over-smoothed images with sub-optimal resolution (2.84 ± 0.37) and only the 50%ASIR-V and DLIR-H could provide the satisfactory diagnostic confidence for all vessels, with DLIR-H being the best reconstruction algorithm (Fig. 2); The DLIR-H provided significantly better diagnostic confidence (4.09 ± 0.30) than that of 50%ASIR-V (3.03 ± 0.18) and 100%ASIR-V (3.00 ± 0.00) (p < 0.001). For the objective measurement results: there was no statistical difference in the vascular CT number and muscle CT number among the reconstruction groups; for the vascular noise and muscle noise, the values with 100%ASIR-V (18.46 ± 3.91HU and 11.22 ± 2.40HU) and DLIR-H (15.58 ± 3.65HU and 12.64 ± 2.71HU) had no statistically significant difference between them, and both significantly lower those (24.96 ± 4.69HU and 17.68 ± 3.52HU) of 50%ASIR-V images. For CNR, the values with 100%ASIR-V (22.54 ± 3.34) and DLIR-H (24.02 ± 4.55) also had no statistically significant difference between them and were both significantly higher than that (15.66 ± 2.37) with 50%ASIR-V. Compared with the FBP images, DLIR-H images reduced image noise by 50.32% in vessels and 49% in soft tissue, and the 50%ASIR-V reduced images noise by 20.41% in vessels and 28.68% in soft tissue; For CNR, DLIR-H increased the value by 101.34% and 50%ASIR-V by 88.94%. Table 2 Results of subjective scores FBP 50%ASIR-V 100%ASIR-V DLIR P value Overall image noise 2.16 ± 0.37 3.09 ± 0.30 4.41 ± 0.50* 4.03 ± 0.18* < 0.001 Display ability of large arteries 3.06 ± 0.25 4.03 ± 0.31* 4.59 ± 0.50# 4.34 ± 0.48# < 0.001 Display ability of small arteries 2.94 ± 0.25# 3.03 ± 0.18† 2.84 ± 0.37#† 3.53 ± 0.51 < 0.001 Diagnostic confidence 2.91 ± 0.30# 3.03 ± 0.18† 3.00 ± 0.00#† 4.09 ± 0.30 < 0.001 #†: without in-group statistical difference Page 5/12 Table 3 Results of objective scores FBP 50%ASIR-V 100%ASIR-V DLIR P value Artery CT value* 391.27 ± 55.23 391.92 ± 54.48 392.28 ± 53.79 393.61 ± 54.19 1.00 Artery SD value 31.36 ± 6.01 24.96 ± 4.69 18.46 ± 3.91* 15.58 ± 3.65* < 0.001 Muscle CT value* 60.46 ± 9.38 60.42 ± 8.74 60.22 ± 8.10 60.54 ± 7.69 1.00 Muscle SD value 24.79 ± 5.16 17.68 ± 3.52 11.22 ± 2.40* 12.64 ± 2.71* < 0.001 Artery CNR 11.93 ± 2.12 15.66 ± 2.37 22.54 ± 3.34* 24.02 ± 4.55* < 0.001 CNR: contrast to noise ratio : without in-group statistical difference 4. Discussion In our study we investigated whether DLIR-H could improve image quality in CTA of pediatric patients with TAK under normal scan conditions, in comparison with the traditional FBP and the state-of-the-art 50%ASIR-V algorithm. From the objective evaluation point of view, our results showed that DLIR-H provided about 50% image noise reduction, while 50%ASIR-V provided about 25% reduction compared with FBP. From the subjective evaluation point of view, our results showed that DLIR-H did not affect the visualization of small arteries and image texture while reducing image noise. The overall appearance of DLIR-H images was similar to that of FBP and 50%ASIR-V images, without "blotchy" feeling, which was conducive to the display of detailed structures such as small arteries. In fact, since the overall image noise was significantly reduced, the ability of displaying small vessels was judged to be even better than that of FBP and 50%ASIR-V (3.53 vs. 2.94 and 3.03). All the subjective image quality scores of DLIR-H were higher than 3.0, and its comprehensive score was better than other reconstruction algorithms. On the other hand, even though 100%ASIR-V also significantly reduced image noise, it also altered image texture making images appeared too "smooth" with some spatial resolution and structure detail loses (Fig. 2), which affected the subjective diagnostic confidence and the diameter measurement for small arteries. CTA is an important evaluation method for TAK (18–19). Since TAK can involve the whole artery system, the scanning range is general much larger (including neck, chest and abdomen simultaneously) than that of conventional CTA. In addition, it is also necessary to observe whether the arterial branches with small arteries are involved, high spatial resolution and thin-layer images are desirable. All these clinical requirements put more pressure on the radiation dose in CTA for TAK patients (22), and efficient solutions are urgently needed to reduce the radiation dose while maintaining or improving image quality. IR algorithms have been widely used to tackle the problem of higher image noise with thin-layer images and/or under reduced radiation conditions. However, IR algorithms with high weights tend to over smooth Page 6/12 the high frequency noise and reduce the average frequency of the noise power spectrum (NPS) of the images. Although the image noise is greatly reduced, images may lose the normal image texture (Fig. 2), making images look "overly smooth", "plastic", or simply "unnatural" (16, 17, 23). Our results indicated that although it did not affect the visualization of large arteries (Fig. 1), it was not conducive to display image details such as small arteries (Fig. 2) using 100%ASIR-V algorithm. Therefore, in general, 50% weight of IR is recommended to maintain the balance between noise and image resolution in clinical routine work. Only when the radiation dose is very low, 100% IR weighted is recommended as a mean to salvage the scans (24). This is the major limitation of low-dose CTA applicated in TAK. DLIR is a new generation of image reconstruction algorithm. The deep learning method is used to analyze the characteristics of image noise generation and distribution in different organizations. In theory, the targeted reduction of noise will have minimum impact on the image resolution. In the process of DLIR reconstruction, the integrity of the image information remains largely unchanged when the image noise is greatly reduced, and there is no artificial addition or loss of image resolution. Our results indicated that when DLIR algorithm was used in the assessment of TAK, the image details were maintained, while image noise was significantly reduced. Ever though our study was focused on image noise reduction and image quality improvement, since at the current dose level with 50%ASIR-V, image quality was acceptable, the lower image noise and better image quality performance of DLIR-H could be traded with radiation dose reduction in the future. There were still some limitations in this study. Firstly, the case number in the study was rather small, because TAK is not a common disease, so it was hard to collect large samples, and impossible to compare children of different ages in groups. In the future, the sample size will be increased to enrich the data information. Secondly, subjective image evaluation was performed using a non-validated scale developed by the authors. Third, our study focused on the image quality improvement over the state-ofthe-art ASIR-V algorithm using the same scan under the standard-dose condition. Although the image quality of some ASIR-V images was different from those of DLIR images, no children had interventional radiology results as the gold standard, so it was impossible to confirm the diagnostic accuracies of the different reconstruction algorithms. Further research is needed to demonstrate the improvement in diagnostic efficacy. In addition, further research is also needed to focus on dose reduction to investigate whether DLIR could achieve the same image quality and diagnostic efficacy as the standard-dose ASIR-V images under lower-radiation dose or lower-contrast agent dose. 5. Conclusion DLIR-H improves the CTA image quality and diagnostic confidence for TAK patients compared with the state-of-the-art 50%ASIR-V, and best balances image noise and spatial resolution compared with 100%ASIR-V. Abbreviations ALARA: as low as reasonably achievable Page 7/12 CNR: contrast-to-noise ratio CTA: CT angiography CTDIvol: CT dose index DLIR: Deep learning image reconstruction DLIR-H: DLIR with high setting DLP: dose-length-product FBP: Filtered Back-Projection NBP: noise power spectrum SD: standard deviation TAK: Takayasu arteritis Declarations Acknowledgments The authors would like to thank Dr. Z.L., a senior cardiovascular radiologist, for helping us to produce the final subjective evaluation results in case of disagreement between the two observers. Author Contribution H.L.5 and Y.P. designed the study, drafted the initial manuscript, and reviewed and revised the manuscript. J.S., H.L.1, J.L., Z.Z., and M.L. coordinated and supervised data collection, and critically reviewed the manuscript for important content. All authors have read and approved the manuscript, and ensure that this is the case. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or notfor-profit sectors. Availability of data and materials The data is available from the corresponding author on reasonable request. Ethics approval and consent to participate Page 8/12 The authors are 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. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). This study was approved by the Ethical Committee of our Hospital. Informed consents were waived by Ethical Committee of our Hospital. Consent for publication Not Applicable. Competing interests Dr. J.L. reports that he is an employee of GE Healthcare, the manufacturer of the CT system used in this study. The other authors have no conflicts of interest to declare. References 1. Tola H, Ozturk E, Yildiz O, et al. 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Radiology 2019;293:491-503. 18. Benz DC, Benetos G, Rampidis G, et al. Validation of Deep-Learning Image Reconstruction for Coronary Computed Tomography Angiography: Impact on Noise, Image Quality and Diagnostic Accuracy. J Cardiovasc Comput Tomogr 2020;13:S1934-5925(19)30464-2. 19. Akagi M, Nakamura Y, Higaki T, et al. Deep Learning Reconstruction Improves Image Quality of Abdominal Ultra-High-Resolution CT. Eur Radiol 2019;29:6163-6171. 20. Tatsugami F, Higaki T, Nakamura Y, et al. Deep Learning-Based Image Restoration Algorithm for Coronary CT Angiography. Eur Radiol 2019;29:5322-5329. 21. Greffier J, Hamard A, Pereira F, et al. Image quality and dose reduction opportunity of deep learning image reconstruction algorithm for CT: a phantom study. Eur Radiol 2020;30:3951-3959. 22. Masuda T, Funama Y, Nakaura T, et al. Radiation Dose Reduction with a low-tube voltage technique for pediatric chest computed tomographic angiography based on the contrast-to-noise ratio index. Can Assoc Radiol J 2018;69:390-396. 23. Geyer LL, Schoepf UJ, Meinel FG, et al. State of the art: iterative CT reconstruction techniques. Radiology 276:339–357. 24. Sun J, Yang L, Zhou Z, et al. Performance evaluation of two iterative reconstruction algorithms, MBIR and ASIR, in low radiation dose and low contrast dose abdominal CT in children. Radiol Med 2020;125:918-925. Figures Page 10/12 Figure 1 Images of a 14-years old girl suffered from Takayasu arteritis. The scan voltage was 100kV, 1A-1D was the 0.625mm image with FBP, 50%ASIR-V, 100%ASIR-V and DLIR-H. respectively. The image noise of 1C and 1D were lower than that of 1A and 1B ( area). All images could show the slight dilation of big arteries (white arrow), and the stenosis of right renal artery (black arrow), 1C and 1D images had the least image noise. Page 11/12 Figure 2 Images of a 14-years old girl suffered from Takayasu’s arteritis. The scan voltage was 100kV, 2A-2D was the 0.625mm image with FBP, 50%ASIR-V, 100%ASIR-V and DLIR-H, respectively. The 100%ASIR-V image (2C) had the least image noise but was also blotchy in the image texture (black arrow), the boundary of muscles was unclear compared to 2D. The margin of the small artery was blurred and not consistent (white arrow), which could induce the misdiagnosis as vascular occlusion. DLIR-H image (2D) had similar low image noise as 2C but without the pixilated appearance seen in 2C and provided the highest diagnostic confidence for the vessels. Page 12/12
https://openalex.org/W2011437501	https://actavet.vfu.cz/media/pdf/avb_2002071030297.pdf	English	null	Effects of Sexually Activated Rams or Ewes on Pulsatile LH Secretion in Anoestrous Sheep	Acta veterinaria Brno	2,002	cc-by	4,850	Abstract Yildiz S., M. Uzun, M. Cenesiz, O. Ucar, M. Kaya, F. Onder: Effects of Sexually Activated Rams or Ewes on Pulsatile LH Secretion in Anoestrous Sheep. Acta Vet. Brno 2002, 71: 297-302. Yildiz S., M. Uzun, M. Cenesiz, O. Ucar, M. Kaya, F. Onder: Effects of Sexually Activated Rams or Ewes on Pulsatile LH Secretion in Anoestrous Sheep. Acta Vet. Brno 2002, 71: 297-302. The aim of this study was to test whether anoestrous ewes respond differentially, in terms of pulsatile LH release, to sexually activated males or females. For that purpose, anoestrous ewes (n = 21) were divided into three equal groups and placed into three separated rooms. The experiment commenced two months before the expected breeding season. Body weights and body condition scores of ewes were recorded prior to the experiment. Into the Female group (n = 7), long-acting progestins plus oestradiol injected ewes (n = 4) were included; into the Male group (n = 7) long- acting testosterone injected rams (n = 4) were included; and into the Control group (n = 7), sexually inactive ewes (n = 4) were introduced. In order to remove the between animal variance, blood samples for LH were taken twice, before and after the introduction, for 8 h at 15 min intervals. The results showed that mean and smoothed mean LH concentrations, LH pulse frequency, LH pulse amplitude and duration were not different between the experimental groups neither before nor after the introduction. However, when the LH data obtained before and after introduction were compared, it was seen that LH pulse frequency tended to decrease following the introduction in Female group (P = 0.078). Body weights and condition scores were not different between the groups but LH pulse frequency was higher in ewes that had condition scores higher than 2.00 units. Additionally, there was a significant positive correlation between condition score and LH pulse frequency (P = 0.004; R2 = 0.368). In conclusion, it appears that although female sheep tend to reduce LH pulse frequency of anoestrous ewes, body energy reserves appear to be the major effector of LH pulse frequency during the anoestrous period studied. Testosterone oestradiol progestin sexual interactions body condition score Photoperiod, nutrition, body energy reserves and social interactions are among the factors that affect the reproductive activity of the sheep (Adam and Robinson 1994; Yildiz et al. 2001; Yildiz et al. 2002a and 2002b). ACTA VET. BRNO 2002, 71: 297–302 ACTA VET. BRNO 2002, 71: 297–302 Address for correspondence: Dr. Sedat Yildiz Kafkas Universitesi, Veteriner Fakültesi Fizyoloji Bolumu 36040 Kars, Turkey Received March 29, 2002 Accepted June 19. 2002 Received March 29, 2002 Accepted June 19. 2002 Phone: +90 474 242 6846 Fax: +90 474 242 6846 e-mail: yildizsedat@hotmail.com http://www.vfu.cz/acta-vet/actavet.htm Animals and experimental protocol p p Fat-tailed Tuj ewes (3 to 4-year-old, n = 21) were divided into three equal groups during anoestrous season. On Day 1 (15th July 2001), they were weighed and condition scored [1-emaciated to 5-obese scale, Russel et al. (1969)] and placed into three rooms in three different buildings of the farm of the Kafkas University, Kars, Turkey (43º E, 40.5º N). The groups were separated so that there was approximately 50 m distance and at least two walls between them. Ventilation of the rooms was through the roof and all other sides were covered with the wall and a door. These rooms were not occupied by any other animals since the establishment of the farm in 1996 and they were cleaned up prior to the experimental study. Additionally, it was made sure that experimental animals had no contact with the ram-lambs grazing approximately 6 km away during the daytime and stayed in the other part of the farm. The anoestrous ewes were kept at their locations for 4 days in order to reduce the stress of the new place. Afterwards, on Day 5, animals in all groups were blood-sampled for LH for 8 h at 15 min intervals starting at 08:30 h. On the other site of the building, four rams were injected intramuscularly with a long-acting testosterone (Sustanon 250, Organon, UK) at the dosis of 1 ml per ram (30 mg testosterone propionate, 60 mg testosterone phenylpropionate, 60 mg testosterone isocaproate, 100 mg testosterone decanoate) and four anoestrous ewes were injected intramuscularly with a long-acting oestradiol and progestin (Mesigyna, Schering, Istanbul, Turkey) at the dosis of 1 ml per ewe (50 mg noretisteroneenantate, 5 mg estradiol valerate) on Day 5. On Day 6, rams were introduced into the room of one group (Male group), Mesigyna injected ewes were introduced into the room of another group (Female group) and four anoestrous ewes were introduced into the room of the remaining group (Control group). On Day 8, the animals in all groups were blood-sampled again for LH for 8 h with 15 min intervals. Blood samples were immediately centrifuged at 3000 g and plasma was separated and stored at –20 ºC pending measurement for LH. All experimental animals were fed twice at 08:00 and 18:00 h with high quality grass. Water was provided ad libitum. Abstract In spite of the difficulties in managing photoperiod, nutrition and body energy reserves, social manipulation of the reproductive activity in farm animals emerges as a good alternative, especially, to reproductive management techniques that facilitate application of reproductive hormones (Rekwot et al. 2001). Some of the pheromonal methods have been in use for many years as a result of their practicality. “Ram effect”, which includes separation of rams from the ewe flock and their reunification after following 3-4 weeks, is an example of these methods (Martin et al. 1980, 1986; Lindsay 1996). By this way, anoestrous ewes are brought to cyclicity and hence reproductive activity of all flock is improved (Lucidi et al. 2001). This male-to- female interaction is known to be accompanied by increased LH secretion in the ewes (Cohen-Tannoudji et al. 1986, 1989). On the other hand, it appears that a female-to- female interaction might also occur in sheep (Zarco et al. 1995; Yildiz et al. 2002a). Zarco et al. (1995) found that anoestrous ewes in pens closer to the oestrous females showed a high degree of ovulation and similarly, in the study of Yildiz et al. (2002a), Phone: +90 474 242 6846 Fax: +90 474 242 6846 e-mail: yildizsedat@hotmail.com http://www.vfu.cz/acta-vet/actavet.htm 298 females that were separated from the rams showed a good degree of synchronisation of their oestrous cycles. The studies carried out in sheep, therefore, suggest that both male-to-female and female- to-female interactions are possible. However, to the best of our knowledge, there is no study in which both types of interactions are investigated in terms of their effect on pulsatile LH secretion. Although ram effect is known to be very strong (Martin et al. 1986; Cohen- Tannoudji et al. 1986, 1989), effect of an ewe on the LH secretion in other ewes is not known. Therefore the aim of the current study was to investigate, during the anoestrous season, the effects of sexually activated males or females on the pulsatile LH secretion characteristics in anoestrous ewes. For that purpose, (i) long-acting testesterone injected rams were used for male effect, (ii) long-acting progestin and oestradiol injected non-cycling ewes were used for female effect, and (iii) anoestrous ewes were used for control group. Materials and Methods Animals and experimental protocol Animals and experimental protocol Immediately following the experiment, male and female effects produced by injection of sex hormones were tested and it was clearly found out that both ewes and rams were sexually active. Signs of oestrus were observable in female sheep and sexual interest (flehmen, mounting) of the rams to these ewes was very high, generally resulting in exhaustion of ewes and rams. Enzyme immunoassay of LH samples Enzyme immunoassay of LH samples y y p A sensitive competitive enzyme immunoassay method developed by Mutayoba et al. (1990) for bovine LH and modified by Yildiz et al. (2002b) was used to analyse LH concentrations. Briefly, D-Biotinyl-ε-aminocaproic acid N-hydroxy-succimidine ester (Biotin-X-NHS, SIGMA, Germany) was used for labelling oLH (NIDDK-oLH- I-4 (AFP-8614B)). Affinity purified goat IgG antirabbit IgG was attached to the solid phase and labelled and non- labelled (sample) oLH were competed against the anti-oLH raised in rabbit (NIDDK-anti-oLH-1 (AFP-192279)). Dilutions of biotinyl LH and oLH antiserum were found to be 1:5,000 for 1:3,200,000, respectively. Standards used in the current study were between 0.39-50 ng oLH/ml. The minimum detection limit of the assay was 0.70 ng/ml. The intra-assay coefficients of variation were 8.5% and 6.4% and inter-assay coefficients of variation were 19.4 % and 19.8 % for the quality controls containing 4.1 and 10.0 ng/ml LH, respectively. Analysis of LH results Analysis of LH results EIA results of LH were analysed for numbers of pulses by using PC pulsar programme developed by Merriam and Wachter (1982). The G parameters (the number of standard deviation by which a peak must exceed the baseline in order to be accepted as pulse) were 3.0, 2.6, 2.2, 1.8, and 1.4 for G1-G5, these being the requirements for pulses composed of 1 to 5 successive samples that exceed the baseline, respectively. Smoothing time used was 480 min. The Baxter parameters describing the parabolic relationship between the concentration of a hormone in a sample and the standard deviation (assay variation) about that concentration, obtained from a pool of samples, were 0.086 (b1, the y intercept), 0.11 (b2, the x coefficient) and 0.0014 (b3, the χ2 coefficient). 299 Statistical analyses y Body weight, BCS, and LH data taken at the first and second samplings were compared separately using Generalised Linear Models within the MINITAB statistical package (MINITAB Inc, Pennsylvania, USA) by using the experimental groups as factors. For comparison of the first and second samplings, however, paired t-test was used without taking into account the experimental groups. Paired t-test was also used separately for each experimental group to find out the differences that might have occurred between the first and second samplings. Linear regressions within MINITAB statistical package were used to find out the relationships between LH data and BCS. Data are presented as mean ± S.E.M. Results Table 1 Pulsatile LH secretion characterisitics of anoestrous ewes before or after the introduction of sexually-activated females (Female group) or males (Male group) or sexually inactive females (Control group). No statistical differences were observed between the groups neither before nor after the introduction. Values represent mean ± S.E.M. Pulsatile LH secretion characterisitics of anoestrous ewes before or after the introduction of sexually-activated females (Female group) or males (Male group) or sexually inactive females (Control group). No statistical differences were observed between the groups neither before nor after the introduction. Values represent mean ± S.E.M. Body weight appeared to be positively correlated to LH pulse frequency at the first sampling (R2 = 0.158; P < 0.08) and this relationship became even much stronger at the second sampling (R2 = 0.339; P < 0.01). Similarly, BCS was positively correlated to LH pulse frequency at the first (R2 = 0.266; P < 0.05) and at the second (R2 = 0.267; P < 0.05) samplings. However, even a stronger relationship was observed between BCS and mean LH pulse frequency of the first and second samplings (Fig. 1). Additionally, ewes having a BCS less or equal to 2.00 units had lower LH pulse frequencies at the first (0.25 ± 0.16 vs 0.62 ± 0.18 pulses / 8 h; P > 0.05) and g p p Control group Female group Male group Before After Before After Before After Mean LH concentration (ng/ml) 1.6 ± 0.1 1.7 ± 0.2 1.4 ± 0.1 1.5 ± 0.1 1.4 ± 0.1 1.1 ± 0.2 Smoothed mean LH conc. (ng/ml) 1.6 ± 0.1 1.6 ± 0.2 1.3 ± 0.1 1.3 ± 0.2 1.4 ± 0.1 1.1 ± 0.2 LH pulse frequency/ 8 h 0.43 ± 0.20 0.71 ± 0.29 0.57 ± 0.30 0.14 ± 0.14 0.43 ± 0.20 0.57 ± 0.43 amplitude (ng/ml) 1.21 ± 0.19 1.89 ± 0.07 1.67 ± 0.15 1.62 ± 0.19 1.33 ± 0.24 1.66 ± 0.68 duration (min) 30 ± 15 23 ± 8 28 ± 9 15 ± 6 30 ± 15 28 ± 13 2,54 2,00 1,50 1,00 0,50 0,00 1,0 1,5 2,0 2,5 3,0 3,5 Fig. 1. The relationship between BCS and mean LH pulse frequency for the first and second sampling periods. Linear regression lines best described the data, n=21 (some points overlap). Body weight and BCS y g Body weights were 55 ± 2.6, 60 ± 2.3 and 59 ± 3.5 kg for Female, Male and Control groups, respectively (P > 0.05). BCS were 2.1 ±0.2, 2.4 ± 0.2, and 2.3 ± 0.1 units for Female, Male and Control groups, respectively (P > 0.05). LH data Mean and smoothed mean LH concentrations, LH pulse frequencies, amplitudes and durations are given in Table 1 for the first (before introduction) and second (after introduction) samplings. Paired t-test was used to find out whether there had been a change in LH data between the samplings carried out before and after the introductions. However, no difference was observed in LH data but LH pulse frequency tended to decrease in Female groups (P = 0.078), whereas it increased non-significantly in Male and Control groups. Body weight appeared to be positively correlated to LH pulse frequency at the first sampling (R2 = 0.158; P < 0.08) and this relationship became even much stronger at the second sampling (R2 = 0.339; P < 0.01). Similarly, BCS was positively correlated to LH pulse frequency at the first (R2 = 0.266; P < 0.05) and at the second (R2 = 0.267; P < 0.05) samplings. However, even a stronger relationship was observed between BCS and mean LH pulse frequency of the first and second samplings (Fig. 1). Additionally, ewes having a BCS less or equal to 2.00 units had lower LH pulse frequencies at the first (0.25 ± 0.16 vs 0.62 ± 0.18 pulses / 8 h; P > 0.05) and Table 1 Pulsatile LH secretion characterisitics of anoestrous ewes before or after the introduction of sexually-activated females (Female group) or males (Male group) or sexually inactive females (Control group). No statistical differences were observed between the groups neither before nor after the introduction. Values represent mean ± S.E.M. Control group Female group Male group Before After Before After Before After Mean LH concentration (ng/ml) 1.6 ± 0.1 1.7 ± 0.2 1.4 ± 0.1 1.5 ± 0.1 1.4 ± 0.1 1.1 ± 0.2 Smoothed mean LH conc. (ng/ml) 1.6 ± 0.1 1.6 ± 0.2 1.3 ± 0.1 1.3 ± 0.2 1.4 ± 0.1 1.1 ± 0.2 LH pulse frequency/ 8 h 0.43 ± 0.20 0.71 ± 0.29 0.57 ± 0.30 0.14 ± 0.14 0.43 ± 0.20 0.57 ± 0.43 amplitude (ng/ml) 1.21 ± 0.19 1.89 ± 0.07 1.67 ± 0.15 1.62 ± 0.19 1.33 ± 0.24 1.66 ± 0.68 duration (min) 30 ± 15 23 ± 8 28 ± 9 15 ± 6 30 ± 15 28 ± 13 2,54 2,00 1,50 1,00 0,50 0,00 1,0 1,5 2,0 2,5 3,0 3,5 Fig. 1. LH data The relationship between BCS and mean LH pulse frequency for the first and second sampling periods. Linear regression lines best described the data, n=21 (some points overlap). Body condition score (units) R-square = 0.368 P = 0.004 y = -1.23 + 0.76 x LH pulse frequency (per 8 h) 2,50 Table 1 Body condition score (units) R-square = 0.368 P = 0.004 y = -1.23 + 0.76 x LH pulse frequency (per 8 h) 2,50 2,54 2,00 1,50 1,00 0,50 0,00 1,0 1,5 2,0 2,5 3,0 3,5 Body condition score (units) R-square = 0.368 P = 0.004 y = -1.23 + 0.76 x LH pulse frequency (per 8 h) 2,50 Body weight appeared to be positively correlated to LH pulse frequency at the first sampling (R2 = 0.158; P < 0.08) and this relationship became even much stronger at the second sampling (R2 = 0.339; P < 0.01). Similarly, BCS was positively correlated to LH pulse frequency at the first (R2 = 0.266; P < 0.05) and at the second (R2 = 0.267; P < 0.05) samplings. However, even a stronger relationship was observed between BCS and mean LH pulse frequency of the first and second samplings (Fig. 1). Additionally, ewes having a BCS less or equal to 2.00 units had lower LH pulse frequencies at the first (0.25 ± 0.16 vs 0.62 ± 0.18 pulses / 8 h; P > 0.05) and Body weight appeared to be positively correlated to LH pulse frequency at the first sampling (R2 = 0.158; P < 0.08) and this relationship became even much stronger at the second sampling (R2 = 0.339; P < 0.01). Similarly, BCS was positively correlated to LH pulse frequency at the first (R2 = 0.266; P < 0.05) and at the second (R2 = 0.267; P < 0.05) samplings. However, even a stronger relationship was observed between BCS and mean LH pulse frequency of the first and second samplings (Fig. 1). Additionally, ewes having a BCS less or equal to 2.00 units had lower LH pulse frequencies at the first (0.25 ± 0.16 vs 0.62 ± 0.18 pulses / 8 h; P > 0.05) and Body weight appeared to be positively correlated to LH pulse frequency at the first sampling (R2 = 0.158; P < 0.08) and this relationship became even much stronger at the second sampling (R2 = 0.339; P < 0.01). Similarly, BCS was positively correlated to LH pulse frequency at the first (R2 = 0.266; P < 0.05) and at the second (R2 = 0.267; P < 0.05) samplings. However, even a stronger relationship was observed between BCS and mean LH pulse frequency of the first and second samplings (Fig. 1). Discussion The present experiment shows that, compared to the female or male effects, body energy reserves appear to be the main regulator of pulsatile LH secretion in ewes during the anoestrous period studied. It has been reported that BCS is positively correlated to timing of LH surge in sheep (Yildiz et al. 2002b) and LH pulse frequencies in sheep (Robinson 1990) and cattle (Yildiz et al. 1997). The link between BCS and hypothalamus, where GnRH secretion is initiated, appear to include leptin secretion from the adipose tissue (Blache et al. 2000; Yildiz et al. 2001) since leptin informs the hypothalamus about the sufficiency of energy stores for the initiation of reproductive activity (Blache et al. 2000). It appears from the current study that, in fat-tailed sheep breed, which might differ in adipose tissue deposition and mobilisation, LH pulse frequency is also well correlated with BCS. p , p q y LH pulse frequencies did not differ between the groups during the second sampling. In other words, there were no significant differences between the Male and Female groups and the control group. Various reasons might be considered in that respect. First, it appears that the current study was carried out during the period that coincided with the start of replenishment of the pituitary LH stores in anoestrous season. However, there were still approximately two months for the commencement of breeding season for that breed (Yildiz et al. 2002a) and LH pulses were therefore low. Probably this prevented the observation of differences between the groups as a result of lower LH pulse frequency. Additionally, Rosa et al. (2000) reported that numbers of ewes ovulating in response to ram effect were higher when the rams were implanted with melatonin. This suggest that season is an important part of sexual interactions. Secondly, for the Male group, it was not possible to induce sex effects due to the short period of introduction. Normally a male effect would be observed after a long period of separation that is 3-4 weeks (Martin et al. 1986). However, female effects are likely to occur within short periods and there might be no need for separation for the observance of female effects. As evidenced in women, the odourless compounds taken at different stages of oestrous cycles have differential effects in the recipient women (Stern and McClintock 1998; Weller 1998). Table 1 Additionally, ewes having a BCS less or equal to 2.00 units had lower LH pulse frequencies at the first (0.25 ± 0.16 vs 0.62 ± 0.18 pulses / 8 h; P > 0.05) and Fig. 1. The relationship between BCS and mean LH pulse frequency for the first and second sampling periods. Linear regression lines best described the data, n=21 (some points overlap). 300 second (0.00 ± 0.00 vs 0.77 ± 0.26 pulses / 8 h; P < 0.05) samplings and when the mean LH pulse frequencies of the first and second samplings were taken into account (0.13 ± 0.08 vs 0.69 ± 0.18 pulses / 8 h; P < 0.05). Discussion Therefore, it appears that female effects might occur within a short period of time. In the current study, in order to be able to compare the male and female effects, we tended to introduce rams after short separation. By this way, short-term results for both male and female effects could be determined. However, the results obtained suggest little or no effect. gg Sampling the ewes twice, before and after the introduction, helped to remove between- animal variances that could occur. The results of the paired t-test revealed that LH pulse frequency tended to drop in the Female group, suggesting a negative role of a cycling ewe on the anoestrous ewes. This is in contrast to the findings of Zarco et al. (1995) who observed a positive effect of cycling ewes on the anoestrous ewes. In fact, two mechanisms might be postulated in terms of female-to-female effect: (1) a ewe might give a positive signal to the other ewes in the flock and hence might advance the timing of their ovulations, or (2) a ewe might give a negative signal to the other ewes to postpone their ovulation and hence they might be within the synchronous cycling group in the next ovulation. The results obtained in the current study are in line with the latter, whereas the study of Zarco et al. (1995) is in line with the first hypothesis. In a study carried out by our group we observed, in female-introduced ewes, a delay in preovulatory LH surges following progestagen sponge removal (unpublished data). This might support the negative signal hypothesis. In fact, under the farm conditions, where the numbers of animals at each stage of oestrous cycle is 301 normally different, both hypotheses might be true. For example, an oestrous ewe might give positive signal to a recipient ewe if she is in late luteal or follicular period but might give negative signals to early or mid-luteal ewes. Similar hypotheses might be postulated for the times preceding the breeding season. However, further studies are required to find out the interactions between ewes at different stages of anoestrous season or oestrous cycle. In conclusion, sexually activated ram introduction for short durations did not yield in significant positive effects on pulsatile LH secretion whereas sexually activated ewe introduction tended to negatively affect LH pulsatility in anoestrous ewes two months before breeding season. Discussion In fact, during this period, body energy reserves appeared to be the main effector of pulsatile LH release. Vliv sexuální aktivace beranÛ nebo bahnic v závislosti na rytmu sekrece LH v anestru ovcí Cílem studie bylo zjistit, zda bahnice v anestru reagují odli‰nû na pﬁítomnost sexuálnû aktivovan˘ch samcÛ a samic. Jejich reakce byla mûﬁena pomocí rytmu pulzatilního uvolÀování LH. Za tímto úãelem byly ovce v anestru (n = 21) rozdûleny do tﬁí stejn˘ch skupin a umístûny do tﬁí oddûlen˘ch místností. Experiment zaãal dva mûsíce pﬁed oãekávanou reprodukãní sezónou. Pﬁed experimentem byla zaznamenána tûlesná hmotnost a kondiãní skóre ovcí. Ke skupinû samic (n = 7) byly zaﬁazeny 4 bahnice, kter˘m byl injekãnû aplikován dlouhodobû pÛsobící progesteron a estradiol; ke druhé skupinû (n = 7) byli pﬁidáni 4 berani, jimÏ byl injekãnû aplikován dlouhodobû pÛsobící testosteron; a ke kontrolní skupinû (n = 7) byly zaﬁazeny 4 pohlavnû neaktivní bahnice. Abychom vylouãili individuální variabilitu namûﬁen˘ch hodnot LH, byly vzorky krve na jeho stanovení odebrány dvakrát, pﬁed a po introdukci do pokusn˘ch skupin, a to po dobu 8 hodin v 15 minutov˘ch intervalech. V˘sledky ukázaly, Ïe mezi experimentálními skupinami nebyly rozdíly v koncentraci LH (prÛmûr a vyhlazen˘ prÛmûr), frekvenci pulzÛ LH, v amplitudû a trvání pulzÛ LH, pﬁed a po vystavení bahnic pﬁítomnosti sexuálnû aktivovan˘ch zvíﬁat. Av‰ak po porovnání získan˘ch v˘sledkÛ LH pﬁed a po introdukci aktivovan˘ch jedincÛ byl ve skupinû samic patrn˘ klesající trend (P = 0.078) ve frekvenci pulzÛ LH. Tûlesná hmotnost ani kondiãní parametry se mezi skupinami neli‰ily, av‰ak frekvence pulzÛ LH byla vy‰‰í v ovcí s tûlesn˘m skóre vy‰‰ím neÏ 2.00 jednotky. Navíc byla nalezena signifikantní pozitivní korelace mezi frekvencí pulzÛ LH a kondiãním stavem (P = 0.004; R2 = 0.368) bahnic. Z v˘sledkÛ studie vypl˘vá, Ïe v pﬁítomnosti aktivovan˘ch ovcí inklinují bahnice v anoestru ke sníÏení frekvenci pulzÛ LH, nejvût‰í vliv na tuto frekvenci bûhem fáze anoestru mají zﬁejmû jejich energetické zásoby. Acknowledgement This study was supported by TUBITAK (The Technical and Scientific Research Council of Turkey, Project No: VHAG-1794/ADP). We wish to thank Dr. A.F. Parlow and NIDDK for the provision of LH antigene and antibody. We would also like to thank to the veterinary students who helped with the design of the experimental units. ADAM, CL, ROBINSON, JJ 1994: The role of nutrition and photoperiod in the timing of puberty. Proc Nutr Soc 23: 89-102 BLACHE, D, TELLAM, RL, CHAGAS, LM, BLACKBERRY, MA, VERCOE, PE, MARTIN, GB 2000: Level of nutrition affects leptin concentrations in plasma and cerebrospinal fluid in sheep. 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https://openalex.org/W3196305196	https://sibmed.elpub.ru/jour/article/download/631/359	Russian	null	Millimeter waves and 5G standard: addition to the review	Sibirskij naučnyj medicinskij žurnal	2,021	cc-by	3,504	С.В. Яргин Российский университет дружбы народов 117198, г. Москва, ул. Миклухо-Маклая, 6 S.V. Jargin Peoples’ Friendship University of Russia 117198, Moscow, Miklukho-Maklay str., 6 Резюме Телекоммуникационные системы пятого поколения (5G) внедряются во всем мире. В настоящей статье при- ведены соображения в пользу того, что миллиметровые волны, используемые в соответствии со стандартом 5G, не обладают большим повреждающим действием на единицу поглощенной энергии, чем инфракрасное излуче- ние. Убедительные доказательства и теоретические соображения в пользу канцерогенного или повреждающего (до уровня термического повреждения) действия миллиметровых волн в литературе отсутствуют. Чрезмерно строгие правила техники безопасности вредны для экономики и создают неудобства в повседневной жизни. Ре- зультаты эпидемиологических исследований важны, но нужно уделять больше внимания уклонам и мешающим факторам. Получить достоверные результаты можно с помощью исследований на животных с регистрацией продолжительности жизни. Для того чтобы выводы были применимыми к использованию мобильной связи или профессиональной деятельности, мощности доз в экспериментах должны быть сравнимыми с таковыми у человека. Ключевые слова: электромагнитное излучение радиочастотного диапазона, миллиметровые волны, стандарт 5G, мобильная связь, канцерогенный эффект. Конфликт интересов. Автор заявляет об отсутствии конфликта интересов. Конфликт интересов. Автор заявляет об отсутствии конфликта интересов. Автор для переписки: Яргин С.В., e-mail: sjargin@mail.ru Для цитирования: Яргин С.В. Миллиметровые волны и стандарт 5G: дополнение к обзору. Сибирский научный медицинский журнал. 2021; 41 (4): 25–29. doi: 10.18699/SSMJ20210403 Для цитирования: Яргин С.В. Миллиметровые волны и стандарт 5G: дополнение к обзору. Сибирский научный медицинский журнал. 2021; 41 (4): 25–29. doi: 10.18699/SSMJ20210403 УДК 616-03:537.86 УДК 616-03:537.86 DOI: 10.18699/SSMJ20210403 25 Conflict of interest. The author declares no conflict of interest. Correspondence author: Jargin S.V., e-mail: sjargin@mail.ru Citation: Jargin S.V. Millimeter waves and 5G standard: addition to the review. Sibirskiy nauchnyy meditsinskiy zhurnal = Siberian Scientific Medical Journal. 2021; 41 (4): 25–29. [In Russian]. doi: 10.18699/SSMJ20210403 СИБИРСКИЙ НАУЧНЫЙ МЕДИЦИНСКИЙ ЖУРНАЛ 2021; 41 (4): 25−29 Abstract С другой стороны, имеются данные, что ЭМИ РЧ защищают от окислительного повреждения живых тканей [7]; подробности и ссылки – в обзорах [1, 2]. Отсутствуют какие-либо доводы в пользу того, что нагрев под действием МВ вызывает более интенсивный окислительный стресс, чем нагрев иным способом. Теоретически нет оснований ожидать от ЭМИ РЧ большего повреждающего действия на единицу поглощенной энергии или температуры, чем от инфракрасных лучей, с которыми МВ соседствуют в спектре. которыми МВ соседствуют в спектре. Согласно заключению Международного агентства по изучению рака (IARC), заболевае- мость опухолями головного мозга не отреагиро- вала на глобальное распространение мобильной связи [7]. По мнению экспертов Научного коми- тета по новым и вновь выявленным рискам для здоровья (SCENIHR) и Международной комиссии по защите от неионизирующего излучения (ICNIRP), эпидемиологические исследования в целом не подтверждают повышения риска опухолей вследствие использования сотовых телефонов [8, 9]. Эпидемиологические данные противоречивы; в них не исключены уклоны (bias): дозозависимый отбор и самоотбор, ошибки памяти и др. [10, 11]. Если бы канцеро- генный эффект ЭМИ РЧ был сколько-нибудь су- щественным, то показатели заболеваемости были бы выше в тех регионах, где давно используются бытовые и промышленные источники ЭМИ РЧ. Частота глиомы в США мало изменилась за пери- од с 1992 по 2008 г. [12, 13], несмотря на быстрый рост популярности мобильных телефонов. Не- которое увеличение заболеваемости в отдельных странах и возрастных группах не имело четкой временной связи с их использованием. Представ- ляется вероятным, что увеличение зарегистриро- ванной заболеваемости обусловлено прогрессом методов визуализации и качества диагностики. Суммарное время использования мобильных телефонов могло быть связано с уровнем дохода [14, 15], который, в свою очередь, ассоциирован с качеством медицинского наблюдения и диагно- стики. Этот уклон может объяснить корреляции доза – эффект. Обычные (термические) реакции получают энергию активации за счет термического воз- буждения в результате случайных столкновений молекул, фотохимические – от поглощения фо- тонов молекулами. Поглощение может вести к электронному возбуждению молекул, но энергия может также рассеиваться в виде тепла. Рассеи- вание преобладает при низких энергиях фотонов (квантов), недостаточных для возбуждения моле- кул. Согласно закону Гротгуса – Дрейпера, только поглощенное излучение может вызывать фотохи- мические реакции. Согласно закону Штарка – Эйнштейна, в процессе возбуждения молекула поглощает один фотон. При воздействии лазера возможно поглощение молекулой нескольких квантов [6]. Как правило, для инициации фото- химических реакций необходимо излучение ви- димого или ультрафиолетового диапазона. Ин- фракрасные фотоны лазерного излучения также могут инициировать реакции. Кванты МВ имеют еще меньшую энергию, чем инфракрасные. Abstract The fifth generation (5G) telecommunication systems are about to be implemented worldwide. It is argued here that millimeter waves used in 5G cannot be more harmful per unit of absorbed energy than infrared radiation. In the literature, there is neither convincing evidence nor theoretical considerations in favor of carcinogenic or damaging (up to the level of thermal damage) effects of millimeter waves. Excessively strict safety regulations are unfavorable for the economy and everyday life. Epidemiological data are important; but more attention should be given to potential bias and confounding factors. Large-scale animal experiments with the registration of average life duration would be a reliable way to determine the net harm. The doses and exposure duration in animals must be comparable to those in related human populations to make results extrapolable to humans. Key words: radiofrequency electromagnetic fields, millimeter waves, 5G standard, mobile phones, carcinogenicity. Key words: radiofrequency electromagnetic fields, millimeter waves, 5G standard, mobile phones, 25 СИБИРСКИЙ НАУЧНЫЙ МЕДИЦИНСКИЙ ЖУРНАЛ 2021; 41 (4): 25−29 Jargin S.V. Millimeter waves and 5G standard: addition to the review Недавно опубликованы обзоры эксперимен- тальных и эпидемиологических исследований предполагаемого канцерогенного и поврежда- ющего действия электромагнитного излучения радиочастотного диапазона (ЭМИ РЧ) [1, 2]. В настоящей статье речь пойдет о миллиметровых волнах (МВ), которые используются в средствах связи нового поколения (стандарт 5G). МВ, которые в электромагнитном спектре соседствуют с инфракрасным излучением, характеризуются малой глубиной проникновения в живые ткани, поглощаются кожей и слизистыми оболочками [3]. Литература на данную тему противоречива, качество многих исследований невысокое [4, 5]; имеется много сообщений сомнительной до- стоверности. С помощью метаанализа для МВ выявлена статистически достоверная (р < 0,05) отрицательная корреляция между показателем качества (quality score) исследований и эффек- тивностью облучения (effect size) [4]. Подобная тенденция неоднократно отмечалась для ЭМИ РЧ в целом [1]. В этих условиях увеличивается зна- чение теоретических аргументов. репарации ДНК и повышении риска опухолей при частом перегреве профессионального или рекреационного характера. С другой стороны, имеются данные, что ЭМИ РЧ защищают от окислительного повреждения живых тканей [7]; подробности и ссылки – в обзорах [1, 2]. Отсутствуют какие-либо доводы в пользу того, что нагрев под действием МВ вызывает более интенсивный окислительный стресс, чем нагрев иным способом. Теоретически нет оснований ожидать от ЭМИ РЧ большего повреждающего действия на единицу поглощенной энергии или температуры, чем от инфракрасных лучей, с которыми МВ соседствуют в спектре. репарации ДНК и повышении риска опухолей при частом перегреве профессионального или рекреационного характера. Abstract Убе- дительные доказательства участия фотохимиче- ских или нетепловых механизмов повреждения тканей или ДНК под действием МВ отсутствуют. Это относится также к образованию свободных радикалов кислорода, которое обсуждается как возможный механизм действия ЭМИ РЧ. Радикалы могут образовываться в результате диссоциации молекул под действием света или тепла, т.е. фотохимических и термических реакций. Гипертермия сама по себе может вызвать образование активных форм кислорода. Сообщалось о нарушении Это относится также к образованию свободных радикалов кислорода, которое обсуждается как возможный механизм действия ЭМИ РЧ. Радикалы могут образовываться в результате диссоциации молекул под действием света или тепла, т.е. фотохимических и термических реакций. Гипертермия сама по себе может вызвать образование активных форм кислорода. Сообщалось о нарушении В отличие от солнечного излучения и инфра- красных обогревателей, МВ в повседневной жиз- ни не вызывают значительного нагрева живых тканей. В литературе обсуждаются канцероген- ные и иные вредные эффекты ЭМИ РЧ нетепло- вой интенсивности [3]. Вместе с тем ультра- и крайне высокочастотная (УВЧ, КВЧ) диатермия широко использовалась для лечения синусита, SIBERIAN SCIENTIFIC MEDICAL JOURNAL 2021; 41 (4): 25−29 26 SIBERIAN SCIENTIFIC MEDICAL JOURNAL 2021; 41 (4): 25−29 Яргин С.В. Миллиметровые волны и стандарт 5G: дополнение к обзору У обезьян даже при таком воздействии катаракты не возникали [21]. Гипотермия предотвращала развитие катаракт у кроликов, что подтверждает термический механизм повреждения хрусталика при микроволновом облучении [23]. Облучение с вышеназванными характеристиками значительно превышает предельно допустимые уровни [24]. тонзиллита и т.п. у детей и взрослых с 1960-х го- дов [16, 17]. О повышении риска онкологических и офтальмологических заболеваний после УВЧ- терапии не сообщалось, хотя избыточная экспо- зиция тканей мозга и глаза в принципе не исклю- чена [8]. Опасения по поводу МВ включают следую- щее. Наличие потовых желез может привести к локальному повышению поглощения энергии [18]. Следует отметить, что первичный пот поч- ти изотоничен плазме крови, а конечный пот, выделяемый на поверхность кожи, становится гипотоническим после реабсорбции ионов Na+ и Cl- в протоке [19]. Соответственно, электро- проводность потовых протоков не должна суще- ственно отличаться от таковой капилляров и дру- гих сосудов. Предпочтительный нагрев потовых протоков, если и имеет место, едва ли вызовет по- вреждение (иначе МВ можно было бы испытать как средство от гипергидроза). Те же соображе- ния относятся к инфракрасному излучению. Воздействие на головной мозг и глаз можно исследовать на крупных животных с имитацией, например, УВЧ-терапии области головы и шеи. Abstract Кроме того, необходимо исследовать возмож- ность появления повреждающих горячих точек вследствие интерференции волн в неподвижном облучаемом теле применительно к УВЧ-терапии или такой «экстремальной» ситуации, как спящий ребенок с прижатым к уху излучающим телефо- ном. Опасения в отношении головного мозга, сет- чатки глаза, других глубоко расположенных орга- нов и тканей не имеют оснований при облучении МВ ввиду малой глубины их проникновения. Наконец, нужно отметить мистификацию вокруг данной темы. Анализировать соответствующую литературу не имеет смысла, приведем лишь несколько цитат: «Воздействие внешнего МВ индуцирует акустоэлектрические волны в клеточных мембранах», «Биологические эффекты не зависят от интенсивности МВ», «Распространение МВ происходит по каналам, которые описаны в традиционной китайской медицине» [25]. Еще один повод для озабоченности состо- ит в том, что беспроводные устройства могут передавать пакеты МВ продолжительностью от нескольких миллисекунд до секунд. Даже если усредненная по времени и площади поглощае- мая мощность находится в допустимых преде- лах, предполагается возможность температурных пиков с повреждающим действием [20]. Данный вопрос следует поставить перед экспериментато- рами: могут ли импульсные ЭМИ РЧ с опреде- ленными характеристиками (плотность потока энергии, отношение пиковой мощности к ее сред- нему значению) повреждать живые ткани. Для МВ исследования будут проще, чем для прочих ЭМИ РЧ, ввиду малой глубины проникновения, так что достаточно будет измерять температуру на поверхности. Заключение Время облучения крыс (9 ч/сут) в исследовании [26] было больше, чем у пользователей мобильных телефонов; у многих животных отмечено измери- мое повышение температуры тела. Для того что- бы выводы были применимыми к использованию мобильной связи или профессиональной деятель- ности, мощности доз в экспериментах должны быть сравнимыми с таковыми у человека. 8. Scientific Committee on Emerging Newly Iden- tified Health Risks. Opinion on potential health effects of exposure to electromagnetic fields. Bioelectromag- netics. 2015; 36 (6): 480–484. doi: 10.1002/bem.21930 9. Swerdlow A.J., Feychting M., Green A.C., Lee- ka Kheifets L.K., Savitz D.A. International commission for non-ionizing radiation protection standing commit- tee on epidemiology. Mobile phones, brain tumours, and the interphone study: Where are we now? Envi- ron. Health Perspect. 2011; 119 (11): 1534–1538. doi: 10.1289/ehp.1103693 10. Jargin S.V. Hormesis and radiation safety norms: Comments for an update. Hum. Exp. Toxicol. 2018; 37 (11): 1233–1243. doi: 10.1177/0960327118765332 Список литературы / References 2006; 98 (23): 1707–1713. doi: 10.1093/jnci/djj464 15. Schüz J., Jacobsen R., Olsen J.H., Boice J.D. Jr., McLaughlin J.K., Johansen C. Cellular telephone use and cancer risk: Update of a nationwide Danish cohort. J. Natl. Cancer Inst. 2006; 98 (23): 1707–1713. doi: 10.1093/jnci/djj464 15. Schüz J., Jacobsen R., Olsen J.H., Boice J.D. Jr., McLaughlin J.K., Johansen C. Cellular telephone use and cancer risk: Update of a nationwide Danish cohort. J. Natl. Cancer Inst. 2006; 98 (23): 1707–1713. doi: 10.1093/jnci/djj464 4. Wood A., Mate R., Karipidis K. Meta-analysis of in vitro and in vivo studies of the biological effects of low-level millimetre waves. J. Expo. Sci. Envi- ron. 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Vestnik otorinolar- ingologii = Bulletin of Otorhinolaryngology. 1966; 28 (6): 31–34. [In Russian]. Nikolaevskaya V.P. The use of microwave therapy in patients with chronic tonsillitis. Vestnik otorinolar- ingologii = Bulletin of Otorhinolaryngology. 1966; 28 (6): 31–34. [In Russian]. 6. Панченко П.А. Фотохимические реакции. М.: РХТУ им. Д.И. Менделеева, 2017. 132 c. 6. Панченко П.А. Фотохимические реакции. М.: РХТУ им. Д.И. Менделеева, 2017. 132 c. Panchenko P.A. Photochemical reactions. Moscow, 2017. 132 p. [In Russian]. 17. Поважная Е.Л., Мамбеталиева А.С. КВЧ- терапия в профилактике острых респираторных заболеваний у детей с хроническими ЛОР- и аллергическими заболеваниями. Вопр. курорт., физиотерапии и лечеб. физ. культуры. 2010; (5): 17–21. 17. Поважная Е.Л., Мамбеталиева А.С. КВЧ- терапия в профилактике острых респираторных заболеваний у детей с хроническими ЛОР- и аллергическими заболеваниями. Вопр. курорт., физиотерапии и лечеб. физ. культуры. 2010; (5): 17–21. Panchenko P.A. Photochemical reactions. Moscow, 2017. 132 p. [In Russian]. 7. IARC Working Group on the Evaluation of Car- cinogenic Risks to Humans. Заключение Убедительные доказательства и теоретичес- кие соображения в пользу канцерогенного или повреждающего (до уровня термического повреждения) действия МВ в литературе отсутствуют. Результаты эпидемиологических исследований важны, но нужно уделять больше внимания уклонам (bias) и мешающим факторам. Большое число наблюдений не предохраняет от систематических ошибок. Получить до- стоверные результаты можно с помощью исследований на животных с регистрацией продолжительности жизни. Неинвазивные экс- перименты такого рода этически приемлемы, технически просты, позволяют объективно оценивать зависимость доза – эффект. Прижиз- ненное и посмертное исследование отдельных животных сопровождается затратами, которые можно направить на увеличение числа наблюдений с целью повышения статистической достоверности. Средняя продолжительность жизни облученных самцов крыс в исследовании высокого качественного уровня была выше, чем в контроле [26], что может отражать благоприятное действие малых доз ЭМИ РЧ в соответствии с Можно с уверенностью предположить, что повреждение кожи и слизистых оболочек под действием МВ будет зависеть от измеряемой тем- пературы как и при любом нагреве. Сообщалось, например, что импульсное и непрерывное облуче- ние МВ с одинаковой эффективностью вызывали катаракты у кроликов [21]. В отношении органа зрения отметим, что развитие катаракты и других повреждений при уровнях ЭМИ РЧ ниже тепло- вого не подтверждается [21, 22]. Эксперимен- ты с облучением всего тела (far field) показали, что у кроликов катаракта образуется только при облучении МВ на уровне, близком к летальному. При локальном облучении (near field) пороговые условия для возникновения катаракты у кроликов были определены как 150 Вт/кг в течение 30 минут, что сопровождалось повышением температуры до 41 °C и выше в хрусталике или рядом с ним. 27 СИБИРСКИЙ НАУЧНЫЙ МЕДИЦИНСКИЙ ЖУРНАЛ 2021; 41 (4): 25−29 Jargin S.V. Millimeter waves and 5G standard: addition to the review Monogr. Eval. Carcinog. Risks Hum. 2013; 102 (Pt 2): 1–460.i концепцией гормезиса. Подробный комментарий и ссылки приведены в обзорах [1, 2]. Очевидно, что средняя продолжительность жизни лучше от- ражает суммарное вредное действие, чем частота связанных с возрастом редких опухолей. Время облучения крыс (9 ч/сут) в исследовании [26] было больше, чем у пользователей мобильных телефонов; у многих животных отмечено измери- мое повышение температуры тела. Для того что- бы выводы были применимыми к использованию мобильной связи или профессиональной деятель- ности, мощности доз в экспериментах должны быть сравнимыми с таковыми у человека. концепцией гормезиса. Подробный комментарий и ссылки приведены в обзорах [1, 2]. Очевидно, что средняя продолжительность жизни лучше от- ражает суммарное вредное действие, чем частота связанных с возрастом редких опухолей. Список литературы / References 11. Vrijheid M., Deltour I., Krewski D., Sanchez M., Cardis E. The effects of recall errors and of selection bias in epidemiologic studies of mobile phone use and cancer risk. J. Expo. Sci. Environ. Epidemiol. 2006; 16 (4): 371–384. doi: 10.1038/sj.jes.7500509 1. Яргин С.В. О биологическом действии элек- тромагнитного излучения радиочастотного диа- пазона. Сиб. науч. мед. ж. 2019; 39 (5): 52–61. doi: 10.15372/ssmj20190506 Jargin S.V. On the biological effects of radiofre- quency electromagnetic fields. Sibirskiy nauchnyy med- itsinskiy zhurnal = Siberian Scientific Medical Jour- nal. 2019; 39 (5): 52–61. [In Russian]. doi: 10.15372/ SSMJ20190506 ( ) j j 12. Inskip P.D., Hoover R.N., Devesa S.S. Brain cancer incidence trends in relation to cellular telephone use in the United States. Neuro-Oncol. 2010; 12 (11): 1147–1151. doi: 10.1093/neuonc/noq077 13. Little M.P., Rajaraman P., Curtis R.E., Deve- sa S.S., Inskip P.D., Check D.P., Linet M.S. Mobile phone use and glioma risk: Comparison of epidemio- logical study results with incidence trends in the United States. BMJ. 2012; 344: e1147. doi: 10.1136/bmj.e1147 2. Jargin S.V. Radiofrequency radiation: carcino- genic and other potential risks. J. Radiat. Oncol. 2020; 9: 81–91. doi: 10.1007/s13566-020-00425-z 2. Jargin S.V. Radiofrequency radiation: carcino- genic and other potential risks. J. Radiat. Oncol. 2020; 9: 81–91. doi: 10.1007/s13566-020-00425-z 3. Григорьев Ю.Г. Стандарт 5G – технологиче- ский скачок вперед в сотовой связи: будет ли про- блема со здоровьем у населения? (погружение в проблему). Радиац. биол. Радиоэкол. 2020; 60 (6): 627–634. doi: 10.31857/S0869803120060181 j 14. Текшева Л.М., Барсукова Н.К., Чумиче- ва О.А., Хатит З.Х. Гигиенические аспекты ис- пользования сотовой связи в школьном возрасте. Гигиена и сан. 2014; (2): 60–65. Grigoriev Yu.G. 5g standard – technological leap ahead for cellular communication. Will there be a prob- lem with the health of the population? (diving in prob- lem). Radiatsionnaya biologiya. Radioekologiya = Ra- diation Biology. Radioecology. 2020; 60 (6): 627–634. [In Russian]. doi: 10.31857/S0869803120060181 ( ) Teksheva L.M., Barsukova N.K., Chumiche- va O.A., Khatit Z.Kh. Hygienic aspects of cellular communication in school age. Gigiena i sanitariya = Hygiene and Sanitation. 2014; (2): 60–65. [In Russian]. Teksheva L.M., Barsukova N.K., Chumiche- va O.A., Khatit Z.Kh. Hygienic aspects of cellular communication in school age. Gigiena i sanitariya = Hygiene and Sanitation. 2014; (2): 60–65. [In Russian]. 15. Schüz J., Jacobsen R., Olsen J.H., Boice J.D. Jr., McLaughlin J.K., Johansen C. Cellular telephone use and cancer risk: Update of a nationwide Danish cohort. J. Natl. Cancer Inst. Список литературы / References Non-ionizing radiation, Part 2: Radiofrequency electromagnetic fields. IARC 7. IARC Working Group on the Evaluation of Car- cinogenic Risks to Humans. Non-ionizing radiation, Part 2: Radiofrequency electromagnetic fields. IARC 28 SIBERIAN SCIENTIFIC MEDICAL JOURNAL 2021; 41 (4): 25−29 Яргин С.В. Миллиметровые волны и стандарт 5G: дополнение к обзору Povazhnaia E.L., Mambetalieva A.S. Extreme- ly high frequency therapy for the prevention of acute respiratory diseases in children with chronic ENT and allergic diseases. Voprosy kurortologii, fizioterapii i lechebnoy fizicheskoy kul’tury = Problems of Balneol- ogy, Physiotherapy, and Exercise Therapy. 2010; (5): 17–21. Kudryashov Yu.B., Perov Yu.F., Rubin A.B. Radiation biophysics. Moscow: Fizmatlit, 2008. 184 p. [In Russian]. 23. Kramar P.O., Emery A.F., Guy A.W., Lin J.C. The ocular effects of microwaves on hypothermic rabbits: a study of microwave cataractogenic mechanisms. Ann. N.Y. Acad. Sci. 1975; 247: 155–165. doi: 10.1111/j.1749-6632.1975.tb35992.x 18. Betzalel N., Ben Ishai P., Feldman Y. The hu- man skin as a sub-THz receiver - Does 5G pose a dan- ger to it or not? Environ. Res. 2018; 163: 208–216. doi: 10.1016/j.envres.2018.01.032 24. Yu Y., Yao K. Non-thermal cellular effects of low-power microwave radiation on the lens and lens epithelial cells. J. Int. Med. Res. 2010; 38 (3): 729–736. doi: 10.1177/147323001003800301 19. Baker L.B. Physiology of sweat gland function: The roles of sweating and sweat composition in human health. Temperature (Austin). 2019; 6 (3): 211–259. doi: 10.1080/23328940.2019.1632145 doi: 10.1177/147323001003800301 25. Резункова О.П. Экологические (биотропные) особенности миллиметрового диапазона. СПб.: СПбГУТ, 2015. 172 c. Rezunkova O.P. Ecological (biotropic) features of the millimeter diapason. Saint-Petersburg, 2015. 172 p. [In Russian]. 20. Neufeld E., Kuster N. Systematic derivation of safety limits for time-varying 5G radiofrequency exposure based on analytical models and thermal dose. Health Phys. 2018; 115 (6): 705–711. doi: 10.1097/ HP.0000000000000930 Rezunkova O.P. Ecological (biotropic) features of the millimeter diapason. Saint-Petersburg, 2015. 172 p. [In Russian]. 26. National Toxicology Program. Toxicology and carcinogenesis studies in Sprague Dawley (Hsd: Sprague Dawley SD) rats exposed to whole-body ra- dio frequency radiation at a frequency (900 MHz) and modulations (GSM and CDMA) used by cell phones. Natl. Toxicol. Program Tech. Rep. Ser. 2018; (595): NTP-TR-595. doi: 10.22427/NTP-TR-595 26. National Toxicology Program. Toxicology and carcinogenesis studies in Sprague Dawley (Hsd: Sprague Dawley SD) rats exposed to whole-body ra- dio frequency radiation at a frequency (900 MHz) and modulations (GSM and CDMA) used by cell phones. Natl. Toxicol. Program Tech. Rep. Ser. 2018; (595): NTP-TR-595. doi: 10.22427/NTP-TR-595 21. Сведения об авторе Сергей Вадимович Яргин, к.м.н., ORCID: 0000-0003-4731-1853, e-mail: sjargin@mail.ru Сергей Вадимович Яргин, к.м.н., ORCID: 0000-0003-4731-1853, e-mail: sjargin@mail.ru Список литературы / References Elder J.A. Ocular effects of radiofrequency energy. Bioelectromagnetics. 2003; (Suppl. 6): S148– S161. doi: 10.1002/bem.10117 22. Кудряшов Ю.Б., Перов Ю.Ф., Рубин А.Б. Ра- диационная биофизика. М.: Физматлит, 2008. 184 c. Сведения об авторе Information about the author Sergei V. Jargin, candidate of medical sciences, ORCID: 0000-0003-4731-1853, e-mail: sjargin@mail.ru Sergei V. Jargin, candidate of medical sciences, ORCID: 0000-0003-4731-1853, e-mail: sjargin Received 28.04.2021 Accepted 19.05.2021 Поступила в редакцию 28.04.2021 Received 28.04.2021 Принята к публикации 19.05.2021 Accepted 19.05.2021 Received 28.04.2021 Accepted 19.05.2021 Поступила в редакцию 28.04.2021 Принята к публикации 19.05.2021 СИБИРСКИЙ НАУЧНЫЙ МЕДИЦИНСКИЙ ЖУРНАЛ 2021; 41 (4): 25−29 29
https://openalex.org/W2013237249	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0120423&type=printable	English	null	Fast or Slow? Compressions (or Not) in Number-to-Line Mappings	PloS one	2,015	cc-by	6,194	RESEARCH ARTICLE Fast or Slow? Compressions (or Not) in Number-to-Line Mappings Victor Candia1,2, Paola Deprez1,2, Jannis Wernery1, Rafael Núñez3* 1 Collegium Helveticum, Zurich, Switzerland, 2 Zurich University of the Arts, FSP Musikalische Interpretation, Zurich, Switzerland, 3 Department of Cognitive Science, University of California San Diego, San Diego, California, United States of America * rnunez@ucsd.edu * rnunez@ucsd.edu Abstract We investigated, in a university student population, spontaneous (non-speeded) fast and slow number-to-line mapping responses using non-symbolic (dots) and symbolic (words) stimuli. Seeking for less conventionalized responses, we used anchors 0–130, rather than the standard 0–100. Slow responses to both types of stimuli only produced linear mappings with no evidence of non-linear compression. In contrast, fast responses revealed distinct patterns of non-linear compression for dots and words. A predicted logarithmic compression was observed in fast responses to dots in the 0–130 range, but not in the reduced 0–100 range, indicating compression in proximity of the upper anchor 130, not the standard 100. Moreover, fast responses to words revealed an unexpected significant negative compres- sion in the reduced 0–100 range, but not in the 0–130 range, indicating compression in proximity to the lower anchor 0. Results show that fast responses help revealing the funda- mentally distinct nature of symbolic and non-symbolic quantity representation. Whole num- ber words, being intrinsically mediated by cultural phenomena such as language and education, emphasize the invariance of magnitude between them—essential for linear map- pings, and therefore, unlike non-symbolic (psychophysical) stimuli, yield spatial mappings that don’t seem to be influenced by the Weber-Fechner law of psychophysics. However, high levels of education (when combined with an absence of standard upper anchors) may lead fast responses to overestimate magnitude invariance on the lower end of word numerals. OPEN ACCESS Citation: Candia V, Deprez P, Wernery J, Núñez R (2015) Fast or Slow? Compressions (or Not) in Number-to-Line Mappings. PLoS ONE 10(3): e0120423. doi:10.1371/journal.pone.0120423 Academic Editor: Matthew Longo, Birkbeck, University of London, UNITED KINGDOM Received: September 17, 2014 Accepted: January 22, 2015 Published: March 27, 2015 Copyright: © 2015 Candia 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 OPEN ACCESS Citation: Candia V, Deprez P, Wernery J, Núñez R (2015) Fast or Slow? Compressions (or Not) in Number-to-Line Mappings. PLoS ONE 10(3): e0120423. doi:10.1371/journal.pone.0120423 Academic Editor: Matthew Longo, Birkbeck, University of London, UNITED KINGDOM Received: September 17, 2014 Accepted: January 22, 2015 Published: March 27, 2015 Copyright: © 2015 Candia 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: Candia V, Deprez P, Wernery J, Núñez R (2015) Fast or Slow? Compressions (or Not) in Number-to-Line Mappings. PLoS ONE 10(3): e0120423. doi:10.1371/journal.pone.0120423 Academic Editor: Matthew Longo, Birkbeck, University of London, UNITED KINGDOM Received: September 17, 2014 Accepted: January 22, 2015 Published: March 27, 2015 Copyright: © 2015 Candia 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. Citation: Candia V, Deprez P, Wernery J, Núñez R (2015) Fast or Slow? Compressions (or Not) in Number-to-Line Mappings. PLoS ONE 10(3): e0120423. doi:10.1371/journal.pone.0120423 Academic Editor: Matthew Longo, Birkbeck, University of London, UNITED KINGDOM Received: September 17, 2014 Accepted: January 22, 2015 Published: March 27, 2015 Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Response Times and Number-to-Line Mappings disabilities [4, 5]. The data, in part, support the idea that numerical estimation follows the do- main-general psychophysical Weber-Fechner law that subjective sensation increases propor- tional to the logarithm of the stimulus intensity (but see [6, 7] for alternative interpretations). It has been documented that with education and mathematical training, the development of mapping patterns starts to shift gradually, between kindergarten and fourth grade, from a loga- rithmic pattern to a primarily linear one [2, 3] (but see [8] for a different interpretation, where children’s responses are better modeled by two separate linear representations, one for units and one for tens). Besides, studies analyzing data with educated western participants have shown that when reporting on a line, participants exhibit a linear pattern in two main situa- tions: [1] when the stimuli are symbolic (e.g., words)—which are intrinsically cultural, and [2] when they are non-symbolic (primarily psychophysical) as long as they are presented visually, and are easy to evaluate (e.g., small numerosity of dots in the 1–10 range). As it is argued else- where [9], the latter case, in fact, reproduces the conditions in which the learning of the num- ber line occurs, that is, via the support of visible, discrete entities (e.g., dots or jumps performed by a bunny), presented in small quantities [9]. By contrast, the mappings have logarithmic compression when stimuli are non-symbolic and are hard to evaluate (e.g., large numerosities of dots, or tones presented acoustically), which by virtue of being primarily psychophysical in nature make them more susceptible to be influenced by the Weber-Fechner law. In line with these results a study has shown that even in the range of small numbers, attention is necessary for producing linear mappings onto the line: when attentional resources are taxed, the resulting mappings are logarithmic, even in educated participants [9]. Taken together, these studies suggest that the main factor responsible for the linearization of the number-to-space mappings is, ultimately, a learned cultural practice. A spe- cific practice that emphasizes the distance invariance between whole numbers and prescribes the use of a line segment as a notational device for spatially characterizing such magnitude in- variance. PLOS ONE \| DOI:10.1371/journal.pone.0120423 March 27, 2015 Introduction Funding: This work was supported by the Collegium Helveticum, a Swiss National Science Foundation award (K-13K1-120706) to VC, and an award of The Cooper-Fonds Commission of ETH Zurich to VC and RN. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Numerical estimation research has shown that, among people in the industrialized world, number-to-space mappings are highly intuitive. Although this intuition is not universal [1], in the West even kindergarteners when asked to locate numbers on a line marked with a 0 on the left and 100 on the right, are reported to promptly place smaller numbers at left of the segment and greater numbers at right [2, 3]. The mapping, however, is not linear, as children allocate more space to small numbers and less to big numbers in a non-linear compressed manner. This non-linear compression is even more pronounced in children with mathematical learning Competing Interests: The authors have declared that no competing interests exist. 1 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0120423 March 27, 2015 Participants Fifty-four students from two large higher education institutions in Zurich (ETH Zurich and University of Zurich), Switzerland, (range 19–40), 26 women (mean age = 24.1, SD = 4.6) and 28 men (mean age = 24.2, SD = 4.3), participated in the study. All participants were self-re- portedly right-handed and their native language was German. They had normal or corrected- to-normal vision, had no disorders or disturbances of the central nervous system (including psychiatric disorders), no disorders of the arm, hand and or fingers, and were not under the in- fluence of any medication. Ethics Statement This study was approved by the ethics committee of the ETH Zurich. All participants gave their written informed consent and were treated according to the Helsinki convention for the treatment of experimental subjects (http://www.wma.net). Participants received monetary re- ward for their participation. Response Times and Number-to-Line Mappings which, being less monitored are relatively more exposed to the Weber-Fechner law of perception. Another factor that seems to influence the shape of number and numerosity mappings is the magnitude of [6], and familiarity with number stimuli and anchors on the line [6, 7], espe- cially the upper anchor [12]. Besides, non-linear compression patterns don’t seem to follow specific canonical magnitudes, but rather they appear to depend on proportional distances be- tween anchors [6, 16]. Thus, reported logarithmical patterns exhibiting compression towards the upper end of a 0–100 scale may not correspond to a canonically compressed mental repre- sentation of the numbers in the vicinity of 100 proper, but rather depend on the magnitude of the upper anchor, which in this case would happen to be 100. In this study we want to further investigate this phenomenon, by using a less familiar upper anchor greater than the standard 100, namely, 130. We predicted that if the compression really depends on the upper anchor (i.e., the magnitude distance between the anchors), then when participants provide responses over the entire 0–130 range the compression of fast response mappings should be detected over the whole 0–130 range, but not within the 0–100 range. To avoid artificially speeded responses we investigated spontaneous responses where ‘fast’ and ‘slow’ responses were categorized following relevant reaction time criteria from the litera- ture in number cognition (e.g., [17]). In order to systematically investigate the role of the vari- ous variables playing a role in number-to-space mappings and the underlying mental representations, in this study we used both, non-symbolic (dots) and symbolic (words) stimuli. We used words rather than Arabic numerals for symbolic stimuli to match the minimal arith- metical affordances of the non-symbolic stimuli (dots): words, unlike Arabic numerals, are not readily available for arithmetical calculations and digit-based algorithms, and therefore reduce the potential of invoking calculation-related confounds [10]. Indeed, educated participants (who have been systematically trained in learned cul- tural practices) by default exhibit linear mappings, but it is only when their attentional resources get taxed—and therefore their culturally learned practices get less monitored—that they begin to exhibit logarithmic compression [10]. This interpretation is further supported by research that has shown that when reporting non-spatially—i.e., via non-culturally de- veloped forms such as squeezing a dynamometer, hitting a bell, or vocalizing at various intensi- ties—university students in the West consistently produce (1) logarithmic mappings for all non-symbolic stimuli, including small number of dots (which when reported on a line it yields linear mappings), but (2) linear mappings for symbolic stimuli (words) in all tested non-spatial reporting forms [10]. By contrast, the mappings have logarithmic compression when stimuli are non-symbolic and are hard to evaluate (e.g., large numerosities of dots, or tones presented acoustically), One possibility for investigating the parameters that influence the forms that take number and numerosity mappings in general, and number-to-space in particular, is to assess partici- pants’ responses at different levels of automatism. For instance, having fast responses from par- ticipants may indicate more native [9], instinctive, less monitored responses than slow responses, which may benefit from meta-cognitive processes as well as calculation and atten- tional resources [9]. But, surprisingly, with rare exceptions [10, 11] most of the literature on numerical estimation research and number-line mappings reports results in which the time given to participants for responding has not been explicitly considered (see for instance [1–3, 9, 12–15]). One of the main goals of the present study is to investigate in an educated western population, the role played by response time in shaping the number-to-space mappings. We hypothesized that, if linearization of number-to-line mapping responses is primarily a product of cultural practices such as education and training, then non-linear mappings, especially with non-symbolic stimuli, should be primarily observed in the more instinctive fast responses, PLOS ONE \| DOI:10.1371/journal.pone.0120423 March 27, 2015 2 / 11 Stimuli and materials For data collection and stimulus presentation, a custom-made software was implemented using Psychtoolbox 3 for MatLab7. Explanations and stimuli were displayed on a computer screen (BenQ GL2450HM) which had a computer mouse connected to its computer (Logitech M-UAS144 LS1 Laser Mouse). For the target task, a black horizontal line was depicted (440 pixel length, 5 pixel thickness) over a white background, with numerical anchors at each side 3 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0120423 March 27, 2015 Response Times and Number-to-Line Mappings above the line, 0 on the left and 130 on the right (using corresponding German words for the symbolic stimuli presentation, and using an empty circle and a circle (radius 90 pixel) with 130 dots, respectively, for the non-symbolic stimuli presentation). Dots had a constant size (radius 5 pixel) and dot patterns were drawn on-line on random positions within the circle. Response time (RT)—i.e. the time between the presentation of a number stimulus and the corresponding response on the line—was recorded automatically. Stimuli were number stimuli between 10 and 130 (inclusive) in steps of 10, presented either symbolically as written German words (Swiss German spelling), or non-symbolically as collections of dots contained within a circle. Number of letters in words varied from 4 (German word for 10) to 15 (German word for 130) [1 number had 4 letters, 7 numbers had 7 letters, 1 number had 8 letters, 1 number had 11 let- ters, 1 number had 14 letters, 1 number had 15 letters; Mean 8.3 letters, SD 3.1]. Procedure Participants were assessed individually, while sitting in an acoustically shielded room in front of a computer screen placed 70 cm away at eye-level. Instructions for the participants were dis- played on the computer screen and, if needed, verbally clarified by the experimenter. Partici- pants were asked to point via a mouse click to the location on the horizontal line Participants were assessed individually, while sitting in an acoustically shielded room in front of a computer screen placed 70 cm away at eye-level. Instructions for the participants were dis- played on the computer screen and, if needed, verbally clarified by the experimenter. Partici- pants were asked to point via a mouse click to the location on the horizontal line corresponding to each of the number stimuli they were presented on the screen, and they were simply instructed to concentrate and answer ‘quickly’. This was done in order to later analyze spontaneous ‘fast’ and ‘slow’ responses not affected by stress associated with artificially forced or speeded responses. The line and the stimuli were presented simultaneously. After each re- sponse, line and number stimuli disappeared right after the participant clicked on the line. To dilute memory effects from one number stimulus presentation to the next, there was, after each response, a short break of 4s displaying a blank screen. We used one experimental block per stimulus condition, each consisting of 3 randomized presentations of each number stimulus (3x13 stimulus presentations in total for each condition). Number of repetitions of stimulus presentations was kept low to avoid lengthening experimental times, which entail the risk of fa- tiguing the participants. Runs of non-symbolic and symbolic stimuli were counterbalanced across subjects and number stimuli were randomized. Previous to the main experimental ses- sion, to familiarize participants with the tasks, there was a short training with five stimuli not included in the experimental set. The total duration of the experiment was ca. 20 min per par- ticipant (comparable to other studies, e.g., [17, 18]). PLOS ONE \| DOI:10.1371/journal.pone.0120423 March 27, 2015 Data preparation To verify the validity of response location for a given type of stimulus, for each participant we calculated multiple regressions with linear and logarithmic predictors on mean response loca- tion (each based on three responses for every number stimulus on the entire range 10–130). We excluded two participants from the analysis of responses to non-symbolic stimuli whose re- sponses showed neither a linear nor a logarithmic pattern (i.e., the weight of neither Blin nor Blog coefficients was statistically significant). To verify the validity of response range—i.e., the use of a sufficiently long extension of the line to locate responses—we calculated, for each par- ticipant, the response range based on median responses. We excluded one participant from the analysis of responses to symbolic stimuli whose responses used less than 75% of the extension of the line. To make sure that participants kept stable levels of engagement and motivation throughout the experiment, we performed a split-half reliability test for each stimulus condi- tion, comparing the mean over median RTs of the ensemble of trials in the first half of the ex- perimental blocks with the corresponding mean in the second half. No differences in RTs were found (dots: t(52) = -0.944, p = 0.349; words: t(52) = 1.779, p = 0.081). 4 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0120423 March 27, 2015 Response Times and Number-to-Line Mappings Spontaneous ‘fast’ and ‘slow’ responses were defined with respect to relevant criteria taken from number cognition studies involving reaction time (e.g., [6, 17, 19, 20]). For non-symbolic stimuli (dots), a response was considered to be fast if its reaction time (RT) was RT 2000 ms and it was considered to be slow if 2000 ms < RT < 3500 ms. For symbolic stimuli (words), a response was considered to be fast if RT 2500 ms and it was considered to be slow if 2500 ms < RT < 4000 ms. Slightly larger RTs cut-offs (+500 ms) were used for symbolic stimuli in order to account for the fact that words require reading (and in German they contain many characters), implying thus longer processing times. The criteria underlying these cut-offs were the following. First, a time window of ca. Data preparation 1400 ms has been used in simple tasks such as in SNARC-related studies (e.g., 1300 ms [17], 1500 ms [19], 1350 ms [20]), where (1) participants simply respond via directly pressing one of two buttons positioned immediately next to corresponding fingers, and (2) where speeded re- sponses are emphasized [17]. Second, in some number-line reports (e.g., [6]), where partici- pants must carefully point (with a finger or cursor manipulated with a computer mouse) on a line segment, 500 ms has been considered to be the minimum for a response to be valid. Given that in the present study extra time was needed to move a computer mouse and that partici- pants were spontaneously providing un-speeded responses, we considered that a cut-off of 2000 ms for non-symbolic stimuli appropriately divided slow from fast spontaneous responses. This de facto left a 1500 ms time window for acceptable fast and slow, responses. Regarding non-symbolic stimuli, we used slightly larger RT cut-offs (+500 ms) in order to account for a number of factors. First, word processing requires longer times. Verbal numerals, for instance, have been shown to go through additional transcoding operations as compared to Arabic nu- merals [17]. Second, it as been discussed that the recognition of printed words require identify- ing symbols from other orthographic competitors, linking the symbols to semantics, which need to be integrated to conceptual content [19]. And third, words with a length of 8 letters or more (as it is the case in the present report) are always fixated—sometimes more than once— and natural reading fixations, which occur between eye saccades, have a range of 200–300 ms with a higher range of variability up to 500 ms [20] and a standard deviation of 70 ms [19]. For a participant to be classified, as having either fast or slow responses for a given stimulus type, and to be eligible for further analysis, s/he had to (1) have at least 10 out of 13 possible number stimuli with data satisfying the time criterion stated above, and (2) have at least a sta- tistically significant weight of either Blin or Blog coefficients in the individual multiple regression with linear and logarithmic regressors on the median time-categorized responses. Data preparation As a result of this procedure, four groups were constituted: A set of responses coming from participants who spontaneously responded fast to non-symbolic stimuli (n = 11), to symbolic ones (n = 13), who spontaneously responded slow to non-symbolic stimuli (n = 34), and to symbolic stimuli (n = 18). Results The mean response locations provided by the participants over the 10–130 range were analyzed by means of ordinary least squares (OLS) multiple regressions with linear and logarithmic pre- dictors (Fig. 1). Slow responses, for both, non-symbolic (dots) and symbolic (words) stimuli, exhibited a linear pattern (dots: Blin = 0.007, st. error = 0.001, t ratio = 10.344, df = 10, p < 0.001; words: Blin = 0.008, st. error < 0.001, t ratio = 16.516, df = 10, p < 0.001) and showed no evidence of a non-linear compression over and above the linear one (dots: Blog = 0.098, st. error = 0.083, t ratio = 1.181, df = 10, p = 0.265; words: Blog = 0.034, st. error = 0.056, t ratio = 0.620, df = 10, p = 0.549). Fast responses, however, exhibited a different pattern. While responses to symbolic 5 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0120423 March 27, 2015 Response Times and Number-to-Line Mappings error = 0.006, t ratio = 4.727, df = 22, p < 0.001), confirming that significant differences in logarithmic compression do exist between fast responses to non- symbolic and symbolic stimuli. Slow responses, on the contrary, did not exhibit such differ- ences in nonlinear compression. Slow responses expectedly shared linear structure (Blin = 0.008, st. error < 0.001, t ratio = 18.578, df = 23, p < 0.001) with no evidence of non-linear compression (Blog = 0.066, st. error = 0.048, t ratio = 1.385, df = 23, p = 0.179). But, important- ly, the weight of the dummy x logarithmic interaction was not significant (BD x log = 0.005, st. error = 0.006, t ratio = 0.88, df = 22, p = 0.388), indicating no evidence that there is a significant difference in logarithmic compression between fast responses to non-symbolic and symbolic stimuli. y In order to analyze the significance of the compression in the range 10–100 (for the responses which had been produced over the whole line segment anchored with 0 and 130), we ran further multiple regressions with linear and logarithmic regressors for this restricted lower range. As in the case of slow responses over the entire 0–130 range, slow responses in the 0–100 range exhibited only a linear pattern for both type of stimuli (dots: Blin = 0.009, st. error = 0.001, t ratio = 1.082, df = 7, p < 0.001; words: Blin = 0.009, st. error = 0.001, t ratio = 14.962, df = 7, p < 0.001) with no evidence of non-linear compression (dots: Blog = -0.073, st. error = 0.052, t ratio = -1.406, df = 7, p = 0.202; words: Blog = -0.072, st. error = 0.058, t ratio = -1.237, df = 7, p = 0.256). Fast responses over the 0–100 range did not provide evidence of a significant non-linear compression when ob- tained with non-symbolic (dots) stimuli (Blin = 0.008, st. error = 0.001, t ratio = 9.44, df = 7, p < 0.001; Blog = 0.063, st. error = 0.078, t ratio = 0.811, df = 7, p = 0.444). However, when obtained with symbolic (words) stimuli they exhibited a significant negative non-linear compression (Blin = 0.01, st. error = 0.001, t ratio = 15.867, df = 7, p < 0.001; Blog = -0.167, st. error = 0.058, t ratio = -2.907, df = 7, p = 0.023). Response Times and Number-to-Line Mappings Fig 1. Responses to the number line task. Mean response locations with standard error of the mean are shown, separated by fast (top) and slow (bottom) responses, as well as by type of stimuli—non-symbolic (dots) in blue and symbolic (red) in red. The fitted regression lines for each case are taken from a multiple regression analysis of response location that included linear and logarithmic regressors. Non-linear compression—positive for dots and negative for words—is only observed in fast responses (see details in text, and in Fig. 2). doi:10.1371/journal.pone.0120423.g001 stimuli (words) showed a linear pattern (Blin = 0.008, st. error < 0.001, t ratio = 17.875, df = 10, p < 0.001) with no evidence of non-linear compression (Blog = -0.076, st. error = 0.055, t ratio = -1.378, df = 10, p = 0.2), responses to non-symbolic stimuli (dots) did exhibit a non- linear compression over and above the linear one (Blin = 0.006, st. error = 0.001, t ratio = 9.78, df = 10, p < 0.001; Blog = 0.179, st. error = 0.075, t ratio = 2.378, df = 10, p = 0.039). g The difference in nonlinear compression between responses to non-symbolic and symbolic stimuli was further corroborated with OLS multiple regressions containing linear and logarith- mic regressors and an added “dummy variable” (D)—a technique widely used in other domains [21]—aimed at evaluating the discriminability between responses to non-symbolic and sym- bolic stimuli with respect to logarithmic compression. When considering the ensemble of fast responses (symbolic and non-symbolic responses combined: 2 stimulus type x 13 number sti- muli = 26 observations) the weight of the linear regressor was significant (Blin = 0.007, st. error = 0.001, t ratio = 11.890, df = 23, p < 0.001), indicating shared linear structure, with no evidence of shared logarithmically compressed structure (Blog = 0.052, st. error = 0.073, t ratio = 0.711, df = 23, p = 0.484). Crucially, however, when adding the dummy variable (with D = 1 if the observation was a response to a non-symbolic stimulus, and D = 0 if it was a re- sponse to a symbolic stimulus) the weight of the dummy x logarithmic interaction was highly significant (BD x log = 0.029, st. PLOS ONE \| DOI:10.1371/journal.pone.0120423 March 27, 2015 6 / 11 Fig 1. Responses to the number line task. Mean response locations with standard error of the mean are shown, separated by fast (top) and slow (bottom) responses, as well as by type of stimuli—non-symbolic (dots) in blue and symbolic (red) in red. The fitted regression lines for each case are taken from a multiple regression analysis of response location that included linear and logarithmic regressors. Non-linear compression—positive for dots and negative for words—is only observed in fast responses (see details in text, and in Fig. 2). Response Times and Number-to-Line Mappings Fig. 2 shows the standardized Beta-log coefficients of these analyses, for both, the 0–100 and 0–130 range. 7 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0120423 March 27, 2015 Discussion The results of this study show that speed of response to number-to-space tasks reveal differ- ences in mapping compression with respect to type of stimuli, and mapping range. Traditional- ly, response time has not been explicitly considered as an important parameter in number estimation and number line studies. The present report points to the importance of doing so, even when studying a university student population. Based on findings from previous studies [10, 13], we predicted that mappings of fast responses to dots (non-symbolic), but not to words (symbolic), would exhibit a logarithmic compression. The results confirm this prediction, and show that the difference in non-linear compression vanishes when responses are slow, since re- sponses to both type of stimuli become linear. The data suggest that fast responses to dots, being more automatic and less monitored than slow responses, are more exposed to the Weber-Fechner law of perception, thus yielding logarithmic mappings. Why do slow responses become linear? It has been suggested that with education and math- ematical training, the development of mapping patterns starts to shift gradually, between kin- dergarten and fourth grade, from a logarithmic pattern to a primarily linear one [2, 3]. Linear mappings are observed in well-educated adults responding to symbolic (words) stimuli and to visual non-symbolic (dots) stimuli [10, 13]. But when responding to a large numerosity of dots [10, 13], or when affected by reduced attentional resources [9], the mappings re-appear as loga- rithmically compressed. This has led to the proposal that linear and logarithmic mappings might co-exist in an individual’s mind [9, 10, 13]—their manifestation depending on factors such as type of stimuli and availability of attentional resources—where the logarithmic ones are taken to be more instinctive or ‘native’ [9]. Our results show that spontaneous slow re- sponses—especially when dots are concerned—seem to get the extra few hundred milliseconds needed to move away from the ‘native’ or instinctive state ruled by the Weber-Fechner law. This extra time allows for adjustments and meta-reflexions, which in the ontogeny of individu- als is mediated and scaffolded via cultural factors such as language and education. This scaf- folding ultimately results in linear mappings that match the ones observed with the culturally shaped symbolic stimuli: words. And why do words yield linear mappings, even when report- ing non-spatially over a variety of media [10]? Response Times and Number-to-Line Mappings Response Times and Number-to-Line Mappings Fig 2. Standardized Beta-log coefficients for fast and slow responses. The values are taken from a multiple regression analysis of response location that included linear and logarithmic regressors. Responses to non-symbolic stimuli (dots) appear on the left, separated by the range under consideration, 0–130 or 0–100, labeled by their upper bound ‘130’ and ‘100’ respectively. Responses to symbolic stimuli (words) appear on the right. Slow responses only produced linear mappings with no evidence of non-linear compression. Fast responses revealed distinct patterns of non-linear compression for non-symbolic and symbolic stimuli. A significant non-linear (logarithmic) compression was observed in responses to non-symbolic stimuli in the entire range 0–130, but not in the reduced 0–100 range, whereas a significant negative compression was observed in responses to symbolic stimuli in the reduced range 0–100, but not in the entire 0–130 range. doi:10.1371/journal.pone.0120423.g002 doi:10.1371/journal.pone.0120423.g002 Response Times and Number-to-Line Mappings PLOS ONE \| DOI:10.1371/journal.pone.0120423 March 27, 2015 8 / PLOS ONE \| DOI:10.1371/journal.pone.0120423 March 27, 2015 8 PLOS ONE \| DOI:10.1371/journal.pone.0120423 March 27, 2015 8 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0120423 March 27, 2015 Discussion First, words for whole numbers bypass psycho- physically-related phenomena such as attentional processes [9], and therefore are somewhat immune to logarithmically inducing pressures. And, most importantly, number words have the crucial property of encapsulating precise quantitative meaning that emphasizes magnitude invariance, between, for instance, the predecessor and the successor of any given counting number. In other words, meaning in number lexica has been culturally created to have a linear structure, property that is taught to individuals via language and education. To minimize conventional responses that might mask compression differences due to re- sponse time and/or type of stimuli, our study used a 0–130 ‘number line’ rather than the stan- dard 0–100. Based on the observation that non-linear compression patterns don’t seem to follow specific canonical magnitudes, but rather they depend on proportional distance between anchors [6, 16], and familiarity with the anchors and number stimuli [12, 14], we predicted that in case of a non-linear compression of fast response mappings, this should only be 9 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0120423 March 27, 2015 Response Times and Number-to-Line Mappings significant when evaluated over the whole 0–130 range, and not when evaluated over the re- stricted 0–100 range. In other words, we expected compression to be detectable at the higher end of the 0–130 range. Our results confirmed this prediction, as fast responses to dots pro- duced a logarithmically compressed mapping detectable at the 0–130 range but not at the 0–100 range, which indicates a compression relative to, and in proximity of, the upper anchor 130, not the standard 100. The results give support to the proposal that non-linear compression in number-to-space mappings is not fixed with respect to specific standards (e.g., numbers in the vicinity of 100) but that it is shaped with respect to the number-line anchors. But our study also produced some unexpected results. Fast responses to words revealed a significant negative compression in the reduced range 0–100, but not in the 0–130 range, indicating compression in proximity to the lower anchor 0. Graphically (Fig. 1), the negative non-linear compression observed for symbolic stimuli (words) at the lower end of the line appears as a mirror opposite of the upper compression exhibited by responses to non-symbolic stimuli (dots). However, while dots are primarily psychophysical stimuli, susceptible to be influenced by the Weber- Fechner law, words are not. Dots and words are different in nature. S1 Dataset. Dots. (XLSX) S1 Dataset. Dots. (XLSX) S2 Dataset. Words. (XLSX) Acknowledgments We thank Samuel Rupprechter for valuable help with data analysis. Author Contributions Conceived and designed the experiments: VC RN. Performed the experiments: VC PD JW. An- alyzed the data: VC PD JW RN. Contributed reagents/materials/analysis tools: JW. Wrote the paper: VC RN. Discussion We speculate that intensely educated university students, when perceiving symbolic stimuli and in the absence of a stan- dard high anchor (which would have been 100) and conventionalized reference points [22, 23], might have been over-cautious in following the invariance magnitude principle. As a result, on the side of origin of the scale they might have over-compensated in leaving enough space for a more abundant number-locations than the standard 100 would have, which resulted in a sig- nificant compression at the lower end of the scale. In slow responses this compression disap- peared as participants operated with more time, which allowed for adjustments leading to a more linear calibration. Further research is needed to test this explanatory proposal, but in doing so, it seems that investigating response time is a fruitful path to follow. Supporting Information S1 Dataset. Dots. (XLSX) S2 Dataset. Words. (XLSX) References 1. Núñez R, Cooperrider K, Wassmann J. Number Concepts without Number Lines in an Indigenous Group of Papua New Guinea. Plos One. 2012; 7(4). 1. Núñez R, Cooperrider K, Wassmann J. Number Concepts without Number Lines in an Indigenous Group of Papua New Guinea. Plos One. 2012; 7(4). 2. Booth JL, Siegler RS. Developmental and individual differences in pure numerical estimation. Dev Psy- chol. 2006; 42(1):189–201. PMID: 16420128 2. Booth JL, Siegler RS. Developmental and individual differences in pure numerical estimation. Dev Psy- chol. 2006; 42(1):189–201. PMID: 16420128 3. Siegler RS, Booth JL. 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https://openalex.org/W4249523657	https://www.qeios.com/read/RR756N/pdf	English	null	SPRR1A wt Allele	Definitions	2,020	cc-by	69	Qeios · Definition, February 2, 2020 Open Peer Review on Qeios SPRR1A wt Allele National Cancer Institute National Cancer Institute Qeios ID: RR756N · https://doi.org/10.32388/RR756N Source National Cancer Institute. SPRR1A wt Allele. NCI Thesaurus. Code C114894. Human SPRR1A wild-type allele is located within 1q21-q22 and is approximately 2 kb in length. This allele, which encodes cornifin-A protein, plays a role in keratinocyte differentiation. Qeios ID: RR756N · https://doi.org/10.32388/RR756N 1/1
https://openalex.org/W2508173339	https://europepmc.org/articles/pmc4993445?pdf=render	English	null	Knowledge, Attitudes, and Practices regarding Diarrhea and Cholera following an Oral Cholera Vaccination Campaign in the Solomon Islands	PLoS neglected tropical diseases	2,016	public-domain	5,118	Background In response to a 2011 cholera outbreak in Papua New Guinea, the Government of the Solo- mon Islands initiated a cholera prevention program which included cholera disease preven- tion and treatment messaging, community meetings, and a pre-emptive cholera vaccination campaign targeting 11,000 children aged 1–15 years in selected communities in Choiseul and Western Provinces. Editor: Edward T. Ryan, Massachusetts General Hospital, UNITED STATES Received: April 28, 2016 Accepted: August 1, 2016 Published: August 22, 2016 Editor: Edward T. Ryan, Massachusetts General Hospital, UNITED STATES Received: April 28, 2016 Accepted: August 1, 2016 Published: August 22, 2016 Methodology and Principal Findings We conducted a post-vaccination campaign, household-level survey about knowledge, atti- tudes, and practices regarding diarrhea and cholera in areas targeted and not targeted for cholera vaccination. Respondents in vaccinated areas were more likely to have received cholera education in the previous 6 months (33% v. 9%; p = 0.04), to know signs and symp- toms (64% vs. 22%; p = 0.02) and treatment (96% vs. 50%; p = 0.02) of cholera, and to be aware of cholera vaccine (48% vs. 14%; p = 0.02). There were no differences in water, sani- tation, and hygiene practices. Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Data Availability Statement: Data are not publicly available for ethical and legal reason, per the Ethics Committee of Solomon Islands. Researchers may contact The Solomon Islands Health Research and Ethics Review Board (SIHRERB) Secretariat at Freda.Pitakaka@simtri.gov.sb to discuss access. Knowledge, Attitudes, and Practices regarding Diarrhea and Cholera following an Oral Cholera Vaccination Campaign in the Solomon Islands Eleanor Burnett1, Tenneth Dalipanda2, Divi Ogaoga2, Jenny Gaiofa2, Gregory Jilini3, Alison Halpin1, Vance Dietz1, Kashmira Date1, Eric Mintz1, Terri Hyde1, Kathleen Wannemuehler1, Catherine Yen1 Eleanor Burnett1, Tenneth Dalipanda2, Divi Ogaoga2, Jenny Gaiofa2, Gregory Jilini3, Alison Halpin1, Vance Dietz1, Kashmira Date1, Eric Mintz1, Terri Hyde1, Kathleen Wannemuehler1, Catherine Yen1 a1111 1 Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America, 2 Ministry of Health and Medical Services, Honiara, Solomon Islands, 3 Gizo Hospital, Gizo, Solomon Islands * wwg7@cdc.gov * wwg7@cdc.gov OPEN ACCESS Citation: Burnett E, Dalipanda T, Ogaoga D, Gaiofa J, Jilini G, Halpin A, et al. (2016) Knowledge, Attitudes, and Practices regarding Diarrhea and Cholera following an Oral Cholera Vaccination Campaign in the Solomon Islands. PLoS Negl Trop Dis 10(8): e0004937. doi:10.1371/journal. pntd.0004937 RESEARCH ARTICLE Conclusions This pre-emptive OCV campaign in a cholera-naïve community provided a unique opportu- nity to assess household-level knowledge, attitudes, and practices regarding diarrhea, chol- era, and water, sanitation, and hygiene (WASH). Our findings suggest that education provided during the vaccination campaign may have reinforced earlier mass messaging about cholera and diarrheal disease in vaccinated communities. Funding: This survey was funded by the World Health Organization. The protocol was reviewed by WHO prior to implementation. The funders had no role in data collection and analysis, decision to publish, or content of the manuscript. 1 / 9 PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004937 August 22, 2016 Knowledge, Attitude, Practices in the Solomon Islands Competing Interests: The authors have declared that no competing interests exist. Author Summary We assessed knowledge, attitudes and practices of diarrhea and cholera disease and pre- vention in two areas of the Solomon Islands near Papua New Guinea. Both areas were ‘at risk’ for cholera disease and received messages about cholera prevention. Later, one of the areas also received vaccination against cholera. This was the first time cholera vaccine was administered to a population that had never reported cholera. Our survey found that peo- ple living in the area were cholera vaccine was administered were more likely to know the signs and symptoms and treatment of cholera, as well as be aware of cholera vaccine. We think this could be related to the extra education provided with vaccination. This was the first knowledge, attitudes, and practices survey about diarrhea and cholera disease and prevention and prevention in a population that had not been exposed to cholera. Introduction From July 2009 until late 2011, an outbreak of cholera in Papua New Guinea (PNG) resulted in >15,500 cases and >500 deaths in 8 of 20 province-level divisions and the Autonomous Region of Bougainville, situated in the western archipelago of the Solomon Islands in the South Pacific (Fig 1) [1]. At the time of the outbreak, no cholera was confirmed in the Solo- mon Islands, a country of approximately 560,000 people and nearly 1,000 islands [3]. How- ever the risk for cholera introduction and transmission was considered high, due to geographical location, frequent travel between Bougainville and the Solomon Islands, and limited access to improved sources of water and improved sanitation infrastructure in the Solomon Islands. As a result, the Government of the Solomon Islands initiated a cholera pre- vention program in the two provinces adjacent to Bougainville: Choiseul and Western Provinces. In April 2011, the Ministry of Health and Medical Services (MHMS) began a messaging campaign in both Provinces about cholera disease, transmission, and prevention via radio programs, newspapers, and community meetings (personal communication, MHMS). Subse- quently, the MHMS, with support from the World Health Organization (WHO), the United Nations Children’s Fund (UNICEF), and the Australian Agency for International Develop- ment (AusAID), conducted a cholera vaccination campaign for 11,000 children 1 to 15 years of age in selected communities in Choiseul Province and the Inner and Outer Shortland Islands of Western Province; children were brought to vaccination sites by their parents, and age was verified by date of birth. For the campaign, the MHMS procured Shanchol (Shantha Biotechnics Ltd., India), an oral, whole-cell, killed vaccine administered in two doses at least two weeks apart [4]. Vaccination teams stationed at health centers and nurse aid posts administered 11,888 doses in May 2012 and 11,318 doses in July 2012 [5] and provided mes- sages about safe water, sanitation, and hygiene practices verbally and through brochures and posters. This cholera vaccination campaign was the first use of Shanchol in a pre-emptive campaign in an area that had never reported cholera and provided a unique opportunity to assess the impact of messaging on cholera-naive communities. To test our hypothesis that education dur- ing the cholera vaccination campaign may have reinforced the earlier, wider messaging cam- paign, we conducted knowledge, attitude, and practices (KAP) survey related to diarrhea and cholera in vaccinated and unvaccinated communities. PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004937 August 22, 2016 Knowledge, Attitude, Practices in the Solomon Islands Fig 1. Map of Papua New Guinea and the Solomon Islands [2]. doi:10.1371/journal.pntd.0004937.g001 g Fig 1. Map of Papua New Guinea and the Solomon Islands [2]. doi:10.1371/journal.pntd.0004937.g001 PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004937 August 22, 2016 Introduction 2 / 9 PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004937 August 22, 2016 Knowledge, Attitude, Practices in the Solomon Islands Data collection Before the survey, team members were trained on data collection methods and survey ques- tions were translated into Pidgin, the main language of the Solomon Islands. For each house- hold, survey teams interviewed the female head of household or an alternate adult 18 years old and collected information regarding household demographic and socioeconomic charac- teristics; recent diarrheal illness; knowledge, attitudes, and practices (KAP) related to diarrhea and cholera; water source, storage, and handling practices; routine and cholera vaccines; hand hygiene and sanitation practices; and vaccine accessibility. Survey teams also made observa- tions regarding water storage and the presence or absence of areas for handwashing, soap, and latrines/toilets. Statistical analyses Data were entered into an Access database and analyzed using SAS v9.3. Frequencies and per- centages were calculated for categorical variables; median and IQR were calculated for continu- ous variables. Univariate logistic regression models were fit to evaluate whether there were significant differences in KAPs between vaccinated and unvaccinated areas using generalized estimating equations to account for the ward-level clustering; generalized score statistic p-val- ues 0.05 were considered significant. This evaluation was reviewed by the National Health Research and Ethics Committee of Sol- omon Islands Ministry of Health and Medical services and Centers for Disease Control and Prevention and considered non-research. Sampling and study population During 6–10 December 2012, approximately 5 months after the cholera vaccination campaign had ended, we conducted a household-level cross-sectional survey in communities targeted for vaccination (Choiseul Province and the Shortland Islands of Western Province), subsequently referred to as “vaccinated areas,” and in communities on nearby islands of Western Province not targeted for cholera vaccination, referred to as “unvaccinated areas.” These populations were chosen because they were both considered at-risk for cholera importation and were both targeted during the initial information campaign. We randomly sampled six wards each in vac- cinated and unvaccinated areas, and then randomly sampled two villages within each ward. Due to small village sizes, we interviewed a convenience sample of 12 households in each selected village. Households that participated in a previous cholera KAP survey were excluded. In areas where there was civil unrest, the survey team approached the village closest to the one originally selected. 3 / 9 PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004937 August 22, 2016 PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004937 August 22, 2016 Results We interviewed 108 households in vaccinated areas and 173 households in unvaccinated areas. Respondents from 46 households reported that they had participated in an earlier cholera KAP survey. Since we could not determine whether specific respondents had participated in the pre- vious survey and had been exposed to similar questions, we excluded them from the analysis. Thus, the final analysis included 89 households in vaccinated and 146 households in unvacci- nated areas (Table 1). In both areas, the respondents’ median age was 39 years, and median household size was six individuals. The reported literacy rate of respondents was high in both vaccinated (91%) and unvaccinated (98%) areas. A higher proportion of households in vacci- nated areas (17%) than in unvaccinated areas (5%) reported at least one household member of any age having had diarrhea in the previous week (p = 0.01). Nearly all respondents correctly named at least one cause (97% in vaccinated areas, 98% in unvaccinated areas) and one treatment (97% in vaccinated areas, 98% in unvaccinated areas) of diarrhea (Table 2). The most commonly mentioned cause was ‘poor hygiene,’ and treatment was ‘go to clinic’. When asked to name a diarrhea prevention measure, 88% of respondents in vaccinated areas provided a correct answer compared with 99% in unvaccinated areas (p = 0.04). In both, the most commonly mentioned prevention strategies were ‘hand washing’, ‘clean cooking utensils’, and ‘cover food to keep away from flies’. Respondents in vaccinated areas were more likely to report recent education about diarrhea (37% in vaccinated, 14% in unvaccinated; p = 0.02). In both areas, the most common source of diarrhea information was a community health worker or clinician. In vaccinated areas, 75% of respondents were aware of cholera compared with 44% in unvaccinated areas (p = 0.09). Among respondents aware of cholera, 55% of respondents in vaccinated areas correctly named at least one cause of cholera transmission, compared with 27% of respondents in unvaccinated areas (p = 0.10); similarly, 67% in vaccinated and 34% in unvaccinated areas named one prevention measure (p = 0.15). Those in vaccinated areas were PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004937 August 22, 2016 4 / 9 (%) in areas not targeted for vaccination p-value (n = 89) (n = 146) Individual-level Age of respondent, median(IQR) 39 (31–44) 39 (30–49) Female respondent 64 (72%) 74 (51%) 0.6 Able to read and write 81 (91%) 142 (98%) 0.1 Primary education or less 51 (57%) 59 (40%) 0.08 Household-level People/hh, median (IQR) 6 (4–8) 6 (4–8) Diarrhea in the last week 15 (17%) 7 (5%) 0.009 Electricity 46 (59%) 112 (78%) 0.06 Cooking gas 1 (1%) 8 (6%) 0.06 Radio 25 (32%) 48 (34%) 0.4 Cell phone 29 (37%) 97 (68%) 0.01 Boat 20 (25%) 18 (13%) 0.06 Fishnet 10 (13%) 19 (13%) 1.0 doi 10 1371/jo rnal pntd 0004937 t001 of individual and household survey respondents in communities targeted and not targeted for vaccination. Table 1. Self-reported characteristics of individual and household survey respondents in communities t Solomon Islands, 2012. more likely than those in unvaccinated areas to correctly name at least one sign or symptom (64% vs. 22%; p = 0.01), identify watery diarrhea as a sign (57% v. 17%; p = 0.01), and name at least one treatment (96% vs. 50%; p = 0.02) for cholera. Persons in vaccinated areas were also Table 2. Knowledge of causes, prevention, and treatment of diarrhea and cholera in areas targeted and not targeted for OCV vaccination. Solomon Islands, 2012. Knowledge, Attitude, Practices in the Solomon Islands more likely than those in unvaccinated areas to correctly name at least one sign or symptom (64% vs. 22%; p = 0.01), identify watery diarrhea as a sign (57% v. 17%; p = 0.01), and name at least one treatment (96% vs. 50%; p = 0.02) for cholera. Persons in vaccinated areas were also Table 1. Self-reported characteristics of individual and household survey respondents in communities targeted and not targeted for vaccination. Solomon Islands, 2012. No. (%) in areas targeted for vaccination No. (%) in areas not targeted for vaccination p-value (n = 89) (n = 146) Individual-level Age of respondent, median(IQR) 39 (31–44) 39 (30–49) Female respondent 64 (72%) 74 (51%) 0.6 Able to read and write 81 (91%) 142 (98%) 0.1 Primary education or less 51 (57%) 59 (40%) 0.08 Household-level People/hh, median (IQR) 6 (4–8) 6 (4–8) Diarrhea in the last week 15 (17%) 7 (5%) 0.009 Electricity 46 (59%) 112 (78%) 0.06 Cooking gas 1 (1%) 8 (6%) 0.06 Radio 25 (32%) 48 (34%) 0.4 Cell phone 29 (37%) 97 (68%) 0.01 Boat 20 (25%) 18 (13%) 0.06 Fishnet 10 (13%) 19 (13%) 1.0 doi:10.1371/journal.pntd.0004937.t001 Table 2. Knowledge of causes, prevention, and treatment of diarrhea and cholera in areas targeted and not targeted for OCV vaccination. Solomon Islands, 2012. doi:10.1371/journal.pntd.0004937.t002 Targeted Not targeted p-value (n = 89) (n = 146) n % n % Diarrhea 1 correct cause named1 86 97% 143 98% 0.5 1 correct prevention measure named2 78 88% 144 99% 0.04 1 correct treatment named3 86 97% 143 98% 0.6 Received education about diarrhea prevention or treatment within the past 6 months 33 37% 20 14% 0.02 Aware of cholera 67 75% 64 44% 0.09 (n = 67) (n = 64) p-value Cholera n % n % 1 correct cause named4 37 55% 17 27% 0.1 1 correct symptom named5 43 64% 14 22% 0.02 1 correct prevention measure named6 45 67% 22 34% 0.1 1 correct treatment named7 64 96% 32 50% 0.02 Received education about cholera prevention or treatment within the past 6 months 22 33% 6 9% 0.04 1 Drinking bad water, eating bad food, unwashed fruits/vegetables, ﬂies/insects, poor hygiene 2 Wash hands with soap and water, cook food thoroughly, boil water, wash fruits/vegetables, clean cooking utensils/vessels, treat water, drink cooled, boiled water, dispose of human waste properly, cover food to keep away from ﬂies 3 Go to clinic/hospital, use oral rehydration solution/salt-sugar solution, go to a traditional healer, coconut-salt solution 4 Drinking bad water, eating bad food, unwashed fruits/vegetables, ﬂies/insects, poor hygiene 5 Fever, vomiting, watery diarrhea, stomach/abdominal pain, bloody diarrhea, dehydration 6 Wash hands with soap and water, cook food thoroughly, boil water, wash fruits/vegetables, clean cooking utensils/vessels, treat water, drink cooled, boiled water, dispose of human waste properly, cover food to keep away from ﬂies Table 1. Self-reported characteristics of individual and household survey respondents in communities targeted and not targeted for vaccination. Solomon Islands, 2012. No. (%) in areas targeted for vaccination No. Targeted Not targeted p-value (n = 89) (n = 146) n % n % Diarrhea 1 correct cause named1 86 97% 143 98% 0.5 1 correct prevention measure named2 78 88% 144 99% 0.04 1 correct treatment named3 86 97% 143 98% 0.6 Received education about diarrhea prevention or treatment within the past 6 months 33 37% 20 14% 0.02 Aware of cholera 67 75% 64 44% 0.09 (n = 67) (n = 64) p-value Cholera n % n % 1 correct cause named4 37 55% 17 27% 0.1 1 correct symptom named5 43 64% 14 22% 0.02 1 correct prevention measure named6 45 67% 22 34% 0.1 1 correct treatment named7 64 96% 32 50% 0.02 Received education about cholera prevention or treatment within the past 6 months 22 33% 6 9% 0.04 1 Drinking bad water, eating bad food, unwashed fruits/vegetables, ﬂies/insects, poor hygiene 2 Wash hands with soap and water, cook food thoroughly, boil water, wash fruits/vegetables, clean cooking utensils/vessels, treat water, drink cooled, boiled water, dispose of human waste properly, cover food to keep away from ﬂies 3 Go to clinic/hospital, use oral rehydration solution/salt-sugar solution, go to a traditional healer, coconut-salt solution 4 Drinking bad water, eating bad food, unwashed fruits/vegetables, ﬂies/insects, poor hygiene 5 Fever, vomiting, watery diarrhea, stomach/abdominal pain, bloody diarrhea, dehydration 6 Wash hands with soap and water, cook food thoroughly, boil water, wash fruits/vegetables, clean cooking utensils/vessels, treat water, drink cooled, boiled water, dispose of human waste properly, cover food to keep away from ﬂies 7 Go to clinic/hospital, use oral rehydration solution/salt-sugar solution, go to a traditional healer, coconut-salt solution doi:10.1371/journal.pntd.0004937.t002 PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004937 August 22, 2016 5 / 9 Table 2. Knowledge of causes, prevention, and treatment of diarrhea and cholera in areas targeted and not targeted for OCV vaccination. Solomon Islands, 2012. Knowledge, Attitude, Practices in the Solomon Islands more likely than those in unvaccinated areas to report any recent education about cholera (33% vs. 9%; p = 0.04). Among cholera-aware respondents in vaccinated areas, the most com- monly mentioned sign of cholera was ‘watery stool’ (57%); the most commonly mentioned cause was ‘poor hygiene’ (39%); the most commonly mentioned prevention was ‘hand washing’ (42%); and the most commonly mentioned treatment was ‘go to clinic’ (90%). In a subanalysis of unvaccinated households, 40 of 58 (69%) unvaccinated households in areas targeted for vaccination reported awareness of cholera while 64 of 144 (44%) unvacci- nated households in areas not targeted for vaccination reported awareness. Though the differ- ence was not statistically significant (p = 0.21), these results suggests that cholera messaging may have reached more households in areas targeted for the OCV campaign, even if they did not include any vaccine recipients. Unvaccinated but cholera-aware households in targeted areas were more likely to report watery diarrhea as sign of cholera than unvaccinated but chol- era-aware households in areas not targeted (48% v. 17%; p = 0.01), to know any signs or symp- toms of cholera (58% v. 21%; p = 0.03), and to know any treatment for cholera (98% v. 50%; p = 0.01). There were no differences in knowledge of causes (p = 0.33), knowledge of preven- tion (p = 0.27), or recent cholera education (p = 0.50) between the two groups. Drinking water sources were similar in vaccinated and unvaccinated areas, with only 17% and 10% respectively reporting an unprotected surface source or well as their main source of drinking water. However, a greater proportion of those surveyed in vaccinated areas reported ever treating their drinking water (53% vaccinated, 11% unvaccinated; p = 0.03) (Table 3). In both groups, fewer than 50% (47% vaccinated, 39% unvaccinated) reported regularly washing their hands. Eighty-five percent of households in vaccinated and 61% in unvaccinated areas reported their usual toilet facilities as the ocean or bush (p = 0.05). Acceptance of routine childhood vaccines was high in both vaccinated and unvaccinated areas: in only one household were all members completely unvaccinated; however, about a quarter of respondents reported having a concern about vaccination for themselves or their child. The most common concerns were related to side effects. In vaccinated areas, more respondents had heard of cholera vaccine compared with unvaccinated areas (48% vs. Targeted Not targeted p-value (n = 89) (n = 146) n % n % Diarrhea 1 correct cause named1 86 97% 143 98% 0.5 1 correct prevention measure named2 78 88% 144 99% 0.04 1 correct treatment named3 86 97% 143 98% 0.6 Received education about diarrhea prevention or treatment within the past 6 months 33 37% 20 14% 0.02 Aware of cholera 67 75% 64 44% 0.09 (n = 67) (n = 64) p-value Cholera n % n % 1 correct cause named4 37 55% 17 27% 0.1 1 correct symptom named5 43 64% 14 22% 0.02 1 correct prevention measure named6 45 67% 22 34% 0.1 1 correct treatment named7 64 96% 32 50% 0.02 Received education about cholera prevention or treatment within the past 6 months 22 33% 6 9% 0.04 and treatment of diarrhea and cholera in areas targeted and not targeted for OCV vaccination. Solomon PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004937 August 22, 2016 5 / 9 Discussion To the best of our knowledge, this is the first household-level evaluation to assess knowledge, attitudes, and practices regarding diarrhea, cholera, and water, sanitation, and hygiene (WASH) in the setting of a pre-emptive OCV campaign in a cholera-naïve community. We found that knowledge of causes and treatments for diarrhea were high both in areas targeted and not targeted for cholera vaccination, while knowledge of cholera vaccine and cholera signs and symptoms and treatments was higher in areas that were targeted for vaccination despite similar levels of cholera awareness in both areas. This was true among only households that were not vaccinated as well as all households that participated. The reason for these findings may be that both vaccinated and unvaccinated areas had previously received messages about cholera disease, prevention and treatment, but key messages were likely reiterated during the vaccination campaign. Survey respondents also reported high acceptance of OCV. This finding is supported by the successful implementation of the vaccination campaign by MHMS, with administrative OCV coverage of 108% and 102% during the first and second rounds of the campaign, respectively (personal communication, MHMS); there was no additional assessment of vaccination coverage associated with this campaign, and the high coverage estimates may be due to population movement unaccounted for in official population estimates. Similar knowl- edge of cholera and attitudes toward OCV have also been reported in Dhaka, Bangladesh, an endemic setting [5]. Of note, use of water treatment practices, hand washing, and use of improved toilet facilities were low both in targeted and non-targeted areas, even though knowl- edge of diarrhea prevention and treatment was high among survey respondents and most households had protected sources of drinking water. Reasons for this are unknown and should be investigated further. Another interesting finding was that knowledge of diarrhea was higher than knowledge of cholera among cholera-aware respondents in vaccinated areas; 97% and 55%, respectively, cor- rectly listed a cause of diarrhea and cholera. Although documentation of health messages pro- vided during the prevention and vaccination campaign is limited, this finding would be expected if cholera prevention messages had been framed as ‘diarrhea’ prevention. A recent KAP survey in Thailand reported that building on diarrhea knowledge while distinguishing cholera was a key challenge in effective communication (personal communication, H. Scobie). Knowledge, Attitude, Practices in the Solomon Islands reported that at least one household member 1–15 years of age had received the cholera vac- cine, though only 21% had an OCV campaign card. In unvaccinated areas, 2 (1%) households reported that at least one member had received OCV. PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004937 August 22, 2016 14%; p = 0.02). Nearly all participants reported they would get the cholera vaccine for themselves, if available (97% in vaccinated, 98% in unvaccinated). In vaccinated areas, 35% of respondents Table 3. Reported and observed water source, storage, and handling practices of an adult household member in areas targeted and not targeted for oral cholera vaccination. Solomon Islands, 2012. Table 3. Reported and observed water source, storage, and handling practices of an adult household member in areas targeted and not targeted for oral cholera vaccination. Solomon Islands, 2012. Targeted Not targeted p-value (n = 89) (n = 146) n % n % Unprotected drinking water source1 15 17% 14 10% 0.7 Unprotected non-drinking water source1 45 51% 29 20% 0.04 Primary water source ever unavailable 35 39% 80 55% 0.3 Ever treats drinking water 47 53% 17 12% 0.3 Regularly washes hands 42 47% 58 39% 0.4 Soap in household2 88 99% 143 98% 0.6 Soap at handwashing area3 21 24% 60 41% 0.1 Ocean or bush toilet facilities4 76 85% 89 61% 0.05 1 Unprotected well, river, stream, or lake 2 Reported 3 Observed 4 Reported household members use ocean or bush as usual toilet facilities doi:10.1371/journal.pntd.0004937.t003 PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004937 August 22, 2016 6 / 9 Table 3. Reported and observed water source, storage, and handling practices of an adult household member in areas targeted and not targeted for oral cholera vaccination. Solomon Islands, 2012. Targeted Not targeted p-value (n = 89) (n = 146) n % n % Unprotected drinking water source1 15 17% 14 10% 0.7 Unprotected non-drinking water source1 45 51% 29 20% 0.04 Primary water source ever unavailable 35 39% 80 55% 0.3 Ever treats drinking water 47 53% 17 12% 0.3 Regularly washes hands 42 47% 58 39% 0.4 Soap in household2 88 99% 143 98% 0.6 Soap at handwashing area3 21 24% 60 41% 0.1 Ocean or bush toilet facilities4 76 85% 89 61% 0.05 1 Unprotected well, river, stream, or lake rce, storage, and handling practices of an adult household member in areas targeted and not targeted ds 2012 Table 3. Reported and observed water source, storage, and handling practices of an adult household memb for oral cholera vaccination. Solomon Islands, 2012. rved water source, storage, and handling practices of an adult household member in areas targeted and not Solomon Islands, 2012. 6 / 9 Acknowledgments We would like to thank Loreta Bakele, Robert Fugui, Venolton Gordon, Rex Igeni, Endrie Kobala, Korina Lapo, George Lui, Moses Mata, Claudence Manetegu Pade, Maritina Penevo- lomo, Gilbert Pitua, Grace Poraiwai, Gabriel Spencer, Mahbub Talukder, and Mathias Tamou for data collection. We also thank Raymond Mauriasi, Lazarus De Neko, Cynthia Joshua, Juliet Fleischl, Chesco Nogaredo, Nick Dutta, Sara Farnbach, Kimberley Fox, Timothy Hare’e, Jeffrey Korini, Malia Rockson, Andrea Tora, Mathias Ronald Hevelao, and Jayaprakash Valiakolleri for their technical and logistical support; Kristin Brown for her data management support; and the people of the Solomon Islands who participated in the survey. Knowledge, Attitude, Practices in the Solomon Islands health messages provided before and during the vaccination campaign prevented more specific comparisons about diarrhea and cholera knowledge. We did find that awareness and specific knowledge was high, however we are unable to determine which specific messages and modes of communication were most effective. Improved documentation of the messaging and other cholera prevention interventions would have allowed us to assess messages for effectiveness and potentially to identify gaps that may have explained our findings. As of this writing, no suspected cholera cases have been reported from any part of Solomon Islands. Therefore, behavior change and improved infrastructure should remain a priority to effectively prevent cholera and other diarrheal disease. Future cholera prevention campaigns in previously unexposed communities should hone in on effective messages that capitalize on diarrheal knowledge. Additionally, learning from the limitations of this survey, future evalua- tions of cholera prevention messaging and vaccination campaigns could be improved by hav- ing clearly documented messages, modes of communications, and timeframes as well as a complete assessment of coverage in the target population when feasible. CDC disclaimer The findings and conclusions of this report are those of the authors and do not necessarily rep- resent the official position of the US Centers for Disease Control and Prevention. Conceived and designed the study: TD DO JG AH VD KD EM TH KW CY GJ. Performed the study: TD DO JG AH CY GJ. Analyzed the data: EB KW CY. Contributed reagents/materials/analysis tools: EB KW CY. Wrote the paper: EB AH VD KD EM TH KW CY. Manuscript review: TD DO JG GJ. Discussion Our finding also suggests an opportunity to improve future cholera awareness campaigns by capitalizing on previous familiarity with diarrheal disease prevention and treatment. This survey had several limitations. Most importantly, the unvaccinated group may not have been comparable to the vaccinated group, as evidenced by the higher proportion of house- holds in vaccinated areas reporting diarrhea in the previous week; this may represent differ- ences in socio-economic status as well as more familiarity with diarrhea and long-term exposure to WASH messages. Interviewers were aware which areas were targeted for vaccina- tion, which may have introduced additional bias during data collection. Additionally, the small size and convenience sampling in a cross-sectional survey limits the generalizability of these findings and limits the precision as well as the power to detect differences among the popula- tion groups. Also, the exclusion of 46 households that had participated in an earlier KAP sur- vey may have biased results. However, a sub-analysis comparing excluded households with included households found no major differences in socioeconomic status or in cholera aware- ness with other households in the same province. Finally, the limited documentation of specific 7 / 9 PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004937 August 22, 2016 Author Contributions Conceived and designed the study: TD DO JG AH VD KD EM TH KW CY GJ. PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004937 August 22, 2016 5. Wahed Tasnuva, et al. "Knowledge of, attitudes toward, and preventive practices relating to cholera and oral cholera vaccine among urban high-risk groups: findings of a cross-sectional study in Dhaka, Bangladesh." BMC public health 13.1 (2013): 242. 4. World Health Organization. Cholera vaccines: WHO position paper. Wkly Epidemiol Rec. 2010; 85:117–28. PMID: 20349546 PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004937 August 22, 2016 Knowledge, Attitude, Practices in the Solomon Islands References 1. Horwood PF, Mueller KS, Jonduo MH et al. Spatio-temporal epidemiology of the cholera outbreak in Papua New Guinea, 2009–2011. BMC Infect Dis. 2014; 14:449. doi: 10.1186/1471-2334-14-449 PMID: 25141942 2. QGIS Development Team. QGIS Geographic Information System. Open Source Geospatial Founda- tion Project. 2015. http://qgis.osgeo.org. 3. World Bank. World development indicators, Solomon Islands. The World DataBank. http://databank. worldbank.org/data/views/reports/tableview.aspx (accessed on 14 May 2014). PLOS Neglected Tropical Diseases \| DOI:10.1371/journal.pntd.0004937 August 22, 2016 8 / 9 4. World Health Organization. Cholera vaccines: WHO position paper. Wkly Epidemiol Rec. 2010; 85:117–28. PMID: 20349546 5. Wahed Tasnuva, et al. "Knowledge of, attitudes toward, and preventive practices relating to cholera and oral cholera vaccine among urban high-risk groups: findings of a cross-sectional study in Dhaka Knowledge, Attitude, Practices in the Solomon Islands 9 / 9
W3185614722.txt	https://rsglobal.pl/index.php/ijite/article/download/600/584	en		ДИВЕРСИФІКАЦІЯ МОДЕЛЕЙ ПОСЕРЕДНИЦТВА НА ГЛОБАЛЬНОМУ РИНКУ ПРОДОВОЛЬСТВА	DOAJ (DOAJ: Directory of Open Access Journals)	2,018	cc-by	4,061	International Journal of Innovative Technologies in Economy ISSN 2412-8368 ДИВЕРСИФІКАЦІЯ МОДЕЛЕЙ ПОСЕРЕДНИЦТВА НА ГЛОБАЛЬНОМУ РИНКУ ПРОДОВОЛЬСТВА Рубан Т. С. Україна, Київ, Київський національний університет імені Вадима Гетьмана, аспірант ARTICLE INFO Received 22 December 2017 Accepted 20 January 2018 Published 10 February 2018 KEYWORDS intermediation models, global food market, intermediary, classification, diversification, international trade © 2018 The Author. ABSTRACT The article researches intermediation models on the global food market using explicative method. The author proposes to diversify intermediation models on the basis of provided classification by thirteen criteria. The article proposes to divide intermediaries according to the activity field into trade, production, logistics, marketing, financial, legal, informational, administrative, and technical intermediaries. Each type has its description in the article. Particular attention is drawn to trade and marketing intermediaries. Moreover, the article groups intermediaries by the following characteristics: on behalf of whom an intermediary works; on whose account an intermediary works; type of influence on product; activity type; territory; exclusiveness; systematicness; market sector. The article provides lists of the most important intermediation models that correspond to the groups. The models can be diversified by one or several of the proposed criteria. Moreover, some models can be indifferent to a definite characteristic or appear in different groups simultaneously. Посередництво на сьогодні є основою глобальної економіки, зокрема, на ринку продовольства. Досліджуючи посередництво варто звернути увагу на різноманітність його моделей. Для потреб даного дослідження оптимальним є експлікативний метод. З огляду на це експлікація моделі являє собою можна її опис. Моделі посередництва як окремий об'єкт зі своїми характеристиками та класифікацією досі є мало дослідженими. Через це необхідно глибше дослідити та диверсифікувати моделі посередництва на глобальному ринку продовольства. Згідно загальновживаного визначення посередниками визначаються усі бізнес суб'єкти, що виникають у процесі збуту між виробником споживчих товарів (обробником) та споживачем. Міжнародна торгова палата (МТП) у регламенті про роботу з агентами, посередниками та іншими третіми сторонами зазначає, що посередники є ефективним інструментом для розбудови, розширення та ведення міжнародного бізнесу. Навіть великі компанії у сучасному глобальному світі звертаються до третіх сторін, щоб покрити всі бажані території та ринкові ніші. [1] Третіми сторонами в даному керівництві вважається широкий перелік юридичних і фізичних осіб, які діють від імені основного підприємства. До даного переліку включені в тому числі агенти, консультанти, торгові представники, митні агенти, субпідрядники, франчайзі, юристи, бухгалтери, інші посередники. [1] Варто звернути увагу, що у визначенні третіх сторін зазначено, що вони діють "від імені" основного підприємства. Це твердження суперечить переліку третіх осіб, наведених у регламенті, адже консультанти, субпідрядники, франчайзі зазвичай діють від власного імені. В даному контексті скоріше малося на увазі, що вони діють згідно домовленостей з основним підприємством та в його інтересах. У регламенті МТП з інтелектуальної власності до посередників включено, зокрема, гуртових та роздрібних торговців, перевізників, провайдерів інтернет послуг [2].У програмі дій на 2017-2018рр. МТП окремо виділяє пункт по роботі з посередниками ланцюжка постачання, перевізниками та інтернет платформами [3]. В той же час, МТП, зокрема, для боротьби з контрафактною продукцією, пропонує наступну класифікацію посередників. Усіх посередників організація поділяє на дві групи: фізичних та онлайн посередників. Фізичні посередники включають постачальників сировини та додаткових матеріалів, перевізників, орендодавців (які володіють приміщеннями, виробничими потужностями та іншими основними засобами). До онлайн посередників належать, по-перше, сайти, платформи та портали; по-друге, компанії, що забезпечують онлайн інфраструктуру; по-третє, пошукові та платіжні сервіси, провайдери та агентства інтернет реклами. До категорії сайтів, платформ та порталів належить широке коло учасників, зокрема, в дану когорту МТП включає інтернет 1(13) February 2018 9 International Journal of Innovative Technologies in Economy ISSN 2412-8368 магазини, мобільні крамниці застосунків, соціальні мережі, файлообмінники тощо. До посередників, що забезпечують онлайн інфраструктуру, МТП відносить провайдерів хостингу, доменів та доступу в інтернет. Третя група онлайн посередників забезпечує економічну спроможність функціонування інтернет послуг, зокрема, за рахунок можливості для клієнта знайти потрібну послугу та оплатити її напряму в інтернеті, можливості для компанії прорекламувати власний продукт. [4] Дана класифікація є оптимальною для дослідження МТП боротьби з контрафактною продукцією, втім, в контексті даного дослідження класифікація є неповною. Через це пропонується альтернативна класифікація посередників, заснована на проведених дослідженнях та на аналізі поточної ринкової ситуації. Класифікація передбачає первинний поділ посередників за 13 характеристиками: за сферою; іменем, від якого працює посередник; за рахунок кого; за власністю на товар; за клієнтом; за системою каналів збуту та комунікації; за наявністю впливу на товар; спрямованістю впливу на товар; проявом роботи посередника; територіальністю; ексклюзивністю; систематичністю, галуззю. В рамках найважливіших для дослідження типів також пропонується подальша класифікація. За сферою діяльності самих посередників пропоную виділити торговельних, виробничих, логістичних, маркетингових, фінансових, юридичних, інформаційних, адміністративних, технічних посередників. Даний перелік можна продовжувати, втім наведені є головними. Логістичні посередники зазвичай забезпечують транспортування товарів та їх зберігання. Насправді на сьогодні їх функції часто спеціалізовані. Певні логістичні посередники займаються перевезеннями по визначеним регіонам певними видами транспорту, інші здійснюють логістику безпосередньо в країні фінального продажу товару. Крім того, окремо зазвичай виділяють компанії, що мають великі складські приміщення, елеватори для тривалого зберігання продукції в специфічних умовах, що дозволяють забезпечити безперебійне постачання певних видів продовольчих товарів, наприклад, яблук, протягом всього року, не зважаючи на сезон врожаю. В свою чергу фінансові посередники надають фінансування (кредити, позики, позички, лізинг, іпотеки тощо), страхують ризики, зменшення зменшують дебіторська заборгованість (факторингу, форфейтинг), обслуговують платежі в тому числі міжнародні розрахунки (інкасо, векселі тощо). Серед фінансових посередників існує значна різноманітність моделей, втім слід виділити основні такі, як: банки, страхові компанії, інвестиційні фонди. Функцію врегулювання відповідності між діяльністю суб'єктів господарювання та законодавством певної країни беруть на себе юридичні посередники. Допомагають у вирішенні спорів між учасниками господарського процесу. Слід зазначити, що з поглибленням глобалізації юридичні посередники все більше задіяні у господарських процесах через необхідність врахування особливостей законодавства різних країн. До юридичних посередників належать юридичні фірми та консультанти, адвокатські контори тощо. Варто відзначити, що до технічних посередників належить цілий ряд різних за сферою діяльності суб'єктів господарювання, оскільки,в залежності від спеціалізації компаній, які вони обслуговують, це будуть різні фірми. Наприклад, до них можуть належати інженерні компанії, ІТ-компанії тощо. Дослідницькі компанії та консультанти, інформаційні бюро, бази даних зазвичай відносять до інформаційних посередників. В залежності від спеціалізації дані посередники можуть одночасно відноситись і до інших сфер, наприклад, маркетингових чи технічних тощо. Адміністративні посередники часто беруть на себе частину адміністративних задач суб’єктів господарювання, наприклад, обслуговування дзвінків, організації відряджень тощо. В рамках підгруп посередників сферою діяльності слід окремо зупинитися на торговельних. Зокрема, за обсягами продажів одного виду товару торгові посередники поділяються на гуртових та роздрібних. Щодо гуртових торгових посередників, то в першу чергу потрібно поділити їх на організовані ринкові інститути; посередників, що допомагають здійснити торговельні операції; та безпосередньо компанії, що беруть участь у торгових операціях (Рис. 1.). Так, до організованих ринкових інститутів належать біржі, ярмарки, аукціони, виставки, інтернет платформи (наприклад, alibaba.com), платформи тендерних закупівель (наприклад, prozorro.gov.ua). До посередників, що допомагають здійснити торговельні операції, але безпосередньо не беруть участі у них належать брокери; маклери; комерційні консультанти, експерти та аналітики; агенти; випадкові посередники (у тлумаченні одного з типових договорів МТП). 10 1(13) February 2018 International Journal of Innovative Technologies in Economy ISSN 2412-8368 Рис. 1. Типи та моделі гуртових торгових посередників. Джерело: розробка автора Варто зупинитися детальніше на агентах та випадкових посередниках. Так, агент сприяє здійсненню угоди купівлі-продажу, але сам у ній (як сторона контракту) не бере участі і не купує за свій рахунок товари, не отримує власність на товари, не має права розпоряджатися товаром, який продає. Цікаво, що у чинному Господарському Кодексі України (глава 31) комерційне посередництво зведено винятково до агентської діяльності, яка полягає у наданні комерційним агентом послуг суб'єктам господарювання при здійсненні ними господарської діяльності шляхом посередництва від імені, в інтересах, під контролем і за рахунок суб'єкта, якого він представляє. За агентським договором одна сторона (комерційний агент) зобов'язується надати другій стороні (суб'єкту, якого представляє агент) послуги щодо укладення угод чи сприяння в їх укладенні від імені цього суб'єкта і за його рахунок. В свою чергу МТП виділяє шість типових договорів торгового посередництва: міжнародної купівлі-продажу готових виробів, призначених для перепродажу; міжнародної купівлі-продажу як такої; дистриб'юторський контракт; договір франчайзингу; агентський договір; договір випадкового посередництва. [5] Випадкове посередництво - це разове надання посередницьких послуг, тобто вчинення фактичних дій, спрямованих на підготовку і укладення замовником однієї чи декількох угод з одним чи декількома контрагентами. До їх числа відносяться надання замовнику доступу до інформації про потенційних контрагентів; пошук (збір) такої інформації; взаємодія (контакт) з підібраними кандидатами, організація і проведення з ними зустрічей, переговорів і вчинення інших дій, спрямованих на підготовку однієї або декількох угод (ознайомлення з товаром, виробничими потужностями, технологією виробництва і зберігання, організація і проведення огляду і випробувань товару і т.п.). [5] Слід зауважити, що гуртові посередники, що безпосередньо беруть участь у торгових операціях включають широкий перелік моделей, основні з яких: дистриб'ютор, дилер, комісіонер, консигнатор, джобер, торговий дім. Дистриб’ютори займаються продажом товарів від власного імені та за власний кошт. Вони самостійно відповідають за всі види ризиків, пов’язаних із псуванням чи втратою товарів, а також з неплатоспроможністю покупця. (6) Дистриб’ютор має тісні зв’язки з виробником, зокрема: здійснює просування та організацію збуту на закріпленій за ним території; виробник втрачає привілейовані позиції на території дистриб’ютора, якому часто надається виключне право збуту; відносини встановлюються на узгоджений період; збут виробів супроводжується обмеженням дистриб'ютора у збуті товарів конкурентів. Зазвичай дистриб'ютори акумулюють портфель споживчих товарів та продають їх або дають на реалізацію роздрібним продавцям. Роль дистриб'юторів полягає у забезпеченні домовленостей з продавцями (наприклад, заведення асортименту в мережу, узгодження цін на послуги мережі (мерчандайзинг, маркетинг тощо), зміна цін на продукцію, узгодження умов поставок та ін.), зменшенні відстрочення платежів від продавців обробникам. Зазвичай великі 1(13) February 2018 11 International Journal of Innovative Technologies in Economy ISSN 2412-8368 мережі супермаркетів, які фактично на даному етапі контролюють основну частину роздрібної збутової мережі, вимагають умови довгого періоду після оплати за продукцію, наприклад, стандартний строк для України - 3 місяці, для ЄС - півроку. Дистриб'ютори беруть на себе дебіторську заборгованість мереж, а обробникові повертають гроші раніше. Часто дистриб'ютори забезпечують транспортну та складську логістики, займаються розширенням покриття продаж товару, забезпечуючи певний рівень нумеричної (кількісної) та зваженої (якісної) дистрибуції. На відміну від дистриб'юторів, дилери є дрібними оптовими покупцями, їх обов'язки і права схожі з правами і обов'язками дистриб'юторів. Найчастіше дилери для охоплення інших регіонів і сегментів ринку знаходяться в безпосередньому підпорядкуванні дистриб'юторів. При збільшенні обсягів реалізації дилер може отримати статус дистриб'ютора і навпаки. Зі свого боку комісіонери отримують право пошуку партнерів і підписання з ними контракту від свого імені, але за рахунок продавця чи покупця (комітента), який бере на себе комерційні ризики. Перед третіми особами комісіонери виступають як продавці та покупці. Вони відповідають за збереження товарів комітентів, що знаходяться в їх розпорядженні, зберігають право власності на ці товари до переходу його на покупців. [6] В свою чергу, консигнатор бере на реалізацію протягом певного терміну товари консигнанта та здійснює платежі консигнанту в міру реалізації товару зі складу. За умови консигнації експортер кредитує посередника на середній термін реалізації товару. Розрізняють наступні способи консигнації, а саме: незворотну, частково зворотну або зворотну. Незворотна консигнація означає, що, якщо якась частина товару, зазначена в договорі консигнації, не буде реалізована, консигнатор бере на себе обов’язок купити її в консигнанта за тверду суму. Частково зворотна консигнація означає, що консигнатор повинен реалізувати товар на певну частину суми, а товари на залишок суми, якщо їх не вдається реалізувати, повернути консигнанту. Зворотна консигнація означає, що всі нереалізовані товари треба повернути консигнанту або подовжити термін консигнації чи дати на ці товари знижку. [6] Посередник, що діє як посередник на біржі та займається скупкою окремих великих партій товару для швидкого перепродажу називається джобером. В свою чергу торговий дім — торговельна фірма, яка закуповує товар у виробників або гуртовиків своєї країни і перепродає їх за кордон; також закуповує іноземні товари за кордоном та перепродає їх місцевим гуртовим і роздрібним торговцям та споживачам, здійснюють операції за свій рахунок і за дорученням клієнтів. Торгові доми працюють з широкою номенклатурою товарів, беруть участь у виробництві продукції. Для виконання своїх функцій у торгових домів часто існує власна мережа магазинів, складські приміщення, транспортні засоби тощо. Діяльність торгового дому характеризується широким діапазоном: здійснення від свого імені та переважно за свій рахунок експортно-імпортних, товарообмінних (бартерних) та інших зовнішньоекономічних операцій; інвестування коштів у виробництво; здавання обладнання в лізинг; надання кредитів; надання послуг у сфері страхування, ремонту і обслуговування, складування, консультацій, інжинірингу, фінансів; торговий дім бере участь у торгах на біржах та в створенні спільних підприємств за участю іноземного капіталу. [7] Роздрібні посередники можуть бути різних типів, яким відповідають певні моделі (Рис.2). З метою даного дослідження пропонується визначити роздрібними продавцями суб'єктів процесу створення продовольчих продуктів, які займаються продажем споживчих продуктів кінцевим покупцям, не залежно від каналу збуту (будь то супермаркет, заклад громадського харчування чи інтернет-магазин або автомат з продуктами). В залежності від спеціалізації та позиціонування роздрібного продавця визначається розташування, асортимент, цінова політика тощо. Так, "магазини біля дому" або автомати з їжею розташовуються якнайближче та якнайзручніше до покупців, щоб задовольнити їх миттєві потреби. Дискаунтери - магазини, що пропонують низькі ціни - зазвичай є великими гіпермаркетами, розташованими на околицях міста або за містом. Фізичні магазини мають обмеження по наявному торговому місцю. Найпоширенішим типом роздрібних посередників є "сучасний роздріб", основною характеристикою якого є самообслуговування в точках продажу. До нього входять такі моделі посередників як супермаркети, гіпермаркети, мінімаркети. "Традиційний роздріб" або його також називають "лінійним роздрібом" характеризується невеликим розміром точки продажу та обслуговуванням продавцем через прилавок. Серед моделей традиційного роздрібу варто виділити магазини біля дому та кіоски. 12 1(13) February 2018 International Journal of Innovative Technologies in Economy ISSN 2412-8368 Рис. 2. Типи та моделі роздрібних торгових посередників. Джерело: розробка автора У свою чергу, франчайзингові торгові точки (фтт) характеризуються організацією роботи за договором франчайзингу, що найчастіше передбачаєрегламентовану назву магазину, асортимент, постачальників, інтер'єр та екстер'єр. Фтт можуть мати різні моделі, найпоширенішими серед яких є магазини біля дому, мінімаркети та кіоски. Найчастіше франчайзингову мережу магазинів будує виробник певного товару в якості каналу збуту для нього (наприклад, франчайзингові точки "Наша Ряба" ПАТ Миронівський Хлібопродукт). Слід зазначити, що канал збуту ХоРеКа включає заклади громадського харчування та проживання, серед яких основними виділяють готелі, кафе, ресторани, бари. В рамках автозаправних комплексів виділяють мінімаркети, супермаркети та кафе. Серед он-лайн посередників варто виділити наступні моделі: інтернет магазини, доставка їжі, доставка з супермаркетів, інтернет платформи, що об'єднують багатьох продавців (наприклад, e-bay, amazon, aliexpress).На сьогодні досить популярний формат інтернет продажу продовольчих товарів. Це може бути як готова їжа на замовлення (піца, суші тощо), так і збір замовлення в супермаркеті на доставку або навіть продаж продуктів довгострокового зберігання через глобальні платформи, такі, як e-bay. У випадку он-лайн торгівлі, продавці обмежені наявними технологіями, можливостями та строками доставки та оплати. Зміна ринку вимагає відповідної зміни форматів. Так, у 2000 р. мережа Kmart була третьою найбільшою мережею супермаркетів у США з оборотом 36 млрд. дол. США [8]. До 2014 р. її доходи впали на дві треті. За аналогічний період річний оборот інтернет магазину Amazon виріс з 2,8 млрд дол. США до 89 млрд. дол. США [ (8)]. Інтернет платформа для бізнесу Alibaba, лідер ринку Китаю, будучи всього 15-річною компанією, у 2014 р. здійснив рекордне за обсягами у світі IPO, оцінене у 25 млрд. дол. США. [8] Існують також посередники, що будують збут через мережу представників "мережевого маркетингу" (наприклад, Herbalife). До них належать такі моделі посередників як агенти, комівояжери тощо. Крім зазначених вище моделей посередників, існують і ті, яких важко об'єднати у певні типи. Серед них автомати (наприклад, автомати зі снеками чи напоями); спеціалізовані магазини (наприклад, спортивного харчування) тощо; аптеки; дрогері магазини (наприклад, "Ватсонс", "Ева", "Космо"). Відносно новою моделлю роздрібної торгівлі є "магазини в магазинах" (shopinshop) - це "острівки" в ТРЦ або супермаркетах чи гіпермаркетах, що продають окремі товари. Моделіроздрібної торгівлі постійно еволюціонують у відповідь на запити покупців. За системою каналів збуту та комунікації можна виділити посередників, що працюють онлайн, оффлай (мають фізичні представництва, магазини), мультиканальні (мають кілька каналів збуту та комунікації онлайн та оффлайн, втім клієнтський досвід у них розрізнений різні ціни, асортимент, умови доставки та ін.); омніканальні (мають онлайн та оффлайн каналів збуту та комунікації з уніфікованим клієнтським досвідом). На сьогодні тенденція у роздрібних торговців до переходу на омніканальну систему. Втім, також значно зростають онлайн 1(13) February 2018 13 International Journal of Innovative Technologies in Economy ISSN 2412-8368 посередники, адже онлайн потребує менше інвестицій, ніж оффлайн, а для омніканальності необхідно мати фізичні точки продажу на рівні з онлайн магазином. Окрему увагу варто приділити зміщенню посередництва в бік виробництва. Зі зростанням спеціалізації все більше посередників залучені у процес створення продукту. Продовольчі товари стали технологічно складними через підвищені вимоги до строків придатності, постійні намагання покращити смакові та харчові властивості продуктів. Через це їх створюють компанії з різними компетенціями, технологічним оснащенням, на різних територіях. Найчастіше стадія фінальної обробки продукту відбувається найближче до покупця. Це може бути фасування продукту в країні фінального продажу або навіть кастомізація продукту безпосередньо в точці продажу під конкретного покупця. Цікаво, що часто роздрібні торгові точки беруть на себе також додаткову виробничу функцію по фінальній передпродажній підготовці продукту. За даних умов важко сказати, яка з компаній є виробником. Фактично кожна компанія, що знаходиться між виробником сировини та фінальним споживачем являється посередником. Посередники, що задіяні у виробничій трансформації продукту називаються виробничими посередниками. Не можна стверджувати, що одні з них є виробниками, а інші постачальниками, аналізуючи увесь ланцюжок створення вартості. Втім, якщо аналізувати його частину, то для певної виробничої компанії вона сама буде виробником, компанія, в якої дана компанія закуповує напівфабрикати буде постачальником, а компанія, якій буде відбуватися збут - клієнтом. Отже розмивається поняття виробника. Воно залежить від частини процесу, який аналізується та точки, з якої проводиться аналіз. Все більшого значення набувають маркетингові посередники. Вони допомагають правильно оцінити потреби ринку та правильно розподілити ресурси, направити їх у найбільш затребувані продукти. Крім того, маркетингові посередники допомагають збільшити нематеріальну складову корисності продукту. В результаті збільшується загальна корисність продукту за рахунок емоційного задоволення споживача. До маркетингових посередників можна віднести дослідницькі компаній, консалтингові, креативні агентства, дизайн бюро, брендингові агентства, медійні агенства, селз хауси, студії виробництва контенту (Юніверсал, Дісней тощо), агентства ліцензійного брендингу, btl-агентства, digital-агентства, мерчендайзингові агентства тощо. Дослідницькі та консалтингові компанії, допомагають краще зрозуміти споживачів. Креативні агентства,дизайн бюро, брендингові агентства, студії виробництва контенту, агентства ліцензійного брендингу тощо збільшують емоційну складову навколо продукту. Медійні агенства, селз хауси, digital-агентства та ін. сприяють донесенні інформації про продукт до споживача та покупця. Це може бути звичайне інформування про характеристики та місця збуту, ціну продукту, а може бути емоційне повідомлення, що, в свою чергу, також збільшує емоційну складову корисності продукту. Btl-агентства сприяють прямій комунікації зі споживачем чи покупцем. Дозволяють їм ознайомитись з продуктом (наприклад, через організацію дегустацій), поринути в емоційний світ бренду продукту (наприклад, на певному тематичному заході від бренду). Мерчандайзингові агентства допомагають розмістити продукти в торговій точці найбільш вигідним способом, забезпечують постійну присутність продукту на полиці та рекламних матеріалів в точці продажу тощо. Якщо розглядати ім’я, від якого діють посередники, їх можна поділити на тих, що працюють від власного імені (наприклад, комісіонери, агенти) та на тих, що діють від імені замовника (дистриб'ютори, дослідницькі та рекламні агентства тощо). Варто зазначити, що серед усіх моделей посередництва переважають ті посередники, що діють від власного імені. Враховуючи, за чий рахунок діють посередники, можна виділити ті моделі посередників, що передбачають роботу за власний рахунок (наприклад, торгові доми, переробники, медійні агенства тощо) та такі, що працюють за рахунок доручителя (агенти, повірені, комісіонери тощо). Зважаючи на власність на товар можна виділити посередників, що забирають товар у власність (наприклад, переробники, супермаркети тощо) та ті, що не отримують власність на товар. В останньому випадку це можуть бути торгові компанії, здійснюють функції товароруху без права власності на товар і одержують комісійну винагороду (комісіонери, агенти тощо) та допоміжні посередники (банки, маркетингові агентства, IT-компанії тощо). Беручи до уваги вплив на товар слід виокремити посередників, що змінюють споживчу цінність товару та тих, що не впливають на неї. Споживчу вартість змінюють, в першу чергу, переробники, адже вони змінюють сам продукт. Впивають на споживчу вартість і дизайн 14 1(13) February 2018 International Journal of Innovative Technologies in Economy ISSN 2412-8368 агентства, креативні агентства тощо, адже вони сприяють росту емоційної корисності товару. Крім того, логістичні компанії та роздрібні торговці забезпечують територіальну доступність та зручність, що також збільшує сукупну цінність товару. Не впливають на цінність товару страхові компанії, кредитори, технічні та адміністративні посередники тощо. Якщо врахувати спрямованість впливу на товар посередників можна поділити на тих, що спричиняють фізичну зміну товару (наприклад, переробники змінюють його склад, форму, колір тощо; в супермаркетах товар можуть запакували; у кафе - прикрасити та сервірування; логістичні посередники товар фізично перемістили з одного місця в інше), та на тих, що здійснюють інтелектуальний вплив (дослідницькі компанії, консультанти, креативні та дизайн агентства, технологічні консультанти тощо). Інтелектуальний вплив може спричинити подальший фізичний вплив або впливати на емоційну корисність продукту. Звертаючи увагу на прояв роботи посередників можна поділити на таких, що здійснюють фізичну, інтелектуальну та формальну роботу. Фізична робота пов'язана зі зміною матеріальних складових продукт та його переміщенні в просторі. Фізично на продукт впливають переробники, логістичні посередники тощо. Інтелектуальний прояв має робота дослідницьких агентств, консультантів, креативних та дизайн агентств тощо. Формальний прояв роботи означає, що дані операції покликані для відповідності процесу певним загально визначеним правилам та не несуть впливу на корисність продукту. До посередників формального прояву належать страхувальники, брокери, маклери тощо. Враховуючи специфіку клієнтів більшість посередників відносяться до В2В сектору тобто обслуговують інші компанії. До них належать усі агентства, дилери, дистриб'ютори, переробники тощо. З кінцевим покупцем (В2С) працюють лише роздрібні торговці (супермаркети, кафе, інтернет магазини готових продуктів тощо). За територіальним принципом можна поділити посередників на національних, іноземних, міжнародних та глобальних. За ексклюзивністю варто виокремити посередників, що мають ексклюзивні права та на тих, що ведуть діяльність на загальних основах. Так, наприклад, ексклюзивні права на продаж продукту часто передають дилеру чи дистрибюторові на певну територію. Зважаючи на систематичність співпраці варто виділити регулярних посередників тобто тих, що тривалий час постійно працюють з одними компаніями,забезпечуючи виконання певної функції господарського процесу; та випадкових, з якими співпраця носить епізодичний характер. Найчастіше регулярними є торгові та логістичні посередники (дистриб'ютори, дилери, супермаркети тощо), а випадковими - консультанти, агентства та ін. підприємства, що виконують проектну роботу. Беручи до уваги галузеву приналежність діяльності посередників глобального продовольчого ринку можна також класифікувати. Так, основними галузями будуть продовольча промисловість (виробники сировини та переробники), послуги (в основному торгові, фінансові, маркетингові, консультаційні), наукова галузь (наукові розробки у всіх сферах, наприклад, у виробників сировини). Отже, моделей посередництва велика різноманітність. Їх можна диверсифікувати на основі наведеної класифікації за одним або декількома з тринадцяти основних напрямів таких, як за сфера; ім'я, від якого працює посередник; за рахунок кого; власність на товар; клієнт; система каналів збуту та комунікації; наявність впливу на товар; спрямованість впливу на товар; прояв роботи посередника; територіальність; ексклюзивність; систематичність; галузь. Модель посередництва відноситься за даними напрямами до певного типу посередників. В рамках деяких типів також виділено підтипи та наведено моделі, які їм відповідають. Не зважаючи на те, що моделі посередництва диверсифікуються за наведеними характеристиками, деякі з них можуть бути нейтральними до певної характеристики і тому бути присутніми у декількох групах одночасно. ЛІТЕРАТУРА 1. 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W3030639362.txt	https://bmcmedgenet.biomedcentral.com/track/pdf/10.1186/s12881-020-01057-3	en		Comprehensive analysis on phenotype and genetic basis of Chinese Fanconi anemia patients: dismal outcomes call for nationwide studies	BMC medical genetics	2,020	cc-by	6,203	Nie et al. BMC Medical Genetics (2020) 21:118 https://doi.org/10.1186/s12881-020-01057-3 RESEARCH ARTICLE Open Access Comprehensive analysis on phenotype and genetic basis of Chinese Fanconi anemia patients: dismal outcomes call for nationwide studies Daijing Nie1,2, Jing Zhang1, Fang Wang1, Wei Zhang1, Lili Liu1, Xue Chen1, Yang Zhang1, Panxiang Cao1, Min Xiong3, Tong Wang1, Ping Wu1, Xiaoli Ma1, Wenjun Tian4, Mangju Wang5, Kylan N. Chen2 and Hongxing Liu1,2,6* Abstract Background: Fanconi anemia (FA) is the most common inherited bone marrow failure (BMF) syndrome with 22 related genes identified. The ALDH2 rs671variant has been proved related to accelerate the progression of BMF in FA patients. The phenotype and genetic basis of Chinese FA patients have not been investigated yet. Methods: We analyzed the 22 FA-related genes of 63 BMF patients suspected to be FA. Clinical manifestations, morphological and cytogenetic feathers, ALDH2 genotypes, treatment, and outcomes of the definite cases were retrospectively studied. Results: A total of 21 patients were confirmed the diagnosis of FA with the median age of BMF onset was 4-yearold. The number of patients manifested as congenital malformations and growth retardation were 20/21 and 14/21, respectively. BM dysplasia and cytogenetic abnormalities were found in 13/20 and 8/19 patients. All the patients with abnormal karyotypes also manifested as BM dysplasia or had evident blasts. Thirty-five different mutations were identified involving six genes and including twenty novel mutations. FANCA mutations contributed to 66.67% of cases. Eight patients harboring ALDH2-G/A genotype have a significantly younger age of BMF onset (p = 0.025). Within the 19 patients adhering to continuous follow-up, 15 patients underwent hematopoietic stem cell transplantations (HSCTs). During the 29 months of follow-up, 8/19 patients died, seven of which were HSCT-related, and one patient who did not receive HSCT died from severe infection. Conclusions: The phenotypic and genetic spectrum of Chinese FA patients is broad. Bone marrow dysplasia and cytogenetic abnormalities are prevalent and highly consistent. The overall outcome of HSCTs is disappointing. Nationwide multicenter studies are needed for the rarity and adverse outcome of this disease. Keywords: Fanconi anemia, Bone marrow failure, Aldehyde dehydrogenase, Hematopoietic stem cell transplantation * Correspondence: starliu@pku.edu.cn 1 Division of Pathology & Laboratory Medicine, Hebei Yanda Lu Daopei Hospital, 6 Sipulan Road, Langfang 065201, China 2 Beijing Lu Daopei Institute of Hematology, Beijing 100176, China Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Nie et al. BMC Medical Genetics (2020) 21:118 Background Fanconi anemia (FA) is a rare genetic disease highly heterogeneous in clinical manifestations and genetics. Clinical features primarily include congenital malformations, progressive bone marrow failure (BMF), and predisposition to hematopoietic and solid malignancies [1, 2]. The most common congenital abnormalities include skin pigmentation, café au lait spots, short stature, and hypoplastic of radii and/or thumbs [2]. The time of BMF onset is variable but usually at pre-school age, with the cumulative incidence of 90% by the age of 40 [3]. The malignancy risk in FA patients is mounting, especially the risks of myelodysplastic syndrome/acute myeloid leukemia (MDS/AML), which are several hundred folds higher than those of the general population [3–6]. Twenty-two genes have been identified related to FA (Table S1) to date. Products of the 22 genes participate in FA-BRCA pathway, which is responsible for correcting interstrand crosslinks (ICLs) and other DNA damage events induced by genotoxic agents. Endogenous aldehyde is a genotoxic agent and is detoxicated by aldehyde dehydrogenases (ALDHs) in vivo [7]. Previous studies have suggested aldehydes are highly toxic in FA deficient cells and could speed up the development of BMF and leukemia in FA deficient mice models [8–10]. The mitochondrial ALDH2 isoform is the most efficient acetaldehyde-detoxifying enzyme in humans [11]. Inactivating ALDH2 variant (rs671 c.1510G > A/p.E504L) is highly prevalent in east Asia and can abolish ALDH2 activity by a dominant-negative effect [12]. ALDH2-A/A and ALDH2-G/A genotypes have been proved related to accelerated progression of BMF and malignant transformation in FA patients [13, 14]. Although the genetic basis, pathological mechanisms, and epidemiology of FA have been extensively studied, few researches focus on Chinese patients [15]. In the present study, we report 21 Chinese FA patients aiming to depict their genetic basis and clinical characteristics. Methods Patient enrollment We retrospectively analyzed 63 BMF patients who were suspected to be inherited BMF in Hebei Yanda Lu Daopei Hospital from May 2012 to Dec. 2017. Detailed disease histories and examination files were retrieved from the electronic medical record system of our institute. BMF is considered with one or more lineages decreased in whole blood cell counts and reduced hematopoiesis with routine Wright-Giemsa staining bone marrow morphological analysis and Hematoxylin-Eosin staining pathological analysis. Categorization of hematopoietic cells, blast percentage, hematopoietic grade, dysplasia, and diagnosis of MDS were according to the 2008 edition of the World Health Organization Classification of Page 2 of 10 Tumors of Hematopoietic and Lymphoid Tissues [16]. All patients enrolled were confirmed BMF and should meet at least two following inclusive criteria: 1) growth retardation; 2) congenital physical malformations; 3) early onset of BMF (≤ 6 years old); 4) chronic onset of BMF with a progressive course (disease course > 6 months); 5) suggestive family history (consanguinity or family history of cancer or hematological disorders); 6) positive for chromosome breakage test (Supplementary methods) (Table S2). Other inherited syndromes manifested as BMF and malformations such as dyskeratosis congenita, Diamond-Blackfan anemia, and Neurofibromatosis-Noonan syndrome diagnosed based on syndromic presentations combined with genetic tests were excluded. The follow-up duration was defined as the time from referral to the last follow-up or loss of follow-up/death. Written informed consents were obtained from the patients or their statutory guardians and all tested family members in accordance with the Declaration of Helsinki. The study was approved by the ethics committee of the Hebei Yanda Lu Daopei hospital. Nucleic acid extraction Peripheral blood (PB), bone marrow (BM), or cryopreserved DNA samples of the patients and their parents were obtained. Genomic DNA was extracted from PB/ BM nucleated cells using silica gel column method. High throughput sequencing, variant calling, and ALDH2 genotyping We carried out Sanger sequencing on the entire coding exons and flank regions of FANCA, FANCC, and FANCG in patients suspected to be inherited BMF from Apr. 2012 to May 2016. Targeted high-throughput sequencing (THS) has been applied since May 2016, FANCD2 and BRCA2 were added in the panel. Whole genomic sequencing (WGS) was carried out using cryopreserved samples for the enrolled cases where the panel test could not find the pathogenic mutations, and all the 22 FA genes were analyzed. The THS process has been described previously [17]. For the WGS, libraries were constructed with NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, US), followed by sequencing on Illumina HiSeq X Ten platform (Illumina, US) using HiSeq X Ten Reagent Kit v2.5 (Illumina, US) running on pairedend 150 bp mode. Reads yielded by the two kinds of sequencing were all aligned to the human reference genome (hg19) with the Burrow-Wheeler Aligner (BWA) mem. Variants were called according to Genome Analysis Toolkit (GATK) best practices using bam files. Final confident variants were annotated using annovar and oncotator. Variants Nie et al. BMC Medical Genetics (2020) 21:118 with minimal allele frequency (MAF) ≥ 1% in general population were filtered out according to 1000 Genomes, EXAC, and gnomAD databases. The pathogenicity of the germline missense mutations was assessed by in silico prediction algorithms, and the putative causal variants were classified according to the standards and guidelines recommended by the American College of Medical Genetics and Genomics (ACMG) [18]. Only pathogenic, likely pathogenic, or uncertain significance variants were considered causative in the present study. The process of CNV analysis based on WGS has been described elsewhere [17]. ALDH2 genotyping was based on WGS data or Sanger sequencing with primers 5′- TGCTATGATGTGTT TGGAGCC-3′ (forward) and 5′-ATTTAGGGTCTCTG CTGGGCG-3′ (reverse). Validation by sanger sequencing Polymerase chain reaction (PCR) and Sanger sequencing performed on the ABI 3500xL Genetic Analyzer (Thermo Fisher, US) were adopted to confirm all the mutations reported in this study. Single nucleotide variants (SNVs) and small insertions/deletions (InDels) were validated by PCR and Sanger sequencing using pedigree’s samples when accessible. For the validation of CNVs, the breakpoints were confirmed by Sanger sequencing using patients’ DNA, and the parental origins were verified through PCR and agarose gel electrophoresis. Results Demography and clinical characteristics A total of 21 patients (six females and fifteen males) from non-related families were finally diagnosed as FA, including one who has already been reported (Case 8) [17]. The median referral age of this cohort was 7 years old, and the median age of BMF onset was 4 years old (range, 1–13 years old). There were 20 Han Chinese and one Uyghur Chinese, and the geographical distribution spread nationwide though over half of the patients came from the south or southwest of China. All patients were referred to our institute because of severe cytopenia except a thirty-year-old boy (Case 10) who was initially diagnosed as MDS for the myeloid dysplasia and increased myeloblasts indicated by BM morphology. Five patients had an indicative family history with two patients had family members died from anemia (Case 3, Case 15), two patients were from consanguineous families (Case 16, Case 21), and one patient was an in vitro fertilize baby whose paternal grandmother died from pancreatic cancer (Case 4) (Table 1). Fourteen (66.67%) patients were growth-retarded, and 20 (95.24%) patients manifested as congenital malformations. Congenital abnormalities in our cohort included Page 3 of 10 skin pigmentation (13/21), café au lait spots (5/21), spin and limbs deformation (11/21), craniofacial malformations (8/21), genitourinary system malformations (7/21), cardiovascular system defects (2/21), nervous system diseases (2/21), and endocrine system defects (2/21) (Table 1). Thoroughly evaluation of the hematologic phenotype is crucial to FA patients since BM dysplasia or pathological cytogenetics relate to disease progression and adverse hematopoietic stem cell transplantations (HSCT) outcomes [5, 14]. Twenty patients’ morphologic test results and nineteen patients’ cytogenetics test results before pre-HSCT conditioning regimen and/or chemotherapy were available. BM dysplasia was found in 13/20 (65%) patients, including one AML with the myeloblast count of 41% (Case 5) and one myelodysplasia with the blast count of 6% (Case 10). Karyotypes were described according to the International System for Human Cytogenetic Nomenclature 2013 [15]; at least 20 metaphases were analyzed for each assay. Cytogenetic abnormalities were found in 8/19 (42.11%) patients with clonality found in five patients, and half of the abnormal karyotypes involved chromosome 7 (− 7, 7q-, or der(7)t(1;7)) (Table 2). The cytogenetic result of Case 5 who was diagnosed as AML was 46, XX, der(7)t(1; 7)(q21;q36) [19], which was confirmed to be nonconstitutional by matched peripheral blood, and the karyotype of patient Case 10 was highly complex (Table 2). All the patients with abnormal karyotypes also manifested as dysplasia on bone marrow smear or had evident blasts, suggesting the initiation of clonal evolution in hematopoietic system. Characteristics of mutations A total of 39 mutations were identified involving six different FA genes and composed of 13 missense mutations, nine large deletions, eight nonsense mutations, seven frameshift mutations, one splicing mutations, and one deep intron mutation (Fig. 1, Table 3). All the large deletions were found within the FANCA gene. 20 (47.73%) mutations identified in our cohort were novel and the majority of mutations were private except FANCA c.367C > T, which was shared by two patients. (Fig. 2, Table 3). We did not find FANCA c.2546delC in our cohort, which accounts for over 30% FANCA mutations in Japanese and Korean patients [22, 24]. Among the 21 patients,15 patients carried compound heterozygous mutations, three patients carried homozygous mutations, two patients harbored hemizygous FANCB mutations, and one patient with a heterozygous FANCE mutation were identified. Biallelic FANCA mutations caused 61.90% (13/21) of the cases, followed by monoallelic FANCB mutations and FANCD2 mutations, which both constituted 9.52% (2/21) of the cases; and Nie et al. BMC Medical Genetics (2020) 21:118 Page 4 of 10 Table 1 Clinical features Case No. Gender Age of referral (years) Age of clinical BMF onset (years) Congenital malformations Growth retardation Family history 1 M 12 2 S, C, M Yes Negative 2 M 7 2 S, C, G Yes Negative 3 M 10 5 S, G Yes One sibling manifested as polydactyly and died from anemia 4 M 11 5 C, G, M, Yes One sibling manifested as polydactyly and died from anemia IVF and paternal grandmother died from pancreatic cancer 5 F 11 10 C, H Yes IVF and paternal grandmother died from pancreatic cancer Negative 6 M 7 5 S, M No Negative 7 M 17 10 S, M No Negative 8 M 7 7 S, C, M, H No Negative 9 M 5 4 S, M No Negative 10 M 13 13 E Yes Negative 11 M 9 6 S Yes Negative 12 F 7 7 M Yes Negative 13 M 7 1 S, M Yes Negative 14 M 5 2 S No Negative 15 M 14 5 C, G Yes Two family members died from anemia 16a M 6 4 S, C, M Yes 2nd degree consanguinity 17 M 4 3 None No Negative 18 F 9 4 S, M No Negative 19 F 7 4 S, G, N Yes Negative 20 F 6 3 S, C, G Yes Negative 21 F 6 4 S, M, H, E, G Yes 2nd degree consanguinity Skin and annex abnormalities include skin pigmentation, café au lait spots, excess hair; craniofacial anomalies include microcephalus, ptosis, hypertelorism, hypotelorism, flat nose bridge; malformations in musculoskeletal system include polydactyly, deformity of thumbs, absence of thumbs, hypoplasia of thenar eminence, and scoliosis; genitourinary system malformations include kidney malformation, hydronephrosis, indirect inguinal hernia, cryptorchidism, ovary absence, and uterine malformation/absence; cardiovascular system defects include patent ductus arteriosus and ventricular septal defect; nervous system abnormalities include encephalatrophy and moyamoya disease; endocrine system defects include hypothyroidism, primary adrenocortical insufficiency, and obesity F female, M male, S skin and annex, C craniofacial anomalies, M musculoskeletal system, G genitourinary system, H cardiovascular system, E endocrine system, N nervous system, IVF in vitro fertilized a Case 16 is of Chinese Uyghur ancestry FANCC, FANCE, and ERCC4 mutations caused one case each (Table 3). We did not find any case attributed to FANCG mutations, which is the second most prevalent responsible gene in East Asian according to Japanese and Korean studies [22, 24]. Despite the limited size of this cohort, we identified two FANCB mutations, making it rank one of the most common causative genes in line with the Japanese study [22]. There were three homozygous mutations, FANCA c.1867C > T, FANCC c.545C > A, and ERCC4 c.257G > A; the latter two mutations were carried by patients both came from consanguineous families, and the FANCC c.545C > A was carried by the only Uyghur patient in our cohort. Rigorous criteria were adopted in the process of criminal variant identification (Table 3, Table S3). Majority of the patients were assigned with compelling mutations with two exceptions. All mutations were classified as pathogenic or likely pathogenic according to the guideline of ACMG. Case 19 carried compound heterozygous FANCE c.1111C > T mutation and FANCE c.1317-237C > G mutation. The c.1111C > T mutation was considered pathogenic, but the c.1317-237C > G mutation is an intron variant and classified as uncertain significance, therefore it was excluded in statistics. ADLH2 rs671 genotype 12/21 (58.33%) patients in our cohort carried ALDH2G/A genotype, and the other patients were all ALDH2-G/G genotype. There was no ALDH2-A/A Nie et al. BMC Medical Genetics (2020) 21:118 Page 5 of 10 Table 2 Bone marrow morphology, karyotype, chromosome breakage tests, and ALDH2 genotypes Case No. BM morphology BM karyotype Chromosome breakage test ALDH2 genotype 1 Dysplasia NA Positive G/A 2 Dysplasia 47,XY,+ 15[1]/46,XY[20] Positive G/A 3 Hypoplasia normal Positive G/A 4 NA NA Positive G/G G/G 5 a AML 46,XX,der(7)t(1;7)(q21;q36)[20] Positive 6 Hypoplasia Normal Positive G/A 7 Dysplasia 46,XY,-7,+ 21[5]/46,XY[16] Positive G/G 8 Hypoplasia Normal Positive G/G 9 Hypoplasia Normal Positive G/A 10b MDS Complex Positive G/G 11 Hypoplasia Normal Positive G/A 12 Dysplasia Normal Positive G/G 13 Dysplasia Normal Positive G/G 14 Dysplasia Normal Positive G/A 15 Dysplasia 46,XY,del(7)(p13)[13]/46,XY[7] Positive G/A 16 Hypoplasia Normal Positive G/A 17 Dysplasia Normal Positive G/A 18 Dysplasia 46,XX,t(1;5)(p36.1;q13)[1]/46,XX[19] Positive G/G 19 Dysplasia 46,XX,del(14)(q24)[1]/46,XX[20] Positive G/G 20 Dysplasia 46,XX,del(7)(q22)[8]/46,XX,del(5)(p11)/46,XX[19] Positive G/A 21 Hypoplasia Normal Positive G/A Chromosome breakage tests were induced by mitomycin C NA not available, AML acute myeloid leukemia, MDS myelodysplastic syndrome a Case 5 is diagnosed as acute myeloid leukemia. The myeloblasts count 41% of the nucleated cells according to morphologic test of bone marrow smears b Case 10 is diagnosed as myelodysplastic syndrome. His bone marrow morphology shows dysplasia was observed in his granulocytic lineage and megakaryocytic lineage with the myeloblasts count 6% of the nucleated cells. The result of his karyotype is:46,XY,dup(1)(q21q23),add(2)(p11.2),add(3)(q27),der(5)t(1;5)(q21;q35),add(20)(p12)[17]/45,XY,der(1)(?::1q42- > 1q21::1p36.3- > 1q32::1q21- > 1q44::?),add(2)(p11.2),add(3)(q27),add(4)(p16),der(5)t(1;5)(q21;q35),-18,add(20)(p12),ace [2]/46,XY [1] Fig. 1 Distribution and composition of the 39 mutations. a. mutation distribution. b. mutation composition Nie et al. BMC Medical Genetics (2020) 21:118 Page 6 of 10 Table 3 Mutation details Case No. Gene 1 Mutation 1 (maternal) Mutation 2 (paternal) Genomic location cDNA/Protein Ref./ Com. Genomic location cDNA/Protein Ref./ Com. FANCA chr16:89833593 c.2557C > T/p.R853X [20] chr16:89877396 c.367C > T/p.Q123X NA 2 FANCA chr16:89815145–89, 815,146 c.3270_3271delCT/ p.C1090RfsX25 Novel chr16:89868906–89, 875,410 c.792 + 761_c.523-635del Novel 3 FANCA chr16:89877396 c.367C > T/p.Q123X NA chr16:89818822 c.2982-192A > G [21] 4a FANCA chr16:89842183 c.1867C > T/p.Q623X Novel chr16:89842183 c.1867C > T/p.Q623X Novel 5 FANCA chr16:89804935–89, 806,139 c.3935-178_4368 + 74del Novel chr16:89819567–89, 839,134 c.c.2014 + 545_2982-937del Novel 6 FANCA chr16:89811185–89, 815,741 c.3239 + 397_3626 + 202del Novel chr16:89858887 c.1074_1075delGT/p.Y359PfsX49 NA 7 FANCA chr16:89826812–89, 919,023 FANCA c.2852 + 1545_SPIRE2 c.646-1671del Novel chr16:89825071 c.2894_2895delCT/p.P965RfsX9 Novel 8 FANCA chr16:89780001–89, 822,000 VPS9D1 c.432-877_FANCA c.2981 + 2985del [16] chr16: 89808940–89, 809,954 c.3627-607_3765 + 268del [16] 9 FANCA chr16:89823177–89, 825,446 c.2853-333_2981 + 1808del Novel chr16:89809270 c.3703C > T/p.Q1235X Novel 10 FANCA chr16:89818619 c.2990_2993delGTTA/ p.S997MfsX28 NA chr16:89862229 c.987_990delTCAC/p.H330AfsX4 [22, 23] 11 FANCA chr16:89816286 c.3091C > T/p.Q1031X NA chr16:89792569–89, 821,767 ZNF276 c.1007-1118_FANCA c.2982-3137del Novel 12 FANCA chr16:89806417 c.3918dupT/p.Q1307SfsX6 [24] chr16:89831438 c.2638C > G/p.R880G NA 13 FANCA chr16:89858941 c.1021C > T/p.Q341X Novel chr16:89811412 c.3581C > T/p.P1194L [24] 14 FANCB chrX:14868651 c.1472 T > A/p.V491E Novel – – – 15 FANCB chrX:14877390 c.1018C > A/p.Q340K Novel – – – 16a FANCC chr9:97912346 c.545C > A/p.S182Y Novel chr9:97912346 c.545C > A/p.S182Y Novel 17 FANCD2 chr3:10084828 c.983G > A/p.R328Q NA chr3:10114634 c.2574 T > G/p.I858M Novel 18 FANCD2 chr3:10132005 c.3713 T > A/p.M1238K NA chr3:10089599 c.1279-2A > T Novel 19 FANCE – – NA chr6:35426215 c.1111C > T/p.R371W [21, 22, 25] 20 FANCE chr6:35423547 c.272C > T/p.P91L Novel chr6:35427467–35, 427,470 c.1246_1249delCAAA/p.T417SfsX7 NA 21a ERCC4 chr16:14015937 c.257G > A/p.R86H NA chr16:14015937 c.257G > A/p.R86H NA NA not available a Case 4, Case 16, and Case 21 carry homozygous variants genotype identified (Table 2). The age of BMF onset of ALDH2-G/A patients was significantly younger than that of the ALDH2-G/G patients (p = 0.025, ttest). Treatment and outcome Within the 21 patients, continuous medical records of 19 patients can be retrieved except Case 8 and Case 21, who only came to us once and were excluded in this section. All the 21 patients were eligible for HSCT for they were all transfusion-dependent, and HSCT was performed on 15 patients (71.43%). The numbers of patients accepted HSCT from HLA-matched unrelated donors (MUD), HLA-unmatched unrelated donors (UUD), HLA-haploidentical related (sibling or parental) donors (HRD), and HLA-matched related donors (MRD) were three, four, six, and one, respectively. Another patient accepted HLA-unmatched unrelated cord blood (UUC) HSCT. The other four patients who did not undergo HSCT accepted androgen, cytokine, and/or intermittent transfusion support. All the patients with abnormal karyotype underwent HSCTs. In the HSCT subgroup, 9/15 (60%) were ALDH2-G/A genotype. The median follow-up duration was 29 months ranged from 1 month to 68 months. By the end of the study, eight patients (38.10%) have been dead. Seven of them were HSCT-related, mainly severe acute graft-versus-host disease (aGVHD) and/or infections, accounting for 46.67% of the subgroup. One patient who did not receive HSCT died from severe infection (Table 4). Nie et al. BMC Medical Genetics (2020) 21:118 Page 7 of 10 Fig. 2 Locations, frequencies, and types of mutations in FANCA, FANCB, FANCC, FANCD2, FANCE, and ERCC4 genes. Exons represent by colored rectangles; mutation types are represented by colored patterns; large deletions are represented by the black horizontal bars Discussion The 21 patients displayed a wide range of clinical phenotype and genetic variation spectrum that all physiological systems were involved (Table 1), and the responsible mutations were detected in six different genes (Fig. 1, Table 3). In keeping with other studies, bone marrow dysplasia and abnormal karyotypes were prevailing (65 and 42.11%, respectively) and highly consistent [14, 26], denoting the risk of hematologic malignant transformation, especially the ones with aberration in chromosome 7, which is the most prevalent cytogenetic abnormality in pediatric MDS and indicates an adverse long-term outcome even after HSCTs in MDS/AML patients [27]. ALDH2-G/A and ALDH-A/A genotypes are confirmed to be associated with more severe hematologic phenotype and more adverse outcomes of FA in Asian patients [13, 14]. The same tendency was observed in our cohort, despite there was no patient of ALDH2-AA genotype. All patients in our cohort presented with a more severe hematologic manifestation and the proportion of patients who received HSCTs was higher than that of most studies [3–6, 14, 28]. Although BMF is the typical and most prevalent feature, our data may not reflect the actual behavior of FA since all the patients were referred to our institute seeking for HSCTs. Studies suggest the high HSCT-related mortality in FA patients, of which infection and aGVHD were the two leading causes [5, 28]. In our cohort, 46.67% of HSCT patients died from HSCT-related acute complications. Studies also suggest the overall dismal outcome that 10 years cumulative risk of death was over 22% and the overall survival after 30 years of diagnosis dropped to below 40%; besides, the long-term survival of HSCT patients and non-HSCT patients were comparable [5, 26, 28, 29], partly because HSCT in the context of FA is explicitly challenging. Therefore, even with the optimized pre-HSCT conditioning regimens like the reduced intensity and the combination of fludarabine, meticulousness is needed in decisionmaking. Whether HSCT is the best treatment strategy depends much on the severity of cytopenia and the hematologic adverse events of a given patient and the type of donor he/she could get. Nie et al. BMC Medical Genetics (2020) 21:118 Page 8 of 10 Table 4 Treatment and outcomes Case No. Therapeutics Donor & HLA matching Pre-HSCT conditioning regimen Outcomes 1 HSCT UUD; 8/10 Bu + CTX + Flu+Alemtuzumab Dead (aGVHD, infections) 2 HSCT UUD; 9/10 Bu + CTX + Flu+ATG + Me-CCNU Alive 3 HSCT MUD; 10/10 Bu + Flu+CTX + ATG Alive 4 HSCT HRD; 8/10 Bu + CTX + Flu+ATG + Me-CCNU Alive 5 HSCT HRD; 6/10 Decitabine+Ara-C + Bu + Flu+ATG + MeCCNU 6 Androgen and transfusion – 7 HSCT HRD; 7/10 – Dead (aGVHD, drug-induced encephalopathy) Alive Dead (aGVHD, MODS) Decitabine+Ara-C + Bu + Flu+ATG + MeCCNU 8 Lost follow-up – – – 9 HSCT MRD; 10/12 Bu + Flu+CTX + ATG 10 HSCT HRD; 5/10 Dead (aGVHD, septic shock) Dead (aGVHD, septic shock) Decitabine+Ara-C + Bu + Flu+ATG + MeCCNU 11 HSCT UUC; 5/8 Bu + Flu+CTX + ATG Dead (aGVHD, pulmonary infection, CMV infection) 12 Androgen, cytokine, transfusion – – Alive 13 Androgen and cytokine – – Alive 14 HSCT HRD; 7/10 Bu + Flu+CTX + ATG Dead (aGVHD, TMA, pulmonary infection) 15 HSCT HRD; 7/10 Bu + Flu+CTX + ATG Alive 16 Androgen and transfusion – – Dead (pulmonary infection, septic shock) 17 HSCT MUD; 10/10 Bu + Flu+CTX + ATG Alive 18 HSCT UUD; 8/10 Bu + Flu+CTX + ATG Alive 19 HSCT UUD; 9/10 TBI + CTX + Flu+ATG Alive 20 HSCT MUD; 10/10 Bu + Flu+CTX + ATG Alive 21 Loss to follow-up – – – HSCT hematologic stem cell transplantation, UUD HLA-unmatched unrelated donor, MUD HLA-matched unrelated donor, HRD HLA-haploidentical related donor, MRD HLA-matched related donors, UUC HLA-unmatched unrelated cord blood, Bu Busulfan, CTX cyclophosphamide, Flu Fludarabine, ATG antithymocyte globulin, Me-CCNU Semustine, TBI total body irradiation, aGVHD acute graft-versus-host disease, CMV cytomegalovirus The cumulative incidence of leukemia and solid tumors in the middle age of FA patients was reported to be ~ 20% and ~ 30%, respectively [4–6, 30]. In our cohort, no patient developed hematologic or solid malignancies during the follow-up up to date except the ones initially diagnosed as AML (Case 5) and MDS (Case 10), but the longest follow-up in our cohort was only 5.5 years, which may not be long enough for the malignant phenotype to emerge. Conclusions Although this study is limited by its cohort size, it is still informative and enriches the knowledge on Chinese FA patients which was nearly a barren. Here we thoroughly investigated the clinical manifestations, morphologic and cytogenetic changes, genetic basis, and outcomes of 21 Chinese FA patients. Our data displayed a broad phenotypic and genetic variant spectrum of Chinese FA patients, the disappointing outcomes which need improving, and highlighted the urgency of nationwide multicenter studies to reveal the mask of Chinese FA patients and optimize the clinical management. Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s12881-020-01057-3. Additional file 1. Supplementary methods. Additional file 2: Table S1. Details of 22 FA-related genes. Additional file 3. Data of hromosome breakage test. Additional file 4: Table S3. Mutation interpretations. Nie et al. BMC Medical Genetics (2020) 21:118 Abbreviations ACMG: American College of Medical Genetics and Genomics; AGE: Agarose gel electrophoresis; aGVHD: Acute graft-versus-host disease; ALDHs: Aldehyde dehydrogenases; AML: Acute myeloid leukemia; BM: Bone marrow; BMF: Bone marrow failure; BWA: Burrow-Wheeler Aligner; CNVs: Copy number variants; FA: Fanconi anemia; HLA: Human leukocyte antigen; HRD: HLA-haploidentical related donors; HSCT: Hematopoietic stem cell transplantations; ICLs: Interstrand crosslinks; InDels: Insertions/deletions; MAF: Minimal allele frequency; MDS: Myelodysplastic syndrome; MRD: HLAmatched related donors; MUD: HLA-matched unrelated donors; PB: Peripheral blood; PCR: Polymerase chain reaction; SNVs: Single nucleotide variants; THS: Targeted high-throughput sequencing; UUC: HLA-unmatched unrelated cord blood; UUD: HLA-unmatched unrelated donors; WGS: Whole genomic sequencing Acknowledgments The authors would like to thank the patients and their families for participating in the study. Authors’ contributions DN reviewed the medical history of the patients, analyzed sequencing data, and wrote the manuscript. JZ, FW, WZ, XM, and LL performed the sequencing process, analyzed the data, and wrote the manuscript. XC and YZ analyzed the morphology and karyotype results, PC designed the bioinformatic analysis process. MX analyzed and interpreted the clinical significance of the genetic variation, guided the clinical diagnosis and treatment. TW and PW carried out the morphologic and cytogenetic study. WT, MW, and KC analyzed the clinical data and supervised the study. HL designed and supervised the study. All authors have read and approved the manuscript. Funding Retrospective WGS, like cost of reagent, was supported by grants from the Shandong Nature Science Fund (ZR2016HP02) and Peking University Medicine Seed Fund for Interdisciplinary Research (BMU2018ME002, supported by the Fundamental Research Funds for the Central Universities). Availability of data and materials The datasets generated during the current study are available in the Sequence Read Archive (SRA) repository of the National Center for Biotechnology Information [accession number: PRJNA631475; https://www. ncbi.nlm.nih.gov/bioproject/631475]. The reference datasets used in this study is human reference genome (hg19). Ethics approval and consent to participate The study was approved by the ethics committee of the Hebei Yanda Lu Daopei hospital. Written informed consent to genetic analysis and participate in this study was obtained from the all of the participants or parents or legal guardians of any participant under the age of 16. Consent for publication Written informed consent for publication of clinical details was obtained from the parents or legal guardians of any participant under the age of 18. Competing interests The authors declare that no competing interests in this study. Author details 1 Division of Pathology & Laboratory Medicine, Hebei Yanda Lu Daopei Hospital, 6 Sipulan Road, Langfang 065201, China. 2Beijing Lu Daopei Institute of Hematology, Beijing 100176, China. 3Department of Hematology, Hebei Yanda Lu Daopei Hospital, Langfang 065201, China. 4Department of Clinical Laboratory Medicine, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250000, China. 5Department of Hematology, Peking University First Hospital, Beijing 100034, China. 6Division of Pathology & Laboratory Medicine, Beijing Lu Daopei Hospital, Beijing 100176, China. Page 9 of 10 Received: 31 August 2019 Accepted: 24 May 2020 References 1. Kimble DC, Lach FP, Gregg SQ, Donovan FX, Flynn EK, Kamat A, Young A, Vemulapalli M, Thomas JW, Mullikin JC, Auerbach AD, Smogorzewska A, Chandrasekharappa SC. A comprehensive approach to identification of pathogenic FANCA variants in Fanconi anemia patients and their families. 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https://openalex.org/W4383369083	https://www.researchsquare.com/article/rs-3056280/latest.pdf	English	null	Analysis and simulation of fractional-order financial models with and without market confidence using triangular basis functions	Research Square (Research Square)	2,023	cc-by	11,327	Research Article Keywords: Financial system, Market con¦dence, Triangular basis function, Operational matrix, Equilibrium points, Lyapunov exponent, chaotic behaviour. Posted Date: July 6th, 2023 Analysis and simulation of fractional-order ﬁnancial models with and without market conﬁdence using triangular basis functions ShwetaDubey1, M.Kundu2 S.Chakraverty3 1,3 Department of Mathematics, National Institute of Technology Rourkela, Odisha 2 Department of Chemical Engineering, National Institute of Technology Rourkela, Odisha, Email : mkundu@nitrkl.ac.in Abstract Researchers have long been interested in the dynamics of the ﬁnancial system, in- cluding its stability, periodic and chaotic behaviour, as well as its eﬀects on market conﬁdence. The government, ﬁnancial institutions, and all other stakeholder groups in a healthy economic system can be better prepared with accurate ﬁnancial trend predictions. The ﬁnancial system appears to be best described as having a frac- tional order because of the long memory eﬀect in market patterns. The study of nonlinear dynamical phenomena, such as equilibrium points, stability, the Lyapunov exponent, the Lyaponov dimension, chaos, the impact of market conﬁdence on ﬁ- nancial systems, etc., enhances overall comprehension. The numerical simulation of the fractional-order model of this system has been done using the triangular basis function (TBF). DOI: https://doi.org/10.21203/rs.3.rs-3056280/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License 1 Introduction Arbitrary order description has been credited with better analyzing and predicting the behavior of the processes possessing memory and chaos. The main distinction between fractional and integer-order models is that fractional order models are mem- ory dependent; i.e., the fractional-order model depends on the history of the system. Diverse techniques including Fourier transform, Laplace transform, wavelet methods based on orthogonal polynomials, semi-analytical methods providing truncated series solution have been harnessed for solving fractional-order models. Techniques includ- ing modiﬁed extended tanh method [1], Sumudu transformation method [2], coupled transformation method [3], Galerkin and collocation methods [4], etc. are some re- cent endeavors in this paradigm. Further few fractional models [5], [6] [7] have been studied extensively in uncertain environment. A dynamic model of ﬁnance, composed of three ﬁrst-order diﬀerential equations has been reported in [8, 9]. The fractional-order ﬁnancial systems exhibit important characteristics including memory and chaos [10]. The ﬁnancial variables such as for- eign exchange rates, gross domestic product (GDP), inﬂation rate, and share and stock market prices can have very long memory, in fact, they can demonstrate a time series behavior. The basic dynamical behaviors such as the equilibrium, and stability of this ﬁnancial system were investigated by Cui et al. [11]. A competent ﬁnancial system merits additional consideration and study [12, 13, 14], especially its stability, as it is an essential component of a robust and healthy economic system. The ﬁnancial system may exhibit a variety of nonlinear dynamical behaviours, including bifurcation, chaos, and fractals [15, 16]. Numerous experts have recently developed an interest in the complicated characteristics of ﬁnancial system , such as bifurcation and chaos [17, 9, 18]. Additionally, some expanded versions of the ﬁnancial system were displayed. These included a fractional form of the ﬁnancial system presented by Chen and Ching [10] in 2008, an uncertain fractal-order form established by Wang et al. [19] in 2012, a delayed form proposed by Mircea et al. [20] in 2012, and a discrete form proposed by Xin et al. [21] in 2010. Moreover, the ﬁnancial system was extended with the average proﬁt margin [22], and an investment incentive has been introduced into ﬁnancial system [23] and a four-dimensional ﬁnan- cial system has been demonstrated. Contrarily, conﬁdence is the most crucial aspects in averting or inﬂuencing the economic catastrophe. Keywords Financial system; Market conﬁdence; Triangular basis function; Operational matrix; Equilibrium points; Lyapunov exponent; chaotic behaviour. 1 1 Introduction Hence, conﬁdence, or the consumer’s conﬁdence, is a crucial aspect that needs to be taken into account while modeling and studying economic and ﬁnancial systems [24]. People lose faith in economic systems, when a ripple eﬀect 2 and series of events result, including decreased productivity, canceled investments, and halted consumption [25, 26] occurs. Because of this, there will be fewer em- ployment, less investment, less conﬁdence, more product stock, more deﬁcits, and more cutbacks. Consumers would not save excessive amounts of money if there is adequate investment conﬁdence and opportunity. Moreover, low interest rates will boost economic system conﬁdence whereas scary interest rates will undermine it [27]. Governments must make every eﬀort to build a framework that balances and encour- ages savings and investments. The encouragement of investment and, as a result, the behaviour of systems are signiﬁcantly inﬂuenced by investment conﬁdence. In this manner, it makes sense to take into account market conﬁdence in ﬁnancial sys- tem modeling. Nonetheless, some researches [28] take market conﬁdence into account while analyzing chaotic behavior in ﬁnancial systems. Although research in this area has advanced signiﬁcantly up to this point, some is- sues relating to economic behaviour remain unresolved. Focused research on fractional- order chaotic ﬁnancial systems without market conﬁdence and with market conﬁdence are needed in order to visualize these systems. The current research is motivated by these problems. This work carefully examines the nonlinear dynamical phenomena, such as equilibrium points, stability, Lyapunov exponent, Lyapunov dimension and chaos, that the ﬁnancial systems with and without market conﬁdence may exhibit. Additionally, we simulate the model using TBF and compare the results with ODE 45 solver (MATLAB, INC) which are in excellent agreement, and also compute the absolute error between them. During nineteenth century, most important set of function used for communica- tion problem was Block pulse function. Recently, it had been drawing attention to the researchers because of its digital technology compatibility. It’s a complete set of orthogonal function with piecewise constant values providing piecewise constant solution and proved to be a useful tool in the paradigm of control and system science. Block pulse function (BPF) was ﬁrst proposed by Harmuth ([29]) and later introduced formally by Chen et al. [30]. Block pulse functions have advantages in solving prob- lems involving diﬀerentials and integrals due to simplicity of their formulation and clarity in expressions. 1 Introduction Here the original problem is converted to their corresponding set of algebraic equations with the help of operational matrices, which are patterned upper triangular in nature and reduces the computational burden drastically. Some of the signiﬁcant contribution in block pulse function include Rao and Srinivasan [31], Kwong and Chen [32], Chen and Lee [33], Chen et al. [30], Sannuti [34] and many others. A new set of triangular orthogonal function evolved out of BPF, unlike providing staircase solution (in BPF), it provides piecewise linear solution with less mean integral square error (DEB et al. [35]). Darmala and Kundu [36, 37, 38] have 3 derived complementary pair of operational matrices for ﬁrst order integration in the TF domain and demonstrated that the traingular function (TF) domain technique for dynamical systems analysis is computationally more eﬀective. Section 2 provides the fundamental introductions pertaining to TBF and the op- erational matrices. In addition, section 3 discusses the convergence of TBF. A math- ematical postulate of the fractional-order ﬁnancial system with and without market conﬁdence as well as its dynamic behaviour has also been provided in section 4. The ﬁnal part, section 5, contains the conclusion. 2 Triangular basis function (TBF) As discussed in [35], a BPF has been splited into two TFs for the sake of the following ϕ0(t) = T10(t) + T20(t). (1) (1) Here, ϕ0(t) is the initial element of m-set BPF and the ith component of left handed triangular function (LHTF) vector T1m(t) is deﬁned as T1i(t) = ( 1 − t−ih h , ih ≤t < (i + 1)h 0, otherwise , i = 0, 1, . . . , m −1 (2) (2) and ith component of right handed triangular function vector (RHTF) T2m(t) is deﬁned as T2i(t) = ( t−ih h , ih ≤t < (i + 1)h 0, otherwise , i = 0, 1, . . . , m −1 (3) (3) In light of this, T1i(t) and T2i(t) can be created as previously mentioned for each ϕi(t), i = 0, 1, . . . , m −1. Therefore, we are able to create two orthogonal TFs viz. T1m(t) and T2m(t) in the way that In light of this, T1i(t) and T2i(t) can be created as previously mentioned for each ϕi(t), i = 0, 1, . . . , m −1. Therefore, we are able to create two orthogonal TFs viz. T1m(t) and T2m(t) in the way that Φm(t) = T1m(t) + T2m(t). (4) (4) We can express m-set TF vectors as We can express m-set TF vectors as T1m(t) = [T10(t)T11(t) · · · T1i(t) · · · T1m−1(t)] T2m(t) = [T20(t)T21(t) · · · T2i(t) · · · T2m−1(t)] (5) (5) 4 Since the elements of T1m(t) and T2m(t) are mutually disjoint, which gives a key characteristic as T1i(t)T1j(t) = ( T1i(t), i = j, 0, i ̸= j , i = 0, 1, . . . , m −1 (6) (6) similarly, T2i(t)T2j(t) = ( T2i(t), i = j, 0, i ̸= j , i = 0, 1, . . . , m −1 (7) (7) By employing above two properties the product of two TFs is given below: T1m(t)T1T m(t) =        T10(t) 0 0 · · · 0 0 T11(t) 0 · · · 0 0 0 T12(t) · · · 0 ... ... ... ... ... 2 Triangular basis function (TBF) 0 0 0 · · · T1m−1(t)        = diagT1(t) (8) (8) T2m(t)T2T m(t) =        T20(t) 0 0 · · · 0 0 T21(t) 0 · · · 0 0 0 T22(t) · · · 0 ... ... ... ... ... 0 0 0 · · · Tm−1(t)        = diagT2(t) (9) T1m(t)T2T m(t) = T2m(t)T1T m(t) = 0m×m (10) T1m(t)T2T m(t) = T2m(t)T1T m(t) = 0m×m (10) (10) where, 0 is the null matrix. where, 0 is the null matrix. where, 0 is the null matrix. 2.1 Operational matrices for integer order integration [36] 2.1 Operational matrices for integer order integration [36] Consider a function g(t) deﬁned in triangular function domain, then we can write Consider a function g(t) deﬁned in triangular function domain, then we can write g(t) = C0T1m(t) + D0T2m(t) (11) (11) where, C0 = c0 c1 c2 · · · cm−1 and D0 = d0 d1 d2 · · · dm−1 where, di = ci+1 and ci = f(ih) , i = 0, 1, 2, · · · , m −1. Z t 0 g(t) = CT + DT (P1T1m(t) + P2T2m(t)) where, C0 = c0 c1 c2 · · · cm−1 and D0 = d0 d1 d2 · · · dm−1 where, di = ci+1 and ci = f(ih) , i = 0, 1, 2, · · · , m −1. Z t 0 g(t) = CT + DT (P1T1m(t) + P2T2m(t)) (12) (12) 5 5 P1 = h 2        0 1 1 · · · 1 0 0 1 · · · 1 0 0 0 · · · 1 ... ... ... ... 1 0 0 0 · · · 0        m×m , P2 = h 2        1 1 1 · · · 1 0 1 1 · · · 1 0 0 1 · · · 1 ... ... ... ... 1 0 0 0 · · · 1        m×m 2.2 Operational matrices for fractional order integration [36] ξ2 0 0 · · · 0 · · · ξ1          m×m where, ξj = jα+1 −(j + α)(j −1)α, j = 1, 2, · · · , m −1. where, ξj = jα+1 −(j + α)(j −1)α, j = 1, 2, · · · , m −1. For the special case of α = 1 , P α 1 = P α 3 = P1, P α 2 = P α 4 = P2 2.2 Operational matrices for fractional order integration [36] The Riemann-Liouville fractional integral of order α of g(t) is The Riemann-Liouville fractional integral of order α of g(t) is Jαg(t) = CTP α 1 + DTP α 3 T1m(t) + CTP α 2 + DTP α 4 T2m(t) (13) Jαg(t) = CTP α 1 + DTP α 3 T1m(t) + CTP α 2 + DTP α 4 T2m(t) (13) (13) P α 1 = hα Γ(α + 2)          0 ζ1 ζ2 ζ3 · · · ζm−1 0 0 ζ1 ζ2 · · · ζm−2 0 0 0 ζ1 · · · ζm−3 ... ... ... ... ... ... 0 0 · · · 0 ... ζ1 0 0 · · · 0 · · · 0          m×m , P α 2 = hα Γ(α + 2)          ζ1 ζ2 ζ3 ζ4 · · · ζm−1 0 ζ1 ζ2 ζ3 · · · ζm−2 0 0 ζ1 ζ2 · · · ζm−3 ... ... ... ... ... ... 0 0 · · · 0 ... ζ2 0 0 · · · 0 · · · ζ1          m×m where, ζj = jα (1 + α −j) + (j −1)α+1 , j = 1, 2, · · · , m −1. Similarly, where, ζj = jα (1 + α −j) + (j −1)α+1 , j = 1, 2, · · · , m −1. Similarly, P α 3 = hα Γ(α + 2)          0 ξ1 ξ2 ξ3 · · · ξm−1 0 0 ξ1 ξ2 · · · ξm−2 0 0 0 ξ1 · · · ξm−3 ... ... ... ... ... ... 0 0 · · · 0 ... ξ1 0 0 · · · 0 · · · 0          m×m , 6 P α 4 = hα Γ(α + 2)          ξ1 ξ2 ξ3 ξ4 · · · ξm−1 0 ξ1 ξ2 ξ3 · · · ξm−2 0 0 ξ1 ξ2 · · · ξm−3 ... ... ... ... ... ... 0 0 · · · 0 ... 2.3 Solution approaches by TBF Let us consider the following fractional-order ordinary diﬀerential equation Dα t ωi(t) = λ X k=1 bkDβk t ωi(t)+F(t, ω1(t), ω2(t), · · · , ωN(t))+fi(t), n−1 ≤α < n, n ∈Z+, (14) (14) (14) where, i = 1, 2, · · · N N ∈Z+, ωi(t) is the ith unknown function, fi(t) is the known function, and F(t, ω1(t), ω2(t), · · · , ωN(t)) can be linear or non linear. ( ) where, i = 1, 2, · · · N N ∈Z+, ωi(t) is the ith unknown function, fi(t) is the known function, and F(t, ω1(t), ω2(t), · · · , ωN(t)) can be linear or non linear. For N = 1, Eq. (14) becomes For N = 1, Eq. (14) becomes For N = 1, Eq. (14) becomes Dα t ω(t) = λ X k=1 bkDβk t ω(t) + F(t, ω(t)) + f(t), n −1 ≤α < n, n ∈Z+, (15) (15) with initial conditions ωj(0) = γj, j = 0, 1, · · · n −1. Here, t ∈[0, T], ω(t) : [0, T] →[0, ∞), F(t, ω(t)) : [0, ∞) →[0, ∞), f(t) : [0, T] → [0, ∞). with initial conditions ωj(0) = γj, j = 0, 1, · · · n −1. Here, t ∈[0, T], ω(t) : [0, T] →[0, ∞), F(t, ω(t)) : [0, ∞) →[0, ∞), f(t) : [0, T] → [0, ∞). Eq. (15) can be written in integral form as given below ω(t) = Ω(t) + λ X k=1 bkJα−βk t ω(t) + Jα t F(t, ω(t)) + V (t), (16) (16) where, Ω(t) = Pn−1 r=0 tr r!ωr(0), V (t) = Jα t f(t) −P⌊βk⌋ k=1 bk tk k!ωk(0). According to Eq. (13), we obtain According to Eq. (13), we obtain According to Eq. (13), we obtain          ω(t) = CTT1m(t) + DTT2m(t), Ω(t) = CT 1 T1m(t) + DT 1 T2m(t), V (t) = CT 2 T1m(t) + DT 2 T2m(t), F(t, ω(t)) = CT 3 T1m(t) + DT 3 T2m(t). (17) (17) Plugging Eq. (17) into Eq. (16) and using the operational matrices of fractional- order integration as described in subsection (2.2), Eq. (16) reduces to Plugging Eq. (17) into Eq. (16) and using the operational matrices of fractional- order integration as described in subsection (2.2), Eq. 2.3 Solution approaches by TBF (16) reduces to CTT1m(t) + DTT2m(t) CTT1m(t) + DTT2m(t) = CT 1 T1m(t) + DT 1 T2m(t) + CT 3 P α 1 + DT 3 P α 3 T1m(t) + CT 3 P α 2 + DT 3 P α 4 T2m(t) + λ X k=1 bk CTP α−βk 1 + DTP α−βk 3 T1m(t) + CTP α−βk 2 + DTP α−βk 4 T2m(t) CTT1m(t) + DTT2m(t) = CT 1 T1m(t) + DT 1 T2m(t) + CT 3 P α 1 + DT 3 P α 3 T1m(t) + CT 3 P α 2 + DT 3 P α 4 T2m(t + λ X k=1 bk CTP α−βk 1 + DTP α−βk 3 T1m(t) + CTP α−βk 2 + DTP α−βk 4 T2m(t) + CT 2 T1m(t) + DT 2 T2m(t). (18) Equating the coeﬃcients of left-hand triangular function vector and right-hand triangular function vector, we get Equating the coeﬃcients of left-hand triangular function vector and right-hand triangular function vector, we get CT = CT 1 + CT 3 P α 1 + DT 3 P α 3 + λ X k=1 bk CTP α−βk 1 + DTP α−βk 3 + CT 2 , (19) (19) DT = DT 1 + CT 3 P α 2 + DT 3 P α 4 + λ X k=1 bk CTP α−βk 2 + DTP α−βk 4 + DT 2 , (20) DT = DT 1 + CT 3 P α 2 + DT 3 P α 4 + λ X k=1 bk CTP α−βk 2 + DTP α−βk 4 + DT 2 , (20) (20) After solving the system of algebraic Eqs. (19) and (20), we obtain the approxi- mate solution of the considered fractional-order ordinary diﬀerential equation (14) . The procedure is also applicable for stiﬀor non-stiﬀsystem of diﬀerential equations. 4 Mathematical modeling of the ﬁnancial systems In this section, the two ﬁnancial models—one without and another with market con- ﬁdence have been illustrated. 3 Convergence analysis of TBF [36] = Mh6 6 (26) (26) Consider, ϵm = Pm−1 j=0 ϵj, which implies Consider, ϵm = Pm−1 j=0 ϵj, which implies Consider, ϵm = Pm−1 j=0 ϵj, which implies Consider, ϵm = Pm−1 j=0 ϵj, which implies 9 9 ∥ϵm∥1 = Z T 0 \|ϵm\|dt = Z T 0 m−1 X j=0 \|ϵj\| ! dt = m−1 X j=0 Z T 0 \|ϵj\| dt = m−1 X j=0 ∥ϵj∥1 = T 4M 6m2 Taking limit, lim m→∞∥ϵm∥1 = lim m→∞ T 4M 6m2 = 0 lim m→∞∥ϵm∥1 = lim m→∞ T 4M 6m2 = 0 Therefore, limm→∞ϵm = 0 3 Convergence analysis of TBF [36] This section demonstrate the convergency of TF approximate solution to the exact solution. 8 Let eω(t) be the TF estimate for the exact solution ω(t) of the fractional order nonlinear ordinary diﬀerential equation in Eq. (14). Suppose that ϵj be the error between exact solution and TF approximate solution on the jth subinterval then we have ϵj = ω(t) −eω(t), t ∈[jh, (j + 1)h], j = 0, 1, · · · , m −1 (21) (21) Approximating ω(t) in the jth subinterval [36], eω(t) = ω(jh) 1 − x −jh h + ω ((j + 1)h) x −jh h , = ω(jh) + ω ((j + 1)h) −ω(jh) h (x −jh) , = ω(jh) + dω(t) dt t=jh (t −jh) ! (22) eω(t) = ω(jh) 1 − x −jh h + ω ((j + 1)h) x −jh h , (( )h) ( h) (22) = ω(jh) + dω(t) dt t=jh (t −jh) ! Now, we write the Taylor series expansion of ω(t) around the center jh as ω(t) = ω(jh)+ dω(t) dt t=jh (t−jh)+ d2ω(t) dt2 t=jh (t −jh)2 2! + d3ω(t) dt3 t=jh (t −jh)3 3! +· · · (23) (23) Truncating the above series to the ﬁrst three terms, we obtain ω(t) ≈ω(jh) + dω(t) dt t=jh (t −jh) + d2ω(t) dt2 t=jh (t −jh)2 2! (24) (24) From Eqs. (22) and (24), we have From Eqs. (22) and (24), we have ϵj = d2ω(t) dt2 t=jh (t −jh)2 2! = ω′′(jh)(t −jh)2 2! (25) (25) Let us assume, M = max [\|ω′′(0), ω′′(h), · · · , ω′′(mh)\|] . Taking norm in Eq. (25) , we get Let us assume, M = max [\|ω′′(0), ω′′(h), · · · , ω′′(mh)\|] . Taking norm in Eq. (25) , we get ∥ϵj∥1 = Z (j+1)h jh \|ϵj\|dt = Z (j+1)h jh \|ω′′(h)\|(t −jh)2 2! = M Z (j+1)h jh (t −jh)2 2! = Mh6 6 (26) ∥ϵj∥1 = Z (j+1)h jh \|ϵj\|dt = Z (j+1)h jh \|ω′′(h)\|(t −jh)2 2! = M Z (j+1)h jh (t −jh)2 2! 4.1 Fractional-order ﬁnancial model without market conﬁ- dence The main component of the model has been puriﬁed and streamlined to make the problem simple to solve. It has been decided to use variable x to represent the interest rate, variable y to represent the investment demand, and variable z to represent the price exponent in the considered model. The contradiction in the investment market speciﬁcally, the excess of investments over savings as well as the structure adjustment from changes in the prices of goods are the key drivers inﬂuencing changes in x. Mathematically, it can be written as, ˙x = ρ1(y −ν1)x + ρ2z, here, ν1 represents the amount of saving, ρ1 and ρ2 serve as constants. The rate at which y changes is inversely correlated to the cost of the investment and the interest rate, and it varies in proportion to the rate at which investments are made. Assume that throughout a particular duration, the return on investment is constant, and we can compute, ˙y = ρ3(Ω−ν2y −ν3x2), 10 therein, Ωis return on investment, ρ3, ν2, and ν3 are constants. therein, Ωis return on investment, ρ3, ν2, and ν3 are constants. The competition between supply and demand in the commercial market controls variations in z on the one hand, while the inﬂation rate has an impact on them on the other. Here, we assume that the quantity of supplies and demands for commercials is constant during a predetermined time period and that it is inversely proportional to pricing. Yet, the real interest rate can really be used to indicate changes in the inﬂation rate, and the inﬂation rate is equal to the normal interest rate minus the real interest rate. Hence, we calculate ˙z = −ρ4z −ρ5x, As a result, we may obtain the following more simpliﬁed model with only the three most crucial parameters: x, y, and z by selecting the proper coordinate system and setting a suitable dimension to every state variable. Huang and Li [8] initially suggested the following intriguing nonlinear integer-order ﬁnancial model. ˙x = z + (y −a) x, ˙y = 1 −by −x2, ˙z = −x −cz (27) (27) In Eq. (27), a(≥0) stands for the saving amount, b(≥0) for cost per investment, and c(≥0) for the elasticity of demand for commercials. In Eq. (27), a(≥0) stands for the saving amount, b(≥0) for cost per investment, and c(≥0) for the elasticity of demand for commercials. Dynamics of fractional-order ﬁnancial system without market conﬁdence This research focuses on a chaotic ﬁnancial system of fractional order that can be characterised as [10] Dα1x(t) = z + (y −a) x, Dα2y(t) = 1 −by −x2, Dα3z(t) = −x −cz (28) (28) 11 The above system is a non linear three dimensional fractional order ﬁnancial model. The model depicts the time-variation of three state variables: the interest rate, x, the investment command, y, and the price index, z. The constants a = 0.9, b = 0.2, and c = 1.2 used in this research are taken from [22]. Table 1: Stability analysis of fractional order chaotic ﬁnancial system Equilibrium points Eigen values Nature E1(0, 5.0, 0) 3.9041, −1.0041, 0.2000 saddle point of index 2 E2(0.8083, 1.7333, −0.6735) 0.1766 ± 1.2937i, −0.9198 saddle point of index 2 E3(−0.8083, 1.7333, −0.6735) 0.1766 ± 1.2937i, −0.9198 saddle point of index 2 Table 1: Stability analysis of fractional order chaotic ﬁnancial system Figure 1: 3-D plot of equilibrium points for the ﬁnancial system (37) Figure 1: 3-D plot of equilibrium points for the ﬁnancial system (37) Fig. 1 represents the equilibrium points E1, E2, and E3 as calculated in Table 1. Fig. 1 represents the equilibrium points E1, E2, and E3 as calculated in Table 1. This section starts with an examination of the stability of fractional-order sys- tems. Because systems with memory are often more stable than those without mem- ory, fractional-order diﬀerential equations are at least as stable as their integer-order counterparts [24]. Utilizing the Matignon [39] ﬁnding, A necessary and suﬃcient condition for a commensurate fractional-order Dαx(t) = Ax system to be asymptotically stable is \|arg(eig(A))\| > απ 2 (29) (29) 12 Figure 2: Stability zone of the linear fractional-order system of order α Figure 2: Stability zone of the linear fractional-order system of order α A saddle point is an equilibrium point in a three-dimensional nonlinear dynamical system where the corresponding linearized model includes at least one eigenvalue in the stable region and one eigenvalue in the unstable region. If one of the eigenvalues in the same system is unstable while the other eigenvalues are stable, the saddle point is referred to as a saddle point of index 1. Moreover, a saddle point with index 2 has one stable eigenvalue and two unstable eigenvalues. It has been observed that scrolls only appear at the saddle points of index 2 in chaotic systems. Dynamics of fractional-order ﬁnancial system without market conﬁdence Beside that, the saddle points of index 1 are exclusively used to join scrolls. Therefore, for the fractional system Dαx(t) = Ax to be chaotic, one of the saddle points of index 2 eigenvalues must be in an unstable region. Fig. 3 depicts the stable and unstable area for this model, from which it follows that \|Imλ\| Reλ < tan απ 2 , =⇒2 πtan−1 \|Imλ\| Reλ < α (30) (30) The equilibrium points E2 and E3 of Table 1 are saddle points of index 2. Hence, The equilibrium points E2 and E3 of Table 1 are saddle points of index 2. Hence, 13 the system (28) contains a 2-scroll attractor. Hence for E2 and E3,we calculate (28) contains a 2-scroll attractor. Hence for E2 and E3,we calculate (31) minj\|arg(Ej)\| = 1.4351 (31) As a result, from Eq. (30) if α < 2 πminj\|arg(Ej)\| ≈0.9136, the ﬁnancial system (28) will behaves normally and for α > 0.9136, the system demonstrate chaos. As a result, from Eq. (30) if α < 2 πminj\|arg(Ej)\| ≈0.9136, the ﬁnancial system (28) will behaves normally and for α > 0.9136, the system demonstrate chaos. The exponential rates of divergence and convergence of neighbouring trajecto- ries in the phase space of the system are measured by the Lyapunov exponents in accordance with the chaos theory. The Lyapunov exponent for the fractional-order ﬁnancial system (28) using the initial conditions x(0) = 1, y(0) = 3, and z(0) = 2 and α = 0.92 is calculated as [see also Fig. (3)] LE = (2.3588, −0.3950, −1.3771) (32) LE = (2.3588, −0.3950, −1.3771) (32) Lemma 1. Let #»f be a map in Rm, m ≥2. Consider an orbit with LEs h1 ≥h2 ≥· · · hm and let r be the largest integer such that r X j=1 hj ≥0 r X j=1 hj ≥0 The Lyapunov dimension DL of the orbits is deﬁned to be ❼0 if r does not exist. ❼0 if r does not exist. ❼m if r = m ❼m if r = m r + 1 \|hr+1\| Pr j=1 hr if r < m ❼r + 1 \|hr+1\| Pr j=1 hr if r < m From Eq. Dynamics of fractional-order ﬁnancial system without market conﬁdence (32), sum of Lyapunov exponents is 0.5867 > 0 which implies Lyapunov dimension DL = 3 14 14 LEs for the 3-D financial system for t [0,300] and =0.92 0 50 100 150 200 250 300 t -3 -2 -1 0 1 2 3 4 5 LEs LE1 LE2 LE3 Figure 3: Dynamical behaviour of LEs with respect to time LEs for the 3-D financial system for t [0,300] and =0.92 0 50 100 150 200 250 300 t -3 -2 -1 0 1 2 3 4 5 LEs LE1 LE2 LE3 LEs for the 3-D financial system for t [0,300] and =0.92 LEs for the 3-D financial system for t [0,300] and =0.92 Figure 3: Dynamical behaviour of LEs with respect to time 15 0.74 0.76 0.78 0.8 0.82 0.84 0.86 0.88 0.9 0.92 x 1.55 1.6 1.65 1.7 1.75 1.8 y (a) α = 0.88 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 x 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 y (b) α = 0.91 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 x 0 0.5 1 1.5 2 2.5 3 3.5 y (c) α = 0.92 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 x -0.5 0 0.5 1 1.5 2 2.5 3 3.5 y (d) α = 0.97 Figure 4: Phase portraits for 3-D fractional-order ﬁnancial system in x −y plane for diﬀerent values of α 0.74 0.76 0.78 0.8 0.82 0.84 0.86 0.88 0.9 0.92 x 1.55 1.6 1.65 1.7 1.75 1.8 y (a) α = 0.88 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 x 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 y (b) α = 0.91 (a) α = 0.88 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 x 0 0.5 1 1.5 2 2.5 3 3.5 y (c) α = 0.92 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 x -0.5 0 0.5 1 1.5 2 2.5 3 3.5 y (d) α = 0.97 (d) α = 0.97 (c) α = 0.92 Figure 4: Phase portraits for 3-D fractional-order ﬁnancial system in x −y plane for diﬀerent values of α 16 Table 2: Error in TBF solution of ﬁnancial model for α1 = α2 = α3 = 1. Dynamics of fractional-order ﬁnancial system without market conﬁdence Absolute error between TBF and ode45 solver t \|ϵ1\| = \|X(t)−˜X(t)\| \|ϵ2\| = \|Y (t) −˜Y (t)\| \|ϵ3\| = \|Z(t) −˜Z(t)\| 0 3.00000e-15 3.00000e-14 9.00000 e-15 0.1 9.76534e-4 3.93559e-3 7.78215e-4 0.2 1.03179e-3 8.25930e-3 1.40392e-3 0.3 5.29174e-3 1.03128e-2 1.45725e-3 0.4 9.59166e-3 8.93452e-3 7.80854e-4 0.5 1.20561e-2 4.98181e-3 4.53832e-4 0.6 1.21993-2 1.69490e-4 1.90272e-3 0.7 1.06112e-2 4.16055e-3 3.24624e-3 0.8 8.18105e-3 7.42614e-3 4.29028e-3 0.9 5.60918e-3 9.60158e-3 4.96774e-3 1.0 3.29151e-3 1.09068e-2 5.29627e-4 17 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Time(t) 0.5 1 1.5 2 2.5 3 3.5 Interest rate (x) 1= 2= 3= 1 1= 0.6, 2= 3= 1 1= 3=1, 2= 0.8 1= 2=1, 3= 0.2 Figure 5: Approximate solution for interest rate (X) using TBF. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Time(t) -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 3 Investment command (y) 1= 2= 3= 1 1= 2= 3= 0.90 1= 2= 3= 0.85 1= 2= 3= 0.80 Figure 6: Approximate solution for investment demand (Y) using TBF. 2.5 1= 2= 3= 1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Time(t) 0.5 1 1.5 2 2.5 3 3.5 Interest rate (x) 1= 2= 3= 1 1= 0.6, 2= 3= 1 1= 3=1, 2= 0.8 1= 2=1, 3= 0.2 Figure 5: Approximate solution for interest rate (X) using TBF. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Time(t) 0.5 1 1.5 2 2.5 3 3.5 Interest rate (x) 1= 2= 3= 1 1= 0.6, 2= 3= 1 1= 3=1, 2= 0.8 1= 2=1, 3= 0.2 Figure 5: Approximate solution for interest rate (X) using TBF. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Time(t) 0.5 1 1.5 2 2.5 3 3.5 Interest rate (x) 1= 2= 3= 1 1= 0.6, 2= 3= 1 1= 3=1, 2= 0.8 1= 2=1, 3= 0.2 Figure 5: Approximate solution for interest rate (X) using TBF. Dynamics of fractional-order ﬁnancial system without market conﬁdence 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Time(t) -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 3 Investment command (y) 1= 2= 3= 1 1= 2= 3= 0.90 1= 2= 3= 0.85 1= 2= 3= 0.80 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Time(t) -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 3 Investment command (y) 1= 2= 3= 1 1= 2= 3= 0.90 1= 2= 3= 0.85 1= 2= 3= 0.80 Figure 6: Approximate solution for investment demand (Y) using TBF. Figure 6: Approximate solution for investment demand (Y) using TBF. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Time(t) -1 -0.5 0 0.5 1 1.5 2 2.5 Investment command (y) 1= 2= 3= 1 1= 2= 3= 0.90 1= 2= 3= 0.85 1= 2= 3= 0.80 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Time(t) -1 -0.5 0 0.5 1 1.5 2 2.5 Investment command (y) 1= 2= 3= 1 1= 2= 3= 0.90 1= 2= 3= 0.85 1= 2= 3= 0.80 Figure 7: Approximate solution for price index (Z) using TBF. Figure 7: Approximate solution for price index (Z) using TBF. 18 18 We investigated the dynamics and numerical simulations of the model with initial conditions x(0) = 1, y(0) = 3, and z(0) = 2 in the fractional-order ﬁnancial system without market conﬁdence (28). Table 1 examines the stability of the system (28), we can conclude that the system behaves chaotically for the fractional value α > 0.9136. Additionally, we have determined the Lyapunov exponent (32), and the Lyapunov dimension at α = 0.92 because the system has a positive LE (32), which guarantees that the system has a chaotic solution. Further, the chaotic solutions for commensurate fractional-order ﬁnancial system (28) has been illustrated by 2D and 3D phase potraits for diﬀerent values of α as shown in Fig. 8 and in appendix Figs. A1, A2, and A3. The phase portraits, shows diﬀerent type of attractor for diﬀerent values of α. Fractional order dynamic simulation of the ﬁnancial system using TBF has been discussed. Absolute errors have been estimated at several domain locations and provided in tabular format to show the accuracy of the consider method. Additionally, Table 3 shows the absolute error between the benchmark ode45 solver and TBF. Dynamics of fractional-order ﬁnancial system without market conﬁdence Further, we go through the fractional order dynamic simulation of the above system of equations using TBF. The value of fractional parameters α1, α2 and α3 (0 < α1 = α2 = α3 ≤1) varying for three classes viz. interest rate (x), investment command (y) and price index (z) are graphically represented in Figs. 9, 10 and 11. Here, Fig. 9 shows the change in interest rate (x) with respect to the time for diﬀerent values of α1, α2, α3. Further, the investment command (y) for various values of α1, α2, α3 has been illustrated in Fig. 10. Simlarly, the variation in price index (z) with respect to time is sketched in Fig. 11. 2 Fractional-order ﬁnancial model with market conﬁdence In general, the addition of the interest rate will result in higher interest costs for investors and consumers. Hence, organizations will either pass some or all of the ad- ditional interest costs onto customers or absorb them in their margins. The most likely outcome is that either organizations will reduce their investment, or that consumers will lower their spending, or both. Because of this, market conﬁdence decreases as interest rates rise. Similar to this, organizations will raise investment and consumers will increase their spending when market conﬁdence rises. In fact, when investment commands rise and purchasing power grows, we require more loans, which raises in- terest rates. So, one might suggest the following assertion: Assertion 1. Market conﬁdence (w) has a positive impact on the interest rate (x), whereas the interest rate (x) has a negative impact on market conﬁdence (w). In general, the higher the investment command, the higher the income expecta- tion, and the greater the potential growth in consumer spending power. As a result, organisations will enhance their investments and consumers will increase their pur- 19 chases, which raises market conﬁdence. Similar to this, the investment command turns stronger the more conﬁdent the market is and the more optimistic its expecta- tions are. As a result, one can make the following assertion: Assertion 2. The market conﬁdence (w) and the investment command (y) both have favorable impact on each other. Under usual conditions, a high price in- dex causes a decline in investment revenue as well as income for consumers. Cutting investments for investors and reducing consumer demand are two crucial ways to re- spond to future uncertainty. As a result, market conﬁdence declines. In similar vein , when market conﬁdence increases, investment and consumption gain more control, driving up the price index. Hence, one might assert the following: Assertion 3. Market conﬁdence (w) has positive impacts on the price index (z), whereas the price index (z) has negative impact on the market conﬁdence (w) . The rising interest rate and high price index generally result in higher investment costs than before. In other words, as the interest rate and price index increase, the investment command declines and market conﬁdence deteriorates. Thus, one might make the following assertion: Assertion 4. The interest rate (x), the investment command (y), and the price index are all crucial to the market conﬁdence (w). 2 Fractional-order ﬁnancial model with market conﬁdence Moreover, there is a pro- portionately negative association between the product xyz and the changing rate of w. The ﬁnancial system may be illustrated as in [28] by introducing the market con- ﬁdence into the system (27) and utilising the dimensionless transformation. ˙x = z + (y −a) x + m1ω, ˙y = 1 −by −x2 + m2ω, ˙z = −x −cz + m3ω, ˙ω = −xyz, (33) (33) where the speciﬁcations of x, y, z, a, b, and c are the same as those in system (27), ω stands for market conﬁdence, and m1, m2, and m3 are impact factors. The values of the parameters have been taken from [28] as a = 3, b = 0.1, c = 1, m1 = −8.4, m2 = 6.4, m3 = −2.2. 20 Dynamics of fractional-order ﬁnancial system with market conﬁdence We utilise fractional calculus to characterize the long-term memory impact in the ﬁnancial system (33). Accordingly, the fractional-order version of system (33) can be expressed as [28], Dα1x(t) = z + (y −a) x + m1ω, Dα2y(t) = 1 −by −x2 + m2ω, Dα3z(t) = −x −cz + m3ω, Dα4ω(t) = −xyz, (34) (34) Table 3: Stability analysis of fractional order chaotic ﬁnancial system Table 3: Stability analysis of fractional order chaotic ﬁnancial system Equilibrium points Eigen values Nature E1(−1, 0, 0, 0) 0.1955 ± 1.6262i, −1.4909, 0 Non-hyperbolic E2(1, 0, 0, 0) −0.5500 ± 1.3407i, 0, 0 Non-hyperbolic E3(0, 1.4209, −1.1260, −0.1340) 0.8823 ± 3.5876i, −0.7218 ± 0.5945 saddle point of index 2 E4(−3.8182, −4.5125, 0, 2.0511) −4.4704 ± 4.3838i, 5.6514, −2.3232 saddle point of index 1 As seen in Table 3, the equilibrium points E1 and E2 are non-hyperbolic, which is the case in which bifurcation exists. Besides that, E3 and E4 are saddle points of index 2 and saddle points of index 1 respectively, indicating that the system has a 2-scroll attractor and is chaotic. Since, only equilibrium point E2 is saddle points of index 2, therefore minj\|arg(Ej)\| = 1.3297 (35) (35) As a result, from Eq. (30) if α < 2 πminj\|arg(Ej)\| ≈0.8465, the ﬁnancial system (34) will behaves normally and for α > 0.8465, the system demonstrate chaos. Using the initial values x(0) = −0.01, y(0) = 0.5, z(0) = 0.004 and ω(0) = −0.003 with α = 0.85, the Lyapunov exponent for the fractional-order ﬁnancial system with market conﬁdence (34) is computed as [see also Fig. 12]. Dynamics of fractional-order ﬁnancial system with market conﬁdence LE = (2.0561, −1.3817, −1.4153, −2.1683) (36) (36) Lemma 1 leads us to the conclusion that r = 2 in this situation, and the Lya- punov dimension can be evaluated as, Lemma 1 leads us to the conclusion that r = 2 in this situation, and the Lya- punov dimension can be evaluated as, 21 DL = r + 1 \|hr+1\| r X j=1 hr = 2.4765 LEs of the 4-D financial system with market confidence for t [0,300] and =0.85 0 50 100 150 200 250 300 t -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 LEs LE1 LE2 LE3 LE4 Figure 8: Dynamical behavior of LEs with respect to time LEs of the 4-D financial system with market confidence for t [0,300] and =0.85 0 50 100 150 200 250 300 t -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 LEs LE1 LE2 LE3 LE4 LEs of the 4-D financial system with market confidence for t [0,300] and =0.85 Figure 8: Dynamical behavior of LEs with respect to time 22 -3 -2.95 -2.9 -2.85 -2.8 -2.75 -2.7 x -0.1 -0.08 -0.06 -0.04 -0.02 0 0.02 0.04 0.06 0.08 0.1 y (a) α = 0.85 -5 -4.5 -4 -3.5 -3 -2.5 -2 -1.5 -1 -0.5 0 x -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 y (b) α = 0.90 -6 -5 -4 -3 -2 -1 0 1 x -3 -2 -1 0 1 2 3 4 y (c) α = 0.94 -6 -5 -4 -3 -2 -1 0 1 x -4 -3 -2 -1 0 1 2 3 4 y (d) α = 0.99 Figure 9: Phase portraits for 4-D fractional-order ﬁnancial system in x −y plane for diﬀerent values of α -5 -4.5 -4 -3.5 -3 -2.5 -2 -1.5 -1 -0.5 0 x -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 y (b) α = 0.90 -3 -2.95 -2.9 -2.85 -2.8 -2.75 -2.7 x -0.1 -0.08 -0.06 -0.04 -0.02 0 0.02 0.04 0.06 0.08 0.1 y (a) α = 0.85 (b) α = 0.90 -6 -5 -4 -3 -2 -1 0 1 x -3 -2 -1 0 1 2 3 4 y (c) α = 0.94 -6 -5 -4 -3 -2 -1 0 1 x -4 -3 -2 -1 0 1 2 3 4 y (d) α = 0.99 (c) α = 0.94 (d) α = 0.99 Figure 9: Phase portraits for 4-D fractional-order ﬁnancial system in x −y plane for diﬀerent values of α 23 Table 4: Error in TBF solution of ﬁnancial model with market conﬁdence for α1 = α2 = α3 = 1. Dynamics of fractional-order ﬁnancial system with market conﬁdence Absolute error between TBF and ode45 solver t \|x(t) −˜x(t)\| \|y(t) −˜y(t)\| \|z(t) −˜z(t)\| \|w(t) −˜y(t)\| 0 3.42475e-12 9.50351e-14 2.51917e-14 1.75596e-11 0.1 2.41124e-5 1.98598e-7 1.26156e-5 6.19168e-8 0.2 3.81479e-5 5.79484e-7 1.88603e-5 9.91398e-8 0.3 4.55985e-5 1.03896e-6 2.09559e-5 1.22050e-7 0.4 4.87750e-5 1.52979e-6 2.04281e-5 1.37401e-7 0.5 4.92119e-5 2.03147e-6 1.83196e-5 1.48634e-7 0.6 4.79301e-5 2.53619e-6 1.53381e-5 1.56889e-7 0.7 4.56083e-5 3.04223e-6 1.19597e-5 1.61954e-7 0.8 4.26963e-5 3.55083e-6 8.49819e-6 1.62965e-7 0.9 3.94899e-5 4.06481e-6 5.15477e-6 1.58860 1.0 3.61815e-5 4.58784e-6 2.05278e-6 1.48645 -2.5 -2 -1.5 -1 -0.5 0 Market Confidence (w) 10-3 0.0125 0.013 0.0135 0.014 0.0145 0.015 0.0155 0.016 0.0165 0.017 Interest Rate (x) Figure 10: Impact of Interest rate (x) on Market conﬁdence (w). 24 Table 4: Error in TBF solution of ﬁnancial model with market conﬁdence for α1 = α2 = α3 = 1. Absolute error between TBF and ode45 solver t \|x(t) −˜x(t)\| \|y(t) −˜y(t)\| \|z(t) −˜z(t)\| \|w(t) −˜y(t)\| 0 3.42475e-12 9.50351e-14 2.51917e-14 1.75596e-11 0.1 2.41124e-5 1.98598e-7 1.26156e-5 6.19168e-8 0.2 3.81479e-5 5.79484e-7 1.88603e-5 9.91398e-8 0.3 4.55985e-5 1.03896e-6 2.09559e-5 1.22050e-7 0.4 4.87750e-5 1.52979e-6 2.04281e-5 1.37401e-7 0.5 4.92119e-5 2.03147e-6 1.83196e-5 1.48634e-7 0.6 4.79301e-5 2.53619e-6 1.53381e-5 1.56889e-7 0.7 4.56083e-5 3.04223e-6 1.19597e-5 1.61954e-7 0.8 4.26963e-5 3.55083e-6 8.49819e-6 1.62965e-7 0.9 3.94899e-5 4.06481e-6 5.15477e-6 1.58860 1.0 3.61815e-5 4.58784e-6 2.05278e-6 1.48645 -2.5 -2 -1.5 -1 -0.5 0 Market Confidence (w) 10-3 0.0125 0.013 0.0135 0.014 0.0145 0.015 0.0155 0.016 0.0165 0.017 Interest Rate (x) Figure 10: Impact of Interest rate (x) on Market conﬁdence (w). -2.5 -2 -1.5 -1 -0.5 0 Market Confidence (w) 10-3 0.0125 0.013 0.0135 0.014 0.0145 0.015 0.0155 0.016 0.0165 0.017 Interest Rate (x) Figure 10: Impact of Interest rate (x) on Market conﬁdence (w). 24 -2.5 -2 -1.5 -1 -0.5 0 Market Confidence (w) 10-3 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 Investment Command (y) Figure 11: Impact of Investment command (y) on Market conﬁdence (w). -2.5 -2 -1.5 -1 -0.5 0 Market Confidence (w) 10-3 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 Investment Command (y) Figure 11: Impact of Investment command (y) on Market conﬁdence (w) Figure 11: Impact of Investment command (y) on Market conﬁdence (w). -2.5 -2 -1.5 -1 -0.5 0 Market Confidence (w) 10-3 -11 -10.5 -10 -9.5 -9 -8.5 -8 -7.5 -7 -6.5 Price Index (z) 10-3 Figure 12: Impact of Price index (z) on Market conﬁdence (w). Dynamics of fractional-order ﬁnancial system with market conﬁdence -2.5 -2 -1.5 -1 -0.5 0 Market Confidence (w) 10-3 -11 -10.5 -10 -9.5 -9 -8.5 -8 -7.5 -7 -6.5 Price Index (z) 10-3 Figure 12: Impact of Price index (z) on Market conﬁdence (w). 25 A key term called market conﬁdence(w) has been included in the fractional-order ﬁnancial system in order to observe the change in the dynamical behaviour of the sys- tem (28). The system (34) exhibit non-linear dynamics that are sensitively dependent on the initial conditions x(0) = −0.01, y(0) = 0.5, z(0) = 0.004 and ω(0) = −0.003 and the contained parameters. The stability analysis of the fractional-order ﬁnancial system with market conﬁdence is discussed in Table ??, which indicates the existence of bifurcation and chaos. We have also calculated the Lyapunov exponent (36) and the Lyapunov dimension at α = 0.85 since the system has a positive LE (36), which ensures that the system has a chaotic solution. Moreover the LEs of the system (34) with repect to time are shown in Fig. 12. Further, 2D and 3D phase portraits for various values of α as depicted in Fig. 9 and in appendix Figs. A4 - A12 have been used to illustrate the chaotic solutions for commensurate fractional-order ﬁnancial system with market conﬁdence (34). As stated in Assertions 1, 2, 3, and 4, Figs 10, 11, and 12 provide a visual representation of how market conﬁdence aﬀects interest rate, investment command, and price index. 5 Conclusion Financial ﬂuctuations are important to be recognized and forecasted apriori since it is related to the stability or imminent instability of economy. The two fractional-order ﬁnancial models with and without market conﬁdence are being presented, which are generalizations of their corresponding integer-order models. Triangular basis function has been utilized for model solution. The impact of fractional-order on the dynam- ics of such systems are investigated, and it has been discovered that an appropriate fractional-order would either boost or suppress the appearance of chaotic or periodic motions. The complicated dynamics of the system is examined using a change in fractional order as appropriate. The lowest order at which chaos can exist in each situation is identiﬁed while the period doubling route to chaos has been estimated. The method is projected to have numerous applications in diﬀerent chaos-based in- formation systems. It is envisaged that the concept of fractional nonlinear systems that also contain chaos will help us better comprehend the complicated behaviour of ﬁnancial systems. 6 Data availability No data from open sources are used for current study. 26 Appendix Appendix 1.55 1.6 1.65 1.7 1.75 1.8 y -0.7 -0.69 -0.68 -0.67 -0.66 -0.65 -0.64 -0.63 -0.62 -0.61 z (a) α = 0.88 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 y -0.85 -0.8 -0.75 -0.7 -0.65 -0.6 -0.55 -0.5 -0.45 z (b) α = 0.91 0 0.5 1 1.5 2 2.5 3 3.5 y -1.5 -1 -0.5 0 0.5 1 1.5 z (c) α = 0.92 -0.5 0 0.5 1 1.5 2 2.5 3 3.5 y -1.5 -1 -0.5 0 0.5 1 1.5 z (d) α = 0.97 Figure A1: Phase portraits for 3-D fractional-order ﬁnancial system in y −z plane for diﬀerent values of α 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 y -0.85 -0.8 -0.75 -0.7 -0.65 -0.6 -0.55 -0.5 -0.45 z (b) α = 0.91 1.55 1.6 1.65 1.7 1.75 1.8 y -0.7 -0.69 -0.68 -0.67 -0.66 -0.65 -0.64 -0.63 -0.62 -0.61 z (a) α = 0.88 (b) α = 0.91 -0.5 0 0.5 1 1.5 2 2.5 3 3.5 y -1.5 -1 -0.5 0 0.5 1 1.5 z (d) α = 0.97 0 0.5 1 1.5 2 2.5 3 3.5 y -1.5 -1 -0.5 0 0.5 1 1.5 z (c) α = 0.92 (d) α = 0.97 (c) α = 0.92 Figure A1: Phase portraits for 3-D fractional-order ﬁnancial system in y −z plane for diﬀerent values of α 27 0.74 0.76 0.78 0.8 0.82 0.84 0.86 0.88 0.9 0.92 x -0.7 -0.69 -0.68 -0.67 -0.66 -0.65 -0.64 -0.63 -0.62 -0.61 z (a) α = 0.88 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 x -0.85 -0.8 -0.75 -0.7 -0.65 -0.6 -0.55 -0.5 -0.45 z (b) α = 0.91 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 x -1.5 -1 -0.5 0 0.5 1 1.5 z (c) α = 0.92 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 x -1.5 -1 -0.5 0 0.5 1 1.5 z (d) α = 0.97 Figure A2: Phase portraits for 3-D fractional-order ﬁnancial system in x −z plane for diﬀerent values of α 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 x -0.85 -0.8 -0.75 -0.7 -0.65 -0.6 -0.55 -0.5 -0.45 z (b) α = 0.91 0.74 0.76 0.78 0.8 0.82 0.84 0.86 0.88 0.9 0.92 x -0.7 -0.69 -0.68 -0.67 -0.66 -0.65 -0.64 -0.63 -0.62 -0.61 z (a) α = 0.88 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 x -1.5 -1 -0.5 0 0.5 1 1.5 z (c) α = 0.92 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 x -1.5 -1 -0.5 0 0.5 1 1.5 z (d) α = 0.97 (c) α = 0.92 (d) α = 0.97 Figure A2: Phase portraits for 3-D fractional-order ﬁnancial system in x −z plane for diﬀerent values of α 28 (a) α = 0.88 (b) α = 0.90 (c) α = 0.92 (d) α = 0.97 Figure A3: Phase portraits for 3-D fractional-order ﬁnancial system in 3-D of state variable x −y −z for diﬀerent values of α (a) α = 0.88 (b) α = 0.90 (b) α = 0.90 (c) α = 0.92 (d) α = 0.97 (d) α = 0.97 (c) α = 0.92 Figure A3: Phase portraits for 3-D fractional-order ﬁnancial system in 3-D of state variable x −y −z for diﬀerent values of α 29 -3 -2.95 -2.9 -2.85 -2.8 -2.75 -2.7 x 0.456 0.458 0.46 0.462 0.464 0.466 0.468 0.47 z (a) α = 0.85 -5 -4.5 -4 -3.5 -3 -2.5 -2 -1.5 -1 -0.5 0 x 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 z (b) α = 0.90 -6 -5 -4 -3 -2 -1 0 1 x 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 z (c) α = 0.94 -6 -5 -4 -3 -2 -1 0 1 x 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 z (d) α = 0.99 Figure A4: Phase portraits for 4-D fractional-order ﬁnancial system in x −z plane for diﬀerent values of α -5 -4.5 -4 -3.5 -3 -2.5 -2 -1.5 -1 -0.5 0 x 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 z (b) α = 0.90 -3 -2.95 -2.9 -2.85 -2.8 -2.75 -2.7 x 0.456 0.458 0.46 0.462 0.464 0.466 0.468 0.47 z (a) α = 0.85 -6 -5 -4 -3 -2 -1 0 1 x 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 z (c) α = 0.94 -6 -5 -4 -3 -2 -1 0 1 x 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 z (d) α = 0.99 (d) α = 0.99 Figure A4: Phase portraits for 4-D fractional-order ﬁnancial system in x −z plane for diﬀerent values of α 30 -3 -2.95 -2.9 -2.85 -2.8 -2.75 -2.7 x 1 1.02 1.04 1.06 1.08 1.1 1.12 1.14 w (a) α = 0.85 -5 -4.5 -4 -3.5 -3 -2.5 -2 -1.5 -1 -0.5 0 x 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 w (b) α = 0.90 -6 -5 -4 -3 -2 -1 0 1 x -0.5 0 0.5 1 1.5 2 2.5 w (c) α = 0.94 -6 -5 -4 -3 -2 -1 0 1 x -0.5 0 0.5 1 1.5 2 2.5 w (d) α = 0.99 Figure A5: Phase portraits for 4-D fractional-order ﬁnancial system in x −w plane for diﬀerent values of α -3 -2.95 -2.9 -2.85 -2.8 -2.75 -2.7 x 1 1.02 1.04 1.06 1.08 1.1 1.12 1.14 w (a) α = 0.85 -5 -4.5 -4 -3.5 -3 -2.5 -2 -1.5 -1 -0.5 0 x 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 w (b) α = 0.90 -6 -5 -4 -3 -2 -1 0 1 x -0.5 0 0.5 1 1.5 2 2.5 w (d) α = 0.99 -6 -5 -4 -3 -2 -1 0 1 x -0.5 0 0.5 1 1.5 2 2.5 w (c) α = 0.94 (d) α = 0.99 (c) α = 0.94 Figure A5: Phase portraits for 4-D fractional-order ﬁnancial system in x −w plane for diﬀerent values of α 31 -0.1 -0.08 -0.06 -0.04 -0.02 0 0.02 0.04 0.06 0.08 0.1 y 0.456 0.458 0.46 0.462 0.464 0.466 0.468 0.47 z (a) α = 0.85 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 y 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 z (b) α = 0.90 -3 -2 -1 0 1 2 3 4 y 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 z (c) α = 0.94 -4 -3 -2 -1 0 1 2 3 4 y 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 z (d) α = 0.99 Figure A6: Phase portraits for 4-D fractional-order ﬁnancial system in y −z plane for diﬀerent values of α -0.1 -0.08 -0.06 -0.04 -0.02 0 0.02 0.04 0.06 0.08 0.1 y 0.456 0.458 0.46 0.462 0.464 0.466 0.468 0.47 z (a) α = 0.85 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 y 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 z (b) α = 0.90 -3 -2 -1 0 1 2 3 4 y 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 z (c) α = 0.94 -4 -3 -2 -1 0 1 2 3 4 y 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 z (d) α = 0.99 (d) α = 0.99 (c) α = 0.94 Figure A6: Phase portraits for 4-D fractional-order ﬁnancial system in y −z plane for diﬀerent values of α 32 -0.1 -0.08 -0.06 -0.04 -0.02 0 0.02 0.04 0.06 0.08 0.1 y 1 1.02 1.04 1.06 1.08 1.1 1.12 1.14 w (a) α = 0.85 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 y 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 w (b) α = 0.90 -3 -2 -1 0 1 2 3 4 y -0.5 0 0.5 1 1.5 2 2.5 w (c) α = 0.94 -4 -3 -2 -1 0 1 2 3 4 y -0.5 0 0.5 1 1.5 2 2.5 w (d) α = 0.99 Figure A7: Phase portraits for 4-D fractional-order ﬁnancial system in y −w plane for diﬀerent values of α -0.1 -0.08 -0.06 -0.04 -0.02 0 0.02 0.04 0.06 0.08 0.1 y 1 1.02 1.04 1.06 1.08 1.1 1.12 1.14 w (a) α = 0.85 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 y 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 w (b) α = 0.90 -3 -2 -1 0 1 2 3 4 y -0.5 0 0.5 1 1.5 2 2.5 w (c) α = 0.94 -4 -3 -2 -1 0 1 2 3 4 y -0.5 0 0.5 1 1.5 2 2.5 w (d) α = 0.99 (c) α = 0.94 (d) α = 0.99 Figure A7: Phase portraits for 4-D fractional-order ﬁnancial system in y −w plane for diﬀerent values of α 33 0.456 0.458 0.46 0.462 0.464 0.466 0.468 0.47 z 1 1.02 1.04 1.06 1.08 1.1 1.12 1.14 w (a) α = 0.85 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 z 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 w (b) α = 0.90 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 z -0.5 0 0.5 1 1.5 2 2.5 w (c) α = 0.94 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 z -0.5 0 0.5 1 1.5 2 2.5 w (d) α = 0.99 Figure A8: Phase portraits for 4-D fractional-order ﬁnancial system in z −w plane for diﬀerent values of α 0.456 0.458 0.46 0.462 0.464 0.466 0.468 0.47 z 1 1.02 1.04 1.06 1.08 1.1 1.12 1.14 w (a) α = 0.85 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 z 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 w (b) α = 0.90 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 z -0.5 0 0.5 1 1.5 2 2.5 w (c) α = 0.94 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 z -0.5 0 0.5 1 1.5 2 2.5 w (d) α = 0.99 (d) α = 0.99 Figure A8: Phase portraits for 4-D fractional-order ﬁnancial system in z −w plane for diﬀerent values of α 34 (a) α = 0.85 (b) α = 0.90 (c) α = 0.94 (d) α = 0.99 Figure A9: Phase portraits for 4-D fractional-order ﬁnancial system in 3-D of state variable x −y −z for diﬀerent values of α (a) α = 0.85 (b) α = 0.90 (b) α = 0.90 (b) α = 0.90 (c) α = 0.94 (d) α = 0.99 (d) α = 0.99 (c) α = 0.94 Figure A9: Phase portraits for 4-D fractional-order ﬁnancial system in 3-D of state variable x −y −z for diﬀerent values of α 35 (a) α = 0.85 (b) α = 0.90 (c) α = 0.94 (d) α = 0.99 Figure A10: Phase portraits for 4-D fractional-order ﬁnancial system in 3-D of state variable x −y −w for diﬀerent values of α (a) α = 0.85 (b) α = 0.90 (b) α = 0.90 (b) α = 0.90 (c) α = 0.94 (d) α = 0.99 (d) α = 0.99 (c) α = 0.94 Figure A10: Phase portraits for 4-D fractional-order ﬁnancial system in 3-D of state variable x −y −w for diﬀerent values of α 36 (a) α = 0.85 (b) α = 0.90 (c) α = 0.94 (d) α = 0.99 Figure A11: Phase portraits for 4-D fractional-order ﬁnancial system in 3-D of state variable x −z −w for diﬀerent values of α (a) α = 0.85 (b) α = 0.90 (c) α = 0.94 (d) α = 0.99 (d) α = 0.99 (c) α = 0.94 Figure A11: Phase portraits for 4-D fractional-order ﬁnancial system in 3-D of state variable x −z −w for diﬀerent values of α 37 (a) α = 0.85 (b) α = 0.90 (c) α = 0.94 (d) α = 0.99 Figure A12: Phase portraits for 4-D fractional-order ﬁnancial system in 3-D of state variable y −z −w for diﬀerent values of α (b) α = 0.90 (a) α = 0.85 (b) α = 0.90 (b) α = 0.90 (d) α = 0.99 (c) α = 0.94 Figure A12: Phase portraits for 4-D fractional-order ﬁnancial system in 3-D of state variable y −z −w for diﬀerent values of α 38 References [1] S. 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Kundu, Numerical solution of multi-order fractional diﬀeren- tial equations using generalized triangular function operational matrices, Applied Mathematics and Computation 263 (2015) 189–203. [37] S. K. Damarla, M. Kundu, Piecewise linear approximate solution of fractional or- der non-stiﬀand stiﬀdiﬀerential-algebraic equations by orthogonal hybrid func- tions, arXiv preprint arXiv:1801.06970 (2018). 41 [38] S. K. Damarla, M. Kundu, Fractional Order Processes: Simulation, Identiﬁca- tion, and Control, CRC Press, 2018. [39] D. Matignon, Stability results for fractional diﬀerential equations with applica- tions to control processing, in: Computational engineering in systems applica- tions, Vol. 2, Lille, France, 1996, pp. 963–968. Corresponding author Correspondence to M. Kundu Correspondence to M. Kundu Department of Mathematics, National Institute of Technology Rourkela, Odisha, India Department of Mathematics, National Institute of Technology Rourkela, Odisha, India Shweta Dubey, and S. Chakraverty Shweta Dubey, and S. Chakraverty Contributions Shweta Dubey conducted research, prepared a manuscript, and analyzed the model. M. Kundu Developed the idea, analyzed the results, and reviewed the manuscript. The manuscript has been supervised and reviewed by S. Chakraverty. Authors and Aﬃliations Department of Mathematics, National Institute of Technology Rourkela, Odisha, India Shweta Dubey, and S. Chakraverty Department of Chemical Engineering, National Institute of Technology Rourkela, Odisha, India M. Kundu Acknowledgment First author acknowledges the GATE fellowship provided by MHRD and second au- thor duly acknowledges the funding provided by MATRICS-SERB (MTR/2019/000612 dated 6 February, 2020). 42 Conﬂict of interest The authors declare that we have no conﬂicts of interests about the publication of this paper. 43 43
https://openalex.org/W2099108899	https://hal.archives-ouvertes.fr/hal-00805510v2/document	English	null	On some implicit and semi-implicit staggered schemes for the shallow water and Euler equations	Modélisation mathématique et analyse numérique	2,014	cc-by	37,909	On some implicit and semi-implicit staggered schemes for the shallow water and Euler equations Raphaele Herbin, Walid Kheriji, Jean-Claude Latché To cite this version: Raphaele Herbin, Walid Kheriji, Jean-Claude Latché. On some implicit and semi-implicit staggered schemes for the shallow water and Euler equations. ESAIM: Mathematical Modelling and Numerical Analysis, 2014, 48 (6), pp.1807-1857. 10.1051/m2an/2014021. hal-00805510v2 To cite this version: Raphaele Herbin, Walid Kheriji, Jean-Claude Latché. On some implicit and semi-implicit staggered schemes for the shallow water and Euler equations. ESAIM: Mathematical Modelling and Numerical Analysis, 2014, 48 (6), pp.1807-1857. 10.1051/m2an/2014021. hal-00805510v2 Distributed under a Creative Commons Attribution 4.0 International License ON SOME IMPLICIT AND SEMI-IMPLICIT STAGGERED SCHEMES FOR THE SHALLOW WATER AND EULER EQUATIONS. R. Herbin1, W. Kheriji2 and J.-C. Latch´e3 Abstract. In this paper, we propose implicit and semi-implicit in time ﬁnite volume schemes for the barotropic Euler equations (hence, as a particular case, for the shallow water equations) and for the full Euler equations, based on staggered discretizations. For structured meshes, we use the MAC ﬁnite volume scheme, and, for general mixed quadrangular/hexahedral and simplicial meshes, we use the discrete unknowns of the Rannacher-Turek or Crouzeix-Raviart ﬁnite elements. We ﬁrst show that a solution to each of these schemes satisﬁes a discrete kinetic energy equation. In the barotropic case, a solution also satisﬁes a discrete elastic potential balance; integrating these equations over the domain readily yields discrete counterparts of the stability estimates which are known for the continuous problem. In the case of the full Euler equations, the scheme relies on the discretization of the internal energy balance equation, which oﬀers two main advantages: ﬁrst, we avoid the space discretization of the total energy, which involves cell-centered and face-centered variables; second, we obtain an algorithm which boils down to a usual pressure correction scheme in the incompressible limit. Consistency (in a weak sense) with the original total energy conservative equation is obtained thanks to corrective terms in the internal energy balance, designed to compensate numerical dissipation terms appearing in the discrete kinetic energy inequality. It is then shown in the 1D case, that, supposing the convergence of a sequence of solutions, the limit is an entropy weak solution of the continuous problem in the barotropic case, and a weak solution in the full Euler case. Finally, we present numerical results which conﬁrm this theory. 1991 Mathematics Subject Classiﬁcation. 35Q31,65N12,76M10,76M12. April 2014. HAL Id: hal-00805510 https://hal.science/hal-00805510v2 Submitted on 5 Mar 2016 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Mathematical Modelling and Numerical Analysis Mod´elisation Math´ematique et Analyse Num´erique Will be set by the publisher 3 Institut de Radioprotection et de Sˆuret´e Nucl´eaire (IRSN), BP 3, 13115 Saint-Paul-lez-Durance cedex, France (jean-claude.latche@irsn.fr) 1 Aix-Marseille Universit´e, CNRS, Centrale Marseille, I2M, UMR 7373, 13453 Marseille, France, raphaele.herbin@univ-amu.fr 1. Introduction We ﬁrst deal with the barotropic Euler equations, which read: We ﬁrst deal with the barotropic Euler equations, which read: ∂t ρ + div(ρ u) = 0, (1a) ∂t (ρ u) + div(ρ u ⊗u) + ∇p = 0, (1b) ρ ≥0, p = ℘(ρ) = ργ, (1c) (1a) (1b) (1c) (1c) where t stands for the time, ρ, u, p, are the density, velocity, pressure in the ﬂow, and γ > 1 is a coeﬃcient speciﬁc to the considered ﬂuid. For p = ργ, γ = 2 and ρ = h, we also get the shallow water (or Saint-Venant) equations. the full Euler equations, which are obtained from the complete Navier–Stokes equations Then, we address the full Euler equations, which are obtained from the complete Navier–Stokes ∂tρ + div(ρ u) = 0, (2a) ∂t(ρ u) + div(ρ u ⊗u) + ∇p −div(τ(u)) = 0, (2b) ∂t(ρ E) + div(ρ E u) + div(p u) = 0, (2c) p = (γ −1) ρ e, E = 1 2\|u\|2 + e, (2d) (2a) (2b) (2d) by neglecting the viscous stress tensor, i.e. setting τ(u) = 0. In this system, E and e stand for the total energy and the internal energy in the ﬂow, respectively. Both problems (1) and (2) are supposed to be posed over Ω× (0, T ), where Ωis an open bounded connected subset of Rd, 1 ≤d ≤3, and (0, T ) is a ﬁnite time interval. System (1) (resp. (2)) is complemented by initial conditions for ρ and u (resp. ρ, e and u); these initial conditions are denoted by ρ0 and u0 (resp. ρ0, e0 and u0), with ρ0 > 0 and e0 > 0. In both cases, we consider the boundary condition u · n = 0 at any time and a.e. on ∂Ω, where n stands for the normal vector to the boundary. This paper is organized as follows. We begin by describing the space discretizations (Section 2). We then address the barotropic case in Section 3; we introduce a fully implicit scheme and a pressure correction scheme (sections 3.1 and 3.2 respectively). We prove the stability of each algorithm and their consistency in the one- dimensional case, in the Lax-Wendroﬀsense. We ﬁnally propose in Section 4 a pressure correction scheme for the full Euler equations. We ﬁrst give the general form of the algorithm (Section 4.2). 1. Introduction The objective pursued in this work is to develop and analyze a class of eﬃcient numerical schemes for the simulation of compressible ﬂows at all Mach number regimes. To this purpose, our basic choice is to extend algorithms that are classical in the incompressible framework, namely pressure correction schemes based on (inf-sup stable) staggered discretizations. Keywords and phrases: ﬁnite volumes, ﬁnite elements, staggered, pressure correction, Euler equations, shallow-water equations, compressible ﬂows, analysis. 1 Aix-Marseille Universit´e, CNRS, Centrale Marseille, I2M, UMR 7373, 13453 Marseille, France, raphaele.herbin@univ-amu.fr 2 Institut de Radioprotection et de Sˆuret´e Nucl´eaire (IRSN), BP 3, 13115 Saint-Paul-lez-Durance cedex, France (walid.kheriji@irsn.fr) 3 Institut de Radioprotection et de Sˆuret´e Nucl´eaire (IRSN), BP 3, 13115 Saint-Paul-lez-Durance cedex, France (jean-claude.latche@irsn.fr) 3 Institut de Radioprotection et de Sˆuret´e Nucl´eaire (IRSN), BP 3, 13115 Saint-Paul-lez-Durance cedex, France (jean-claude.latche@irsn.fr) c ⃝EDP Sciences, SMAI 1999 2 TITLE WILL BE SET BY THE PUBLISHER The fractional step strategy involving an elliptic pressure correction step has been recognized to yield algo- rithms which are not limited by stringent stability conditions (such as CFL conditions based on the celerity of the fastest waves) since the ﬁrst attempts to build ”all ﬂow velocity” schemes in the late sixties [23] or in the early seventies [24]; these algorithms may be seen as an extension to the compressible case of the celebrated MAC scheme, introduced some years before [25]. These seminal papers have been the starting point for the develop- ment of numerous schemes, using staggered ﬁnite volume space discretizations [4,6,34,35,38,41,47,64–69,71], colocated ﬁnite volumes [2, 10, 32, 33, 36, 37, 39, 43, 48–51, 54, 57, 59, 61, 70] or ﬁnite elements [3, 46, 52, 72]. Al- gorithms proposed in these works may be essentially implicit-in-time, and the pressure correction step is then an ingredient of a SIMPLE-like iterative procedure, or only semi-implicit, with a single (or a limited number of) prediction and correction step(s), as in projection methods for incompressible ﬂows (see [7,60] for seminal works and [19] for a review of most of the variants). The schemes which we propose in the present paper fall in this latter class. 1. Introduction The scheme solves the internal energy balance, which oﬀers three advantages: ﬁrst, we avoid the space discretization of the total energy, which involves cell-centered and face-centered variables; second, we obtain an algorithm which boils down to a usual pressure correction scheme in the incompressible limit; third, this relation implies that the internal energy remains positive. Consistency (again in the Lax-Wendroﬀsense) with the original total energy conservative equation is obtained thanks to corrective terms in the internal energy balance, designed to TITLE WILL BE SET BY THE PUBLISHER 3 compensate numerical dissipation terms appearing in the discrete kinetic energy inequality. It is then shown in the 1D case (Section 4.3), that, supposing the convergence of a sequence of solutions, the limit is a weak solution of the continuous problem. Finally, we present some numerical tests in Section 4.4 for the case of the Euler equations (note that the numerical study of the correction pressure scheme in the barotropic case was performed in [27]). In several theoretical developments, we are lead to use a derived form of a discrete ﬁnite volume convection operator (for instance, typically, a convection operator for the kinetic energy, possibly with residual terms, obtained from the ﬁnite volume discretization of the convection of the velocity components); the proofs of various related discrete identities are given in the Appendix. 2. Meshes and unknowns Let M be a decomposition of the domain Ω, supposed to be regular in the usual sense of the ﬁnite element literature (see e.g. [8]). The cells may be: - for a general domain Ω, either convex quadrilaterals (d = 2) or hexahedra (d = 3) or simplices, both types of cells being possibly combined in a same mesh, - for a general domain Ω, either convex quadrilaterals (d = 2) or hexahedra (d = 3) or simplices, both types of cells being possibly combined in a same mesh, - for a domain whose boundaries are hyperplanes normal to a coordinate axis, rectangles (d = 2) or rectangular parallelepipeds (d = 3) (the faces of which, of course, are then also necessarily normal to a coordinate axis). - for a domain whose boundaries are hyperplanes normal to a coordinate axis, rectangles (d = 2) or rectangular parallelepipeds (d = 3) (the faces of which, of course, are then also necessarily normal to a coordinate axis). By E and E(K) we denote the set of all (d−1)-faces σ of the mesh and of the element K ∈M respectively. The set of faces included in Ω(resp. in the boundary ∂Ω) is denoted by Eint (resp. Eext); a face σ ∈Eint separating the cells K and L is denoted by σ = K\|L. The outward normal vector to a face σ of K is denoted by nK,σ. For K ∈M and σ ∈E, we denote by \|K\| the measure of K and by \|σ\| the (d −1)-measure of the face σ. For 1 ≤i ≤d, we denote by E(i) ⊂E and E(i) ext ⊂Eext the subset of the faces of E and Eext respectively which are perpendicular to the ith unit vector of the canonical basis of Rd. By E and E(K) we denote the set of all (d−1)-faces σ of the mesh and of the element K ∈M respectively. The set of faces included in Ω(resp. in the boundary ∂Ω) is denoted by Eint (resp. Eext); a face σ ∈Eint separating the cells K and L is denoted by σ = K\|L. The outward normal vector to a face σ of K is denoted by nK,σ. For K ∈M and σ ∈E, we denote by \|K\| the measure of K and by \|σ\| the (d −1)-measure of the face σ. 2. Meshes and unknowns For 1 ≤i ≤d, we denote by E(i) ⊂E and E(i) ext ⊂Eext the subset of the faces of E and Eext respectively which are perpendicular to the ith unit vector of the canonical basis of Rd. The space discretization is staggered. In the case of rectangular or orthogonal parallelepipedic meshes, we use the Marker-And Cell (MAC) scheme [24,25]. For mixed simplicial or quadrilateral/hexahedral meshes, we use the discrete unknowns of the Crouzeix-Raviart [9] and Ranacher-Turek [58] ﬁnite element spaces; however, the associated ﬁnite element formulation is not used here (but it would readily provide a discretization of the diﬀusion terms, in the Navier-Stokes case [1,16]). For all these space discretizations, the degrees of freedom for the scalar unknowns are associated with the cells of the mesh M; these are the discrete pressure and density unknowns, and, for the full Euler equations, the internal energy unknowns, which are denoted by: pK, ρK, eK, K ∈M . t us then turn to the degrees of freedom for the velocity (i.e. the discrete velocity unknowns). - Rannacher-Turek or Crouzeix-Raviart discretizations – The discrete velocity unknowns are located at the center of the faces of the mesh, and represent the average of the velocity through the face. The set of discrete velocity unknowns reads: {uσ,i, σ ∈E, 1 ≤i ≤d}. - MAC discretization – The degrees of freedom for the ith component of the velocity are located at the center of the faces σ ∈E(i), so that the set of discrete velocity unknowns reads: uσ,i, σ ∈E(i), 1 ≤i ≤d . uσ,i, σ ∈E(i), 1 ≤i ≤d . We now introduce a dual mesh, which will be used for the ﬁnite volume approximation of the time derivative and convection terms in the momentum balance equation. TITLE WILL BE SET BY THE PUBLISHER 4 Dσ Dσ′ σ′ = K\|M K L M \|σ\| σ = K\|L ǫ = Dσ\|Dσ′ Dσ K L σ = K\|L σ′ ǫ = Dσ\|Dσ′ Figure 1. Notations for control volumes and dual cells – Left: Finite Elements (the present sketch illustrates the possibility, implemented in our software (CALIF3S [5]), of mixing simpli- cial (Crouzeix-Raviart) and quadrangular (Rannacher-Turek) cells) – Right: MAC discretiza- tion, dual cell for the y-component of the velocity. Figure 1. 2. Meshes and unknowns Notations for control volumes and dual cells – Left: Finite Elements (the present sketch illustrates the possibility, implemented in our software (CALIF3S [5]), of mixing simpli- cial (Crouzeix-Raviart) and quadrangular (Rannacher-Turek) cells) – Right: MAC discretiza- tion, dual cell for the y-component of the velocity. Rannacher-Turek or Crouzeix-Raviart discretizations – For the RT or CR discretizations, the dual mesh is the same for all the velocity components. When K ∈M is a simplex, a rectangle or a cuboid, for σ ∈E(K), we deﬁne DK,σ as the cone with basis σ and with vertex the mass center of K (see Figure 1). We thus obtain a partition of K in m sub-volumes, where m is the number of faces of the mesh, each sub-volume having the same measure \|DK,σ\| = \|K\|/m. We extend this deﬁnition to general quadrangles and hexahedra, by supposing that we have built a partition still of equal-volume sub-cells, and with the same connectivities; note that this is of course always possible, but that such a volume DK,σ may be no longer a cone; indeed, if K is far from a parallelogram, it may not be possible to build a cone having σ as basis, the opposite vertex lying in K and a volume equal to \|K\|/m. The volume DK,σ is referred to as the half-diamond cell associated with K and σ. For σ ∈Eint, σ = K\|L, we now deﬁne the diamond cell Dσ associated with σ by Dσ = DK,σ ∪DL,σ; for an external face σ ∈Eext ∩E(K), Dσ is just the same volume as DK σ. - MAC discretization – For the MAC scheme, the dual mesh depends on the component of the velocity. For the ith component, the dual cells are associated to the faces perpendicular to the ith unit vector of the canonical basis of Rd, i.e. to the faces of E(i) (which is, of course, consistent with the location of the velocity discrete unknowns). A MAC dual cell only diﬀers from the corresponding RT or CR one by the choice of the half-diamond cell, which, for K ∈M and σ ∈E(K), is now the rectangle or rectangular parallelepiped of basis σ and of measure \|DK,σ\| = \|K\|/2. - MAC discretization – For the MAC scheme, the dual mesh depends on the component of the velocity. 2. Meshes and unknowns For the ith component, the dual cells are associated to the faces perpendicular to the ith unit vector of the canonical basis of Rd, i.e. to the faces of E(i) (which is, of course, consistent with the location of the velocity discrete unknowns). A MAC dual cell only diﬀers from the corresponding RT or CR one by the choice of the half-diamond cell, which, for K ∈M and σ ∈E(K), is now the rectangle or rectangular parallelepiped of basis σ and of measure \|DK,σ\| = \|K\|/2. We denote by \|Dσ\| the measure of the dual cell Dσ, by ǫ = Dσ\|Dσ′ the face separating two diamond cells Dσ and Dσ′, and by ¯E(Dσ) the set of the faces of Dσ. Finally, we need to deal with the impermeability (i.e. u · n = 0) boundary condition. Since the velocity unknowns lie on the boundary (and not inside the cells), these conditions are taken into account in the deﬁnition of the discrete spaces. To avoid technicalities in the expression of the schemes, we suppose throughout this paper TITLE WILL BE SET BY THE PUBLISHER 5 5 TITLE WILL BE SET BY THE PUBLISHER TITLE WILL BE SET BY THE PUBLISHER 5 that the boundary is a.e. normal to a coordinate axis, (even in the case of the RT or CR discretizations), which allows to simply set to zero the corresponding velocity unknowns: for i = 1, . . . , d, ∀σ ∈E(i) ext, uσ,i = 0. (3) (3) Therefore, there are no discrete velocity unknowns on the boundary for the MAC scheme, and there are only d −1 discrete velocity unknowns on each boundary face for the CR and RT discretizations, which depend on the orientation of the face. In order to be able to write a unique expression of the discrete equations for both MAC and CR/RT schemes, we introduce the set of faces E(i) S associated with the degrees of freedom of each component of the velocity (S stands for “scheme”): Therefore, there are no discrete velocity unknowns on the boundary for the MAC scheme, and there are only d −1 discrete velocity unknowns on each boundary face for the CR and RT discretizations, which depend on the orientation of the face. 2. Meshes and unknowns In order to be able to write a unique expression of the discrete equations for both MAC and CR/RT schemes, we introduce the set of faces E(i) S associated with the degrees of freedom of each component of the velocity (S stands for “scheme”): E(i) S = E(i) \ E(i) ext for the MAC scheme, E \ E(i) ext for the CR or RT schemes. E(i) S = E(i) \ E(i) ext for the MAC scheme, E \ E(i) ext for the CR or RT schemes. Similarly, we unify the notation for the set of dual faces for both schemes by deﬁning: ¯E(i) S = ¯E(i) \ ¯E(i) ext for the MAC scheme, ¯E \ ¯E(i) ext for the CR or RT schemes, where the symbol ˜ refers to the dual mesh; for instance, ¯E(i) is thus the set of faces of the dual mesh associated with the ith component of the velocity, and ¯E(i) ext stands for the subset of these dual faces included in the boundary. Note that, for the MAC scheme, the faces of ¯E(i) are perpendicular to a unit vector of the canonical basis of Rd, but not necessarily to the ith one. where the symbol ˜ refers to the dual mesh; for instance, ¯E(i) is thus the set of faces of the dual mesh associated with the ith component of the velocity, and ¯E(i) ext stands for the subset of these dual faces included in the boundary. Note that, for the MAC scheme, the faces of ¯E(i) are perpendicular to a unit vector of the canonical basis of Rd, but not necessarily to the ith one. Note that general domains can easily be addressed (of course, with the CR or RT discretizations) by redeﬁning, through linear combinations, the degrees of freedom at the external faces, so as to introduce the normal velocity as a new degree of freedom. 3. Implicit and semi-implicit schemes for the barotropic equations We study two schemes for the numerical solution of System (1) which diﬀer by the time discretization: the ﬁrst one is implicit, and the second one is a non-iterative pressure-correction scheme introduced in [14]. This latter algorithm (and, by an easy extension, also the ﬁrst one) was shown in [14] to have at least one solution, to provide solutions satisfying ρ > 0 (and therefore also p > 0) and to be unconditionally stable, in the sense that its (their) solution(s) satisﬁes an inequality corresponding to the control in L∞(0, T ) of the integral of the discrete entropy over the domain. In this section, we complement this work in several directions. For the implicit scheme, we obtain the following results. - First we pass from a (discrete) global (i.e. integrated over Ω) entropy balance to (discrete) local balance equations. Precisely speaking, a discrete kinetic energy balance is established on dual cells, while a discrete potential elastic balance is established on primal cells. - First we pass from a (discrete) global (i.e. integrated over Ω) entropy balance to (discrete) local balance equations. Precisely speaking, a discrete kinetic energy balance is established on dual cells, while a discrete potential elastic balance is established on primal cells. These equations yield the stability of the scheme (i.e. the same global entropy conservation as in [14]) by These equations yield the stability of the scheme (i.e. the same global entropy conservation a a simple integration in space (i.e. summation over the primal and dual control volumes). - Second, in one space dimension, the limit of any convergent sequence of solutions to the scheme is shown to be a weak solution to the continuous problem, and thus to satisfy the Rankine-Hugoniot conditions. - Second, in one space dimension, the limit of any convergent sequence of solutions to the scheme is shown to be a weak solution to the continuous problem, and thus to satisfy the Rankine-Hugoniot conditions. - Finally, passing to the limit in the discrete kinetic energy and elastic potential balances, such a limit is - Second, in one space dimension, the limit of any convergent sequence of solutions to the scheme is shown to be a weak solution to the continuous problem, and thus to satisfy the Rankine-Hugoniot conditions. 3.1. An implicit scheme 3.1.1. The scheme 3. Implicit and semi-implicit schemes for the barotropic equations - Finally, passing to the limit in the discrete kinetic energy and elastic potential balances, such a limit is also shown to satisfy the usual entropy inequality. - Finally, passing to the limit in the discrete kinetic energy and elastic potential balances, such a limit is also shown to satisfy the usual entropy inequality. For the pressure correction scheme, the results are essentially the same: the scheme is unconditionally stable, and the passage to the limit in the scheme shows that if a sequence of approximate solutions obtained with the scheme is assumed to converge to some limit, then the predicted and end-of-step velocities necessarily tend to TITLE WILL BE SET BY THE PUBLISHER 6 the same function, and that the limit is a weak solution to the problem, satisfying the entropy inequality. The numerical study of this scheme (which is the only one implemented in practice) is performed in [27]. It conﬁrms the present theoretical study: in particular, the scheme is observed to converge to weak entropy solutions of Riemann problems, with an approximately ﬁrst order rate; in addition, it yields qualitatively correct solutions for CFL numbers much larger than one. the same function, and that the limit is a weak solution to the problem, satisfying the entropy inequality. The numerical study of this scheme (which is the only one implemented in practice) is performed in [27]. It conﬁrms the present theoretical study: in particular, the scheme is observed to converge to weak entropy solutions of Riemann problems, with an approximately ﬁrst order rate; in addition, it yields qualitatively correct solutions for CFL numbers much larger than one. 3.1.1. The scheme Let us consider a uniform partition 0 = t0 < t1 < . . . < tN = T of the time interval (0, T ), and let δt = tn+1 −tn for n = 0, 1, . . ., N −1 be the constant time step. We consider an implicit-in-time scheme, which reads in its fully discrete form, for 0 ≤n ≤N −1: ∀K ∈M, \|K\| δt (ρn+1 K −ρn K) + X σ∈E(K) F n+1 K,σ = 0, (4a) (4a) For 1 ≤i ≤d, ∀σ ∈E(i) S , \|Dσ\| δt (ρn+1 Dσ un+1 σ,i −ρn Dσun σ,i) + X ǫ∈¯E(Dσ) F n+1 σ,ǫ un+1 ǫ,i −\|Dσ\| (∆Mu)n+1 σ,i + \|Dσ\| (∇p)n+1 σ,i = 0, (4b) ∀K ∈M, pn+1 K = ℘(ρn+1 K ) = (ρn+1 K )γ, (4c) (4b) (4c) where the terms introduced for each discrete equation are deﬁned hereafter. where the terms introduced for each discrete equation are deﬁned hereafter. where the terms introduced for each discrete equation are deﬁned hereafter. Equation (4a) is obtained by discretizing the mass balance (1a) over the primal mesh, and F n+1 K,σ stands for the mass ﬂux across σ outward K, which, because of the impermeability condition, vanishes on external faces and is given on the internal faces by: ∀σ = K\|L ∈Eint, F n+1 K,σ = \|σ\| ρn+1 σ un+1 K,σ , an approximation of the normal velocity to the face σ outward K. This latter quantity is deﬁned by: un+1 K,σ = un+1 σ,i e(i) · nK,σ for σ ∈E(i) in the MAC case, un+1 σ · nK,σ in the CR and RT cases, (5) (i) d un+1 = un+1 σ,i e(i) · nK,σ for σ ∈E(i) in the MAC case, (5) un+1 K,σ = un+1 σ,i e(i) · nK,σ for σ ∈E(i) in the MAC case, un+1 σ · nK,σ in the CR and RT cases, (5) (5) u + K,σ = un+1 σ · nK,σ in the CR and RT cases, (5) uK,σ = un+1 σ · nK,σ in the CR and RT cases, where e(i) denotes the i-th vector of the orthonormal basis of Rd. The density at the face σ = K\|L is approxi- mated by the upwind technique: n+1 n+1 where e(i) denotes the i-th vector of the orthonormal basis of Rd. 3.1.1. The scheme The density at the face σ = K\|L is approxi- mated by the upwind technique: +1 +1 where e(i) denotes the i-th vector of the orthonormal basis of Rd. The density at the face σ = K\|L is approxi- mated by the upwind technique: +1 +1 ρn+1 σ = ρn+1 K if un+1 K,σ ≥0, ρn+1 L otherwise. (6) (6) Note that the positivity of the density in (1c) is not enforced in the scheme but results from the above upwind choice, see e.g. [16, Lemma 2.1]. We now turn to the discrete momentum balance (4b). In order to obtain the desired estimates on the approximate solution such as a discrete kinetic energy inequality, the discretization of the momentum balance must be performed in a way that is compatible with the discretization of the mass balance equation. Hence we must carefully choose the values ρn+1 Dσ and ρn Dσ as functions of the primal unknowns (ρn+1 K )K∈M and (ρn K)K∈M and the ﬂuxes on the dual faces F n+1 σ,ǫ as functions of the ﬂuxes on the primal faces F n+1 K,σ . The values ρn+1 Dσ and ρn Dσ, which approximate the density on the face σ at time tn+1 and tn respectively, are given by the following weighted average: for σ = K\|L ∈Eint, for k = n and k = n + 1, \|Dσ\| ρk Dσ = \|DK,σ\| ρk K + \|DL,σ\| ρk L. ( (7) 7 TITLE WILL BE SET BY THE PUBLISHER Let us then detail the discretization of the convection term. The ﬁrst task is to deﬁne the discrete mass ﬂux through the dual face ǫ outward Dσ, denoted by F n+1 σ,ǫ ; the guideline for its construction is that a ﬁnite volume discretization of the mass balance equation over the diamond cells, of the form Let us then detail the discretization of the convection term. The ﬁrst task is to deﬁne the discrete mass ﬂux through the dual face ǫ outward Dσ, denoted by F n+1 σ,ǫ ; the guideline for its construction is that a ﬁnite volume discretization of the mass balance equation over the diamond cells, of the form ∀σ ∈E, \|Dσ\| δt (ρn+1 Dσ −ρn Dσ) + X ǫ∈E(Dσ) F n+1 σ,ǫ = 0 (8) (8) must hold in order to be able to derive a discrete kinetic energy balance (see Section 3.1.1 below). 3.1.1. The scheme For the MAC scheme, the ﬂux on a dual face which is located on two primal faces is the mean value of the sum of the ﬂuxes on these two primal faces, and the ﬂux of a dual face located between two primal faces is again the mean value of the sum of the ﬂuxes on these two primal faces [28]. In the case of the CR and RT schemes, for a dual face ǫ included in the primal cell K, this ﬂux is computed as a linear combination (with constant coeﬃcients, i.e. independent of the cell) of the mass ﬂuxes through the faces of K, i.e. the quantities (F n+1 K,σ )σ∈E(K) appearing in the discrete mass balance (4a). We refer to [1,15] for a detailed construction of this approximation. Let us remark that a dual face lying on the boundary is then also a primal face, and the ﬂux across that face is zero. Therefore, the values un+1 ǫ,i are only needed at the internal dual faces; we choose them to be centered (in fact, the upwind choice is also covered by our analysis, see the comments on the numerical diﬀusion below); so, for ǫ = Dσ\|Dσ′, un+1 ǫ,i reads: un+1 ǫ,i = un+1 σ,i + un+1 σ′,i 2 . (9) (9) 2 The quantity (∆Mu)n+1 σ,i stands for a possible stabilizing diﬀusion term which may be written under a ﬁnite volume form over any diamond cell Dσ associated with the ith component of the velocity: The quantity (∆Mu)n+1 σ,i stands for a possible stabilizing diﬀusion term which may be written under a ﬁnite volume form over any diamond cell Dσ associated with the ith component of the velocity: −\|Dσ\| (∆Mu)n+1 σ,i = X ǫ=Dσ\|Dσ′ ν hd−2 ǫ (un+1 σ,i −un+1 σ′,i ), (10) (10) where hǫ is a characteristic dimension of the face ǫ, and ν stands for a non-negative coeﬃcient, possibly depending on a power of hǫ. Note that external faces are excluded in the sum at the right-hand side, which means that the possible associated diﬀusion ﬂux is set to zero. Note also that (∆Mu)n+1 σ,i is usually (i.e. for general meshes) not consistent with a Laplace operator. 3.1.1. The scheme - On the other hand, this formalism may prepare for a stabilization strategy which could be less diﬀusive than the upwind choice, choosing for instance ν on the basis of an a posteriori analysis of the local regularity of the solution [20,21,40]. The last term (∇p)n+1 σ,i stands for the i-th component of the discrete pressure gradient at the face σ. The gradient operator is built as the transpose of the natural discrete divergence operator which is deﬁned by \|K\| (divu)K = X σ∈E(K) \|σ\| uK,σ. (13) (13) the CR and RT case, the duality between the divergence and gradient operators simply reads: In the CR and RT case, the duality between the divergence and gradient operators simply read X K∈M \|K\| pK (divu)K + X σ∈E \|Dσ\| uσ · (∇p)σ = 0. X K∈M \|K\| pK (divu)K + X σ∈E \|Dσ\| uσ · (∇p)σ = 0. This duality relation may be rewritten so as to ﬁt both the CR/RT scheme and the MAC scheme as follows: This duality relation may be rewritten so as to ﬁt both the CR/RT scheme and the MAC scheme as follows: X K∈M \|K\| pK(divu)K + d X i=1 X σ∈E(i) S \|Dσ\| uσ,i (∇p)σ,i = 0. (14) (14) Therefore, on any internal face, the components of the gradients are given by: Therefore, on any internal face, the components of the gradients are given by: for σ = K\|L ∈Eint, (∇p)n+1 σ,i = \|σ\| \|Dσ\| (pn+1 L −pn+1 K ) nK,σ · e(i). Note that, because of the impermeability boundary conditions, the discrete gradient is not deﬁned at the external faces. Finally, the initial approximations for ρ and u are given by the average of the initial conditions ρ0 and u0 on the primal and dual cells respectively: ∀K ∈M, ρ0 K = 1 \|K\| Z K ρ0(x) dx, for 1 ≤i ≤d, ∀σ ∈E(i) S , u0 σ,i = 1 \|Dσ\| Z Dσ (u0(x))i dx. (15) ∀K ∈M, ρ0 K = 1 \|K\| Z K ρ0(x) dx, (15) (15) for 1 ≤i ≤d, ∀σ ∈E(i) S , u0 σ,i = 1 \|Dσ\| Z Dσ (u0(x))i dx. (15) 3.1.1. The scheme Moreover, the upwind scheme un+1 ǫ,i = un+1 σ,i if F n+1 σ,ǫ ≥0, un+1 σ′,i otherwise, can also be written under this form, since in this case, the convection term may be written as: can also be written under this form, since in this case, the convection term may be written as: (F n+1 σ,ǫ un+1 ǫ,i )(up) = F n+1 σ,ǫ un+1 σ,i + un+1 σ′,i 2 + 1 2 \|F n+1 σ,ǫ \| (un+1 σ,i −un+1 σ′,i ) for ǫ = Dσ\|Dσ′. (1 (11) nd choice is included in the formulation (4b),(9), with a numerical diﬀusion term deﬁned by ν hd−2 ǫ = 1 2 \|F n+1 σ,ǫ \|, (12) 2 and ν behaves as hǫ in this case (provided that the density and the velocity are uniformly bounded). and ν behaves as hǫ in this case (provided that the density and the velocity are uniformly bounded). nd ν behaves as hǫ in this case (provided that the density and the velocity are uniformly bounded The introduction of a numerical diﬀusion of the form (10) presents two advantages: - On one hand, if we assume that the coeﬃcient ν is such that C1hα ǫ ≤ν ≤C2hα ǫ with C1, C2 ∈R+ and 0 < α < 2, we obtain a weak L2(H1) control of the velocity which is suﬃcient, at least in one space dimension, to pass to the limit in the scheme (see Section 3.1.3). This is not the the case for 8 TITLE WILL BE SET BY THE PUBLISHER a pure (i.e. without additional diﬀusion term) upwind discretization, because ν vanishes with the mass ﬂux (i.e. the normal velocity). a pure (i.e. without additional diﬀusion term) upwind discretization, because ν vanishes with the mass ﬂux (i.e. the normal velocity). Note however that the situation is diﬀerent if we now assume that the approximate velocity u satisﬁes a BV estimate: the convergence analysis of Section 3.1.3 then still holds in the centered or upwind case, without requiring any additional diﬀusion. Note however that the situation is diﬀerent if we now assume that the approximate velocity u satisﬁes a BV estimate: the convergence analysis of Section 3.1.3 then still holds in the centered or upwind case, without requiring any additional diﬀusion. 3.1.2. Estimates A solution to the system (4) satisﬁes the following equality, for 1 ( gy , p ) on to the system (4) satisﬁes the following equality, for 1 ≤i ≤d, σ ∈E(i) S and 0 ≤n ≤N −1: 1 2 \|Dσ\| δt h ρn+1 Dσ (un+1 σ,i )2 −ρn Dσ(un σ,i)2i + 1 2 X ǫ=Dσ\|Dσ′ F n+1 σ,ǫ un+1 σ,i un+1 σ′,i + \|Dσ\| (∇p)n+1 σ,i un+1 σ,i = −Rn+1 σ,i , (17) (17) where where Rn+1 σ,i = \|Dσ\| 2 δt ρn Dσ un+1 σ,i −un σ,i 2 + h X ǫ=Dσ\|Dσ′ ν hd−2 ǫ (un+1 σ,i −un+1 σ′,i ) i un+1 σ,i . (18) (18) Proof. Let us multiply equation (4b) by the corresponding velocity unknown un+1 σ,i ; this yields T conv σ,i + T ∆ σ,i + T ∇ σ,i = 0 T conv σ,i + T ∆ σ,i + T ∇ σ,i = 0 with: with: T conv σ,i = h\|Dσ\| δt ρn+1 Dσ un+1 σ,i −ρn Dσun σ,i + X ǫ=Dσ\|Dσ′ F n+1 σ,ǫ un+1 ǫ,i i un+1 σ,i , T ∆ σ,i = h X ǫ=Dσ\|Dσ′ ν hd−2 ǫ (un+1 σ,i −un+1 σ′,i ) i un+1 σ,i , T ∇ σ,i = \|Dσ\| (∇p)n+1 σ,i un+1 σ,i . T conv σ,i = h\|Dσ\| δt ρn+1 Dσ un+1 σ,i −ρn Dσun σ,i + X ǫ=Dσ\|Dσ′ F n+1 σ,ǫ un+1 ǫ,i i un+1 σ,i , T ∆ σ,i = h X ǫ=Dσ\|Dσ′ ν hd−2 ǫ (un+1 σ,i −un+1 σ′,i ) i un+1 σ,i , T ∇ σ i = \|Dσ\| (∇p)n+1 σ i un+1 σ i . T ∆ σ,i = h X ǫ=Dσ\|Dσ′ ν hd−2 ǫ (un+1 σ,i −un+1 σ′,i ) i un+1 σ,i , T ∇ σ,i = \|Dσ\| (∇p)n+1 σ,i un+1 σ,i . Applying Lemma A.2 with P = Dσ, we get from the identity (88) that T conv σ,i = 1 2 \|Dσ\| δt h ρn+1 Dσ (un+1 σ,i )2 −ρn Dσ(un σ,i)2i + 1 2 X ǫ=Dσ\|Dσ′ F n+1 σ,ǫ un+1 σ,i un+1 σ′,i + \|Dσ\| 2 δt ρn Dσ un+1 σ,i −un σ,i 2. We then note that T ∆ σ,i + \|Dσ\| 2 δt ρn Dσ un+1 σ,i −un σ,i 2 = Rn+1 σ,i , where Rn+1 σ,i is deﬁned by (18), which concludes the proof of (17). □ □ where Rn+1 σ,i is deﬁned by (18), which concludes the proof of (17). 3.1.2. Estimates We begin with an estimate on the velocity which is a discrete equivalent of the kinetic energy balance. Recall that in the continuous setting, the kinetic energy balance is formally obtained by multiplying the ith component of the momentum balance equation (1b) by the ith component ui of u; this yields for 1 ≤i ≤d, using the mass balance equation (1a) twice: ∂t(1 2ρu2 i ) + div (1 2ρu2 i ) u + (∂xip) ui = 0, TITLE WILL BE SET BY THE PUBLISHER TITLE WILL BE SET BY THE PUBLISHER 9 TITLE WILL BE SET BY THE PUBLISHER 9 and thus, summing over the components: and thus, summing over the components: ∂t(ρ Ek) + div ρ Ek u + ∇p · u = 0, with Ek = 1 2 \|u\|2. (16) (16) In the discrete setting, this multiplication must be performed on the dual mesh, since the velocity unknowns are deﬁned on the faces. This is the reason why we chose the ﬂuxes on the faces of the dual mesh in such a way that a discrete mass balance equation holds on the dual grid cells, thus allowing us to use Lemma A.2 (which performs the discrete equivalent of the above formal computations) on the dual mesh. In the discrete setting, this multiplication must be performed on the dual mesh, since the velocity unknowns are deﬁned on the faces. This is the reason why we chose the ﬂuxes on the faces of the dual mesh in such a way that a discrete mass balance equation holds on the dual grid cells, thus allowing us to use Lemma A.2 (which performs the discrete equivalent of the above formal computations) on the dual mesh. Lemma 3.1 (Discrete kinetic energy balance, implicit scheme). A solution to the system (4) satisﬁes the following equality, for 1 ≤i ≤d, σ ∈E(i) S and 0 ≤n ≤N −1: Lemma 3.1 (Discrete kinetic energy balance, implicit scheme). 3.1.2. Estimates A solution to the system (4) satisﬁes the following equality, for K ∈M and 0 ≤n ≤N −1: \|K\| δt h H(ρn+1 K ) −H(ρn K) i + X σ∈E(K) \|σ\| H(ρn+1 σ ) un+1 K,σ + \|K\| pn+1 K (divun+1)K = −Rn+1 K , (22) (22) with: with: Rn+1 K = \|K\| 2 δt H′′(ρ n+ 1 2 K ) (ρn+1 K −ρn K)2 + 1 2 X σ=K\|L \|σ\| (un+1 K,σ )−H′′(ρn+1 σ ) (ρn+1 σ −ρn+1 K )2, (23) Rn+1 K = \|K\| 2 δt H′′(ρ n+ 1 2 K ) (ρn+1 K −ρn K)2 + 1 2 X σ=K\|L \|σ\| (un+1 K,σ )−H′′(ρn+1 σ ) (ρn+1 σ −ρn+1 K )2, (23) (23) where ρ n+ 1 2 K ∈[min(ρn+1 K , ρn K), max(ρn+1 K , ρn K)], ρn+1 σ ∈[min(ρn+1 σ , ρn+1 K ), max(ρn+1 σ , ρn+1 K )] for all σ ∈E(K), and, for a ∈R, a−≥0 is deﬁned by a−= −min(a, 0). Note that, since the function H is convex, Rn+1 K is non-negative. Proof. Let us multiply the discrete mass balance (4a) by H′(ρn+1 K ). The result is then a consequence of Lemma A.1 with P = K, using the fact that zH′(z) −H(z) = ℘(z) and that ρn+1 σ is the upwind choice between ρK and ρL in the remainder term RK,δt. □ Summing (16) and (21) yields: Summing (16) and (21) yields: ∂tη + div (η + p) u = 0, where η = ρ Ek + H(ρ) is the entropy of the system. This relation is only valid for regular solutions, and should be replaced by an inequality to take into account the presence of shocks (see relations (37)-(38)). Integrating over Ωand using the boundary conditions yields: d dt Z Ω η(x, t) dx ≤0 (for regular solutions, d dt Z Ω η(x, t) dx = 0), d dt Z Ω η(x, t) dx ≤0 (for regular solutions, d dt Z Ω η(x, t) dx = 0), and, for t ∈(0, T ), and, for t ∈(0, T ), Z Ω η(x, t) dx ≤ Z Ω η(x, 0) dx. The following proposition states a discrete analogue to this relation. Proposition 3.3 (Global discrete entropy inequality, existence of a solution). Proposition 3.3 (Global discrete entropy inequality, existence of a solution). Assume that the initial density ρ0 is positive. 3.1.2. Estimates where Rn+1 σ,i is deﬁned by (18), which concludes the proof of (17). where Rn+1 σ,i is deﬁned by (18), which concludes the proof of (17). □ Let us now deﬁne the elastic potential P: Let us now deﬁne the elastic potential P: P(z) = Z z 0 ℘(s) s2 ds i.e. P(z) =      zγ−1 γ −1 if γ > 1, ln(z) if γ = 1, P(z) = Z z 0 ℘(s) s2 ds i.e. P(z) =      zγ−1 γ −1 if γ > 1, ln(z) if γ = 1, (19) (19) TITLE WILL BE SET BY THE PUBLISHER TITLE WILL BE SET BY THE PUBLISHER 10 and let H be the function deﬁned over (0, +∞) by and let H be the function deﬁned over (0, +∞) by and let H be the function deﬁned over (0, +∞) by and let H be the function deﬁned over (0, +∞) by H(z) = z P(z) =      zγ γ −1 if γ > 1, z ln(z) if γ = 1. . (20) H(z) = z P(z) =      zγ γ −1 if γ > 1, z ln(z) if γ = 1. . (20) (20) It may easily be checked that zH′(z)−H(z) = ℘(z); therefore, by a formal computation detailed in the appendix (see (83)), multiplying (1a) by H′(ρ) yields: It may easily be checked that zH′(z)−H(z) = ℘(z); therefore, by a formal computation detailed in the appendix (see (83)), multiplying (1a) by H′(ρ) yields: ∂t H(ρ) + div H(ρ) u + p div(u) = 0. (21) (21) We now derive a discrete analogue of this relation. We now derive a discrete analogue of this relation. Lemma 3.2 (Discrete potential balance). Let H be deﬁned by (20). 3.1.2. Estimates Then, there exists a solution (un, ρn) 0≤n≤N to the scheme (4), and, for 1 ≤n ≤N, ρn > 0 and the following inequality holds: Proposition 3.3 (Global discrete entropy inequality, existence of a solution). Assume that the initial density ρ0 is positive. Then, there exists a solution (un, ρn) 0≤n≤N to t and, for 1 ≤n ≤N, ρn > 0 and the following inequality holds: 1 2 d X i=1 X σ∈E(i) S \|Dσ\| ρn Dσ (un σ,i)2 + X K∈M \|K\| H(ρn K) + Rn ≤C0, (24) (24) 11 TITLE WILL BE SET BY THE PUBLISHER 11 TITLE WILL BE SET BY THE PUBLISHER TITLE WILL BE SET BY THE PUBLISHER 1 where C0 ∈R+ only depends on the initial conditions, and Rn is the following non-negative remainder which depends on the space and time translates of the unknowns: where C0 ∈R+ only depends on the initial conditions, and Rn is the following non-negative remainder which depends on the space and time translates of the unknowns: Rn = d X i=1 n X k=1 h1 2 X σ∈E(i) S \|Dσ\| ρk−1 Dσ (uk σ,i −uk−1 σ,i )2 + δt X ǫ=Dσ\|Dσ′ ∈¯E(i) S ν hd−2 σ (uk σ,i −uk σ′,i)2i +γ 2 n X k=1 δt X σ=K\|L∈Eint \|σ\| (ρk σ,γ)γ−2 \|uk K,σ\| (ρk K −ρk L)2, (25) Rn = d X i=1 n X k=1 h1 2 X σ∈E(i) S \|Dσ\| ρk−1 Dσ (uk σ,i −uk−1 σ,i )2 + δt X ǫ=Dσ\|Dσ′ ∈¯E(i) S ν hd−2 σ (uk σ,i −uk σ′,i)2i n (25) with ρk σ,γ equal to either ρk K or ρk L and such that (ρk σ,γ)γ−2 = min (ρk K)γ−2, (ρk L)γ−2 . Remark 3.4. For γ > 1, the function H is positive and increasing over (0, +∞). The inequality (24) thus readily provides an estimate on the unknowns. For γ > 1, the function H is positive and increasing over (0, +∞). The inequality (24) thus s an estimate on the unknowns. This is still true also for γ = 1, since in this case H(z) = z ln z and therefore H(z) ≥−1/e, ∀z ∈(0, +∞), and H is increasing over (1/e, +∞). 3.1.2. Estimates In fact, in order to get the usual formulation of an estimate, we may rephrase the inequality (24) by changing the expression of H to H(z) = max(z log(z), 0) and adding \|Ω\|/e to the constant C at the right-hand side. Proof. Let us give the proof of Proposition 3.3. The positivity of the density is a consequence of the properties of the upwind choice (6) for ρ [16, Lemma 2.1]; note that it may also be proved applying Lemma A.1 with ψ(s) = 1 2(s−)2 and P = K. Let us then sum Equation (17) over the components i and the faces σ ∈E(i) S , Equation (22) over K ∈M, and, ﬁnally, the two obtained relations. Since the discrete gradient and divergence operators are dual with respect to the L2-inner product (see (14)), noting that the conservative ﬂuxes vanish in the summation, we get, for 1 ≤k ≤N: 1 2 d X i=1 X σ∈E(i) S \|Dσ\| δt h ρk Dσ(uk σ,i)2 −ρk−1 Dσ (uk−1 σ,i )2i + X K∈M \|K\| δt (H(ρk K) −H(ρk−1 K )) = − d X i=1 X σ∈E(i) S Rk σ,i − X K∈M Rk K. (26) (26) (26) Summing (26) for k = 1 to n, and using the fact that H′′(z) = γ zγ−2 for any γ ≥1 yields (24), with Rn given by (25) and Summing (26) for k = 1 to n, and using the fact that H′′(z) = γ zγ−2 for any γ ≥1 yields (24), with Rn given by (25) and C0 = 1 2 d X i=1 X σ∈E(i) S \|Dσ\| ρ0 σ \|u0 σ,i\|2 + X K∈M \|K\| H(ρ0 K). Finally, the existence of a solution may be inferred by the Brouwer ﬁxed point theorem, by an easy adaptation of the proof of [12, Proposition 5.2]. This proof relies on the following set of mesh-dependent estimates: the conservativity of the mass balance discretization, together with the fact that the density is positive, yields an estimate for ρ in the L1-norm, and so, by a norm equivalence argument, of the pressure in any norm; the discrete momentum balance equation then provides a control on the velocity. 3.1.3. Passing to the limit in the scheme 3.1.3. Passing to the limit in the scheme The objective of this section is to show, in the one dimensional case, that if a sequence of solutions is controlled in suitable norms and converges to a limit, this latter necessarily satisﬁes a (part of the) weak formulation of the continuous problem. The 1D version of the scheme which is studied in this section may be obtained from Scheme (4) by taking the MAC variant, with only one horizontal stripe of grid cells, supposing that the vertical component of the velocity (the degrees of freedom of which are located on the top and bottom boundaries) vanishes, and that the measure of the vertical faces is equal to 1. For the sake of readability, however, we completely rewrite this 1D scheme, and, to this purpose, we ﬁrst introduce some adaptations of the notations to the one dimensional case. For any K ∈M, we denote by hK its length (so hK = \|K\|); when we write K = [σσ′], this means that either K = (xσ, xσ′) or K = (xσ′, xσ); if we need to specify the order, i.e. K = (xσ, xσ′) with xσ < xσ′, then we write K = [−→ σσ′]. For an interface σ = K\|L between two cells K and L, we deﬁne hσ = (hK + hL)/2, so, by deﬁnition of the dual mesh, hσ = \|Dσ\|. If we need to specify the order of the cells K and L, say K is left of L, then we write σ = −−→ K\|L. TITLE WILL BE SET BY THE PUBLISHER TITLE WILL BE SET BY THE PUBLISHER 12 3.1.3. Passing to the limit in the scheme With these notations, the implicit scheme (4) may be written as follows in the one dimensional setting: ∀K ∈M, ρ0 K = 1 \|K\| Z K ρ0(x) dx, ∀σ ∈Eint, u0 σ = 1 \|Dσ\| Z Dσ u0(x) dx, (27a) ∀K = [ −→ σσ′] ∈M, \|K\| δt (ρn+1 K −ρn K) + F n+1 σ′ −F n+1 σ = 0, (27b) ∀ −−→ K\|L E ∀K ∈M, ρ0 K = 1 \|K\| Z K ρ0(x) dx, ∀σ ∈Eint, u0 σ = 1 \|Dσ\| Z Dσ u0(x) dx, (27a) (27a) ∀K = [ −→ σσ′] ∈M, \|K\| δt (ρn+1 K −ρn K) + F n+1 σ′ −F n+1 σ = 0, (27b) \|K\| δt (ρn+1 K −ρn K) + F n+1 σ′ −F n+1 σ = 0, (27b) (27b) ∀σ = −−→ K\|L ∈Eint, ∀σ = −−→ K\|L ∈Eint, \|Dσ\| δt (ρn+1 Dσ un+1 σ −ρn Dσun σ) + F n+1 L un+1 L −F n+1 K un+1 K −\|Dσ\|(∆Mu)n+1 σ + pn+1 L −pn+1 K = 0, (27c) ∀σ = −−→ K\|L ∈Eint, \|Dσ\| δt (ρn+1 Dσ un+1 σ −ρn Dσun σ) + F n+1 L un+1 L −F n+1 K un+1 K −\|Dσ\|(∆Mu)n+1 σ + pn+1 L −pn+1 K = 0, (27c) (27c) ∀K ∈M, pn+1 K = ℘(ρn+1 K ) = (ρn+1 K )γ. ∀K ∈M, pn+1 K = ℘(ρn+1 K ) = (ρn+1 K )γ. (27 (27d) The mass ﬂux in the discrete mass balance equation is given, for σ ∈Eint, by: The mass ﬂux in the discrete mass balance equation is given, for σ ∈Eint, by: F n+1 σ = ρn+1 σ un+1 σ , (28) (28) where the upwind approximate density ρn+1 σ at the face σ is deﬁned by (6). In the momentum balance equa- tion, the application of the procedure described in Section 3.1.1 yields the following expression for the density associated with the dual cell Dσ with σ = K\|L and for the mass ﬂuxes at the dual face located at the center of the cell K = [σσ′]: ρn+1 Dσ = 1 2 \|Dσ\| (\|K\| ρn+1 K + \|L\| ρn+1 L ), F n+1 K = 1 2 (F n+1 σ + F n+1 σ′ ), (29) (29) and the approximation of the velocity at this face is centered: un+1 K = (un+1 σ + un+1 σ′ )/2. 3.1.2. Estimates Therefore, computing (i) ρ from the mass balance for ﬁxed u, (i) ρ from the mass balance for ﬁxed u, (ii) p from ρ by the equation of state, (iii) and ﬁnally u from the momentum balance equation with ﬁxed ρ and p, (iii) and ﬁnally u from the momentum balance equation with ﬁxed ρ a yields an iteration in a bounded convex subset of a ﬁnite dimensional space. (iii) and ﬁnally u from the momentum balance equation with ﬁxed ρ and p, yields an iteration in a bounded convex subset of a ﬁnite dimensional space □ □ yields an iteration in a bounded convex subset of a ﬁnite dimensional space. 3.1.3. Passing to the limit in the scheme Finally, for a face σ = −−→ K\|L with K = [−→ σ′σ] and L = [−−→ σσ′′], the stabilization diﬀusion term reads: −\|Dσ\| (∆Mu)n+1 σ = ν h 1 hK (un+1 σ −un+1 σ′ ) + 1 hL (un+1 σ −un+1 σ′′ ) i . (30) (30) TITLE WILL BE SET BY THE PUBLISHER 13 Deﬁnition 3.5 (Regular sequence of discretizations). ( ( ) ( ) ( )) Deﬁnition 3.5 (Regular sequence of discretizations). ( ) We deﬁne a regular sequence of discretizations (M(m), δt(m), ν(m))m∈N as a sequence of meshes, time steps and numerical diﬀusion coeﬃcients satisfying the following assertions: (i) both the time step δt(m) and the size h(m) of the mesh M(m), deﬁned by h(m) = supK∈M(m) hK, tend to zero as m →+∞, (ii) there exists θ > 0 such that: (ii) there exists θ > 0 such that: θ ≤hK hL ≤1 θ, ∀m ∈N and K, L ∈M(m) sharing a face, (iii) the sequence of numerical diﬀusion coeﬃcients (ν(m))m∈N is such that: lim m→+∞ν(m) = 0, lim m→+∞ (h(m))2 ν(m) = 0. Let such a regular sequence of discretizations be given, and ρ(m), p(m) and u(m) be a solution given by the scheme (27) with the mesh M(m), the time step δt(m) and the numerical diﬀusion coeﬃcient ν(m). To the discrete unknowns, we associate piecewise constant functions on time intervals and on primal or dual meshes, so that the density ρ(m), the pressure p(m) and the velocity u(m) are deﬁned almost everywhere on Ω× (0, T ) by: ρ(m)(x, t) = N−1 X n=0 X K∈M (ρ(m))n+1 K XK(x) X(n,n+1](t), p(m)(x, t) = N−1 X n=0 X K∈M (p(m))n+1 K XK(x) X(n,n+1](t), u(m)(x, t) = N−1 X n=0 X σ∈E (u(m))n+1 σ XDσ(x) X(n,n+1](t), where XK, XDσ and X(n,n+1] stand for the characteristic functions of the intervals K, Dσ and (tn, tn+1] respec- tively. where XK, XDσ and X(n,n+1] stand for the characteristic functions of the intervals K, Dσ and (tn, tn+1] respec- tively. 3.1.3. Passing to the limit in the scheme A weak solution to the continuous problem satisﬁes, for any ϕ ∈C∞ c Ω× [0, T ) : − Z T 0 Z Ω h ρ ∂tϕ + ρ u ∂xϕ i dx dt − Z Ω ρ0(x) ϕ(x, 0) dx = 0, (31a) − Z T 0 Z Ω h ρ u ∂tϕ + (ρ u2 + p) ∂xϕ i dx dt − Z Ω ρ0(x) u0(x) ϕ(x, 0) dx = 0, (31b) p = ργ. (31c) − Z T 0 Z Ω h ρ ∂tϕ + ρ u ∂xϕ i dx dt − Z Ω ρ0(x) ϕ(x, 0) dx = 0, (31a) − Z T 0 Z Ω h ρ u ∂tϕ + (ρ u2 + p) ∂xϕ i dx dt − Z Ω ρ0(x) u0(x) ϕ(x, 0) dx = 0, (31b) (31a) (31b) p = ργ. (31c) (31c) Note that these relations are not suﬃcient to deﬁne a weak solution to the problem, since they do not imply anything about the boundary conditions. However, they allow to derive the Rankine-Hugoniot conditions; hence if we show that they are satisﬁed by the limit of a sequence of solutions to the discrete problem, this implies, loosely speaking, that the scheme computes correct shocks (i.e. shocks where the jumps of the unknowns and of the ﬂuxes are linked to the shock speed by Rankine-Hugoniot conditions). This is the result we are seeking and which we state in Theorem 3.7. In order to prove this theorem, we need some deﬁnitions of interpolates of regular test functions on the primal and dual mesh. 14 TITLE WILL BE SET BY THE PUBLISHER Deﬁnition 3.6 (Interpolates on one-dimensional meshes). Let Ωbe an open bounded interval of R, let ϕ ∈ C∞ c (Ω× [0, T )), and let M be a mesh of Ω. The interpolate ϕM of ϕ on the primal mesh M is deﬁned by: Deﬁnition 3.6 (Interpolates on one-dimensional meshes). Let Ωbe an open bounded interval of R, let ϕ ∈ C∞ c (Ω× [0, T )), and let M be a mesh of Ω. 3.1.3. Passing to the limit in the scheme The interpolate ϕM of ϕ on the primal mesh M is deﬁned by: ϕM = N−1 X n=0 X K∈M ϕn K XK X[tn,tn+1), ϕM = N−1 X n=0 X K∈M ϕn K XK X[tn,tn+1), where, for 0 ≤n ≤N and K ∈M, we set ϕn K = ϕ(xK, tn), with xK the mass center of K. The time and space discrete derivatives of the discrete function ϕM are deﬁned by: ðtϕM = N−1 X n=0 X K∈M ϕn+1 K −ϕn K δt XK X[tn,tn+1), and ðxϕM = N−1 X n=0 X σ=−−→ K\|L∈Eint ϕn L −ϕn K hσ XDσ X[tn,tn+1). Let ϕE be an interpolate of ϕ on the dual mesh, deﬁned by: Let ϕE be an interpolate of ϕ on the dual mesh, deﬁned by: Let ϕE be an interpolate of ϕ on the dual mesh, deﬁned by: Let ϕE be an interpolate of ϕ on the dual mesh, deﬁned by: ϕE = N−1 X n=0 X σ∈E ϕn σ XDσ X[tn,tn+1), where, for 1 ≤n ≤N and σ ∈E, we set ϕn σ = ϕ(xσ, tn), with xσ the abscissa of the interface σ. We also deﬁne the time and space discrete derivatives of this discrete function by: ðtϕE = N−1 X n=0 X σ∈E ϕn+1 σ −ϕn σ δt XDσ X[tn,tn+1), and ðxϕE = N−1 X n=0 X K=[ −−→ σσ′]∈M ϕn σ′ −ϕn σ hK XK X[tn,tn+1). nally, we deﬁne ðxϕM,E by: ðxϕM,E = N−1 X n=0 X K=[ −−→ σσ′]∈M ϕn K −ϕn σ hK/2 XDK,σ X[tn,tn+1), +ϕn σ′ −ϕn K hK/2 XDK,σ′ X[tn,tn+1). ðtϕE = N−1 X n=0 X σ∈E ϕn+1 σ −ϕn σ δt XDσ X[tn,tn+1), and ðxϕE = N−1 X n=0 X K=[ −−→ σσ′]∈M ϕn σ′ −ϕn σ hK XK X[tn,tn+1). Finally, we deﬁne ðxϕM,E by: Finally, we deﬁne ðxϕM,E by: ðxϕM,E = N−1 X n=0 X K=[ −−→ σσ′]∈M ϕn K −ϕn σ hK/2 XDK,σ X[tn,tn+1), +ϕn σ′ −ϕn K hK/2 XDK,σ′ X[tn,tn+1). Theorem 3.7 (Consistency of the one-dimensional implicit scheme). ( ) Let Ωbe an open bounded interval of R. We suppose that the initial data satisﬁes ρ0 ∈L∞(Ω), 1/ρ0 ∈L∞(Ω) and u0 ∈L∞(Ω). Let (M(m), δt(m), ν(m))m∈N be a regular sequence of discretizations in the sense of Deﬁnition 3.5, and (ρ(m), p(m), u(m))m∈N be the corresponding sequence of solutions. 3.1.3. Passing to the limit in the scheme We suppose that this sequence converges in Lp(Ω× (0, T ))3, for 1 ≤p < +∞, to (¯ρ, ¯p, ¯u) ∈L∞(Ω× (0, T ))3. We suppose in addition that both sequences (ρ(m))m∈N and (1/ρ(m))m∈N are uniformly bounded in L∞(Ω× (0, T )). Then the limit (¯ρ, ¯p, ¯u) satisﬁes the system (31). Then the limit (¯ρ, ¯p, ¯u) satisﬁes the system (31). Proof. With the assumed convergence for the sequence of solutions, the limit clearly satisﬁes the equation of state (note that in reality this is the diﬃcult point to prove with the estimates at hand, see e.g. [12]). The proof of this theorem is thus obtained by passing to the limit in the scheme, ﬁrst for the mass balance equation and then for the momentum balance equation. Thanks to the assumption on the initial condition, the stability estimate of Proposition 3.3 is uniform with respect to m, and thus provides uniform bounds for some space translates of the solution (see the expression (25) of the remainder term), which are used all along the proof. In particular, using in addition the assumption that both sequences (ρ(m))m∈N and (1/ρ(m))m∈N are uniformly bounded in L∞(Ω× (0, T )), exploiting the last part of the remainder term, we get the following weak BV TITLE WILL BE SET BY THE PUBLISHER 15 estimate for ρ: estimate for ρ: ∀m ∈N, N (m)−1 X n=0 δt X σ=K\|L∈E(m) int \|(u(m))n+1 σ \| h (ρ(m))n+1 K −(ρ(m))n+1 L i2 ≤C, (32) where C stands for a real number which is independent of m. ∀m ∈N, N (m)−1 X n=0 δt X σ=K\|L∈E(m) int \|(u(m))n+1 σ \| h (ρ(m))n+1 K −(ρ(m))n+1 L i2 ≤C, (32) h C d f l b hi h i i d d f ∀m ∈N, N (m)−1 X n=0 δt X σ=K\|L∈E(m) int \|(u(m))n+1 σ \| h (ρ(m))n+1 K −(ρ(m))n+1 L i2 ≤C, (32) (32) where C stands for a real number which is independent of m. where C stands for a real number which is independent of m. Mass balance equation – Let ϕ ∈C∞ c (Ω× [0, T )). Let m ∈N, M(m), δt(m) and ν(m) be given. Dropping for short the superscript (m), let ϕM be the interpolate of ϕ on the primal mesh and let ðtϕM and ðxϕM be its time and space discrete derivatives in the sense of Deﬁnition 3.6. Thanks to the regularity of ϕ, these functions respectively converge in Lr(Ω× (0, T )), for r ≥1 (including r = +∞), to ϕ, ∂tϕ and ∂xϕ respectively. In addition, ϕM(m)(·, 0) converges to ϕ(·, 0) in Lr(Ω) for r ≥1 as m →+∞. Then the limit (¯ρ, ¯p, ¯u) satisﬁes the system (31). Since the support of ϕ is compact in Ω× [0, T ), for m large enough, ϕM(m) vanishes at the boundary cells and at the ﬁnal time; hereafter, we systematically assume that this indeed the case. Let us multiply the discrete mass balance equation (27b) by δt ϕn K, and sum the result on n ∈{0, ..., N −1} and K ∈M, to obtain T (m) 1 + T (m) 2 = 0 with T (m) 1 = N−1 X n=0 X K∈M \|K\| (ρn+1 K −ρn K) ϕn K, T (m) 2 = N−1 X n=0 δt X K=[ −−→ σσ′]∈M h F n+1 σ′ −F n+1 σ i ϕn K. (In the above expressions and in the remainder of the proof, we whall omit the superscript (m) in the summations for the sake of clarity.) Reordering the sums in T (m) 1 yields: (In the above expressions and in the remainder of the proof, we whall omit the superscript (m) in the summations for the sake of clarity.) Reordering the sums in T (m) 1 yields: T (m) 1 = − N−1 X n=0 δt X K∈M \|K\| ρn+1 K ϕn+1 K −ϕn K δt − X K∈M \|K\| ρ0 K ϕ0 K, = − Z T 0 Z Ω ρ(m) ðt ϕM(m) dx dt − Z Ω ρ0(x) ϕM(m)(x, 0) dx. Since, by assumption, the sequence of discrete solutions converges in Lr(Ω× (0, T )) for r ≥1, we get: Since, by assumption, the sequence of discrete solutions converges in Lr(Ω× (0, T )) for r ≥1, we get: lim m−→+∞T (m) 1 = − Z T 0 Z Ω ¯ρ ∂tϕ dx dt − Z Ω ρ0(x) ϕ(x, 0) dx. Reordering the sums in T (m) 2 , we get: T (m) 2 = − N−1 X n=0 δt X σ=−−→ K\|L∈Eint \|Dσ\| ρn+1 σ un+1 σ ϕn L −ϕn K hσ , T (m) 2 = − N−1 X n=0 δt X σ=−−→ K\|L∈Eint \|Dσ\| ρn+1 σ un+1 σ ϕn L −ϕn K hσ , where hσ (which is equal to \|Dσ\|) is by deﬁnition equal to \|xL −xK\| and we recall that ρn+1 σ is the upwind approximation of ρn+1 at the face σ. Then the limit (¯ρ, ¯p, ¯u) satisﬁes the system (31). Using the fact that \|Dσ\| = (\|K\|+\|L\|)/2, we may write T (m) 2 = T (m) 2 +R(m) 2 where hσ (which is equal to \|Dσ\|) is by deﬁnition equal to \|xL −xK\| and we recall that ρn+1 σ is the upwind approximation of ρn+1 at the face σ. Using the fact that \|Dσ\| = (\|K\|+\|L\|)/2, we may write T (m) 2 = T (m) 2 +R(m) 2 TITLE WILL BE SET BY THE PUBLISHER 16 with: with: T (m) 2 = − N−1 X n=0 δt X σ=−−→ K\|L∈Eint h\|K\| 2 ρn+1 K + \|L\| 2 ρn+1 L i un+1 σ ϕn L −ϕn K hσ , T (m) 2 = − N−1 X n=0 δt X σ=−−→ K\|L∈Eint h\|K\| 2 ρn+1 K + \|L\| 2 ρn+1 L i un+1 σ ϕn L −ϕn K hσ , R(m) 2 = − N−1 X n=0 δt X σ=−−→ K\|L∈Eint h\|K\| 2 (ρn+1 K −ρn+1 L )(un+1 σ )−+ \|L\| 2 (ρn+1 K −ρn+1 L )(un+1 σ )+iϕn L −ϕn K hσ . \| R(m) 2 = − N−1 X n=0 δt X σ=−−→ K\|L∈Eint h\|K\| 2 (ρn+1 K −ρn+1 L )(un+1 σ )−+ \|L\| 2 (ρn+1 K −ρn+1 L )(un+1 σ )+iϕn L −ϕn K hσ Therefore we get T (m) 2 = − Z T 0 Z Ω ρ(m) u(m) ðxϕM(m) dx dt, and lim m−→+∞T (m) 2 = − Z T 0 Z Ω ¯ρ ¯u ∂xϕ dx dt, and the remainder term R(m) 2 is bounded as follows: and the remainder term R(m) 2 is bounded as follows: and the remainder term R(m) 2 is bounded as follows: \|R(m) 2 \| ≤Cϕ N−1 X n=0 δt X σ=K\|L∈Eint \|Dσ\| \|ρn+1 K −ρn+1 L \| \|un+1 σ \| ≤Cϕ h1/2 N−1 X n=0 δt h X σ=K\|L∈Eint \|un+1 σ \| \|ρn+1 K −ρn+1 L \|2i1/2 h X σ=K\|L∈Eint \|Dσ\| \|un+1 σ \| i1/2 , ≤Cϕ h1/2 N−1 X n=0 δt h X σ=K\|L∈Eint \|un+1 σ \| \|ρn+1 K −ρn+1 L \|2i1/2 h X σ=K\|L∈Eint \|Dσ\| \|un+1 σ \| i1/2 , where the notation Cϕ means that this real number only depends on the function ϕ. Thanks to the stability estimate (32), this term tends to zero when m tends to +∞. where the notation Cϕ means that this real number only depends on the function ϕ. Then the limit (¯ρ, ¯p, ¯u) satisﬁes the system (31). From the deﬁnition (27a) of the initial conditions and the assumed regularity of ρ0 and u0, the sequences (ρ(m))0 and u(m))0 converge in Lr(Ω), for 1 ≤r < +∞, to ρ0 and u0 respectively. From the convergence assumption for the sequence of discrete solutions, we thus get: From the deﬁnition (27a) of the initial conditions and the assumed regularity of ρ0 and u0, the sequence (ρ(m))0 and u(m))0 converge in Lr(Ω), for 1 ≤r < +∞, to ρ0 and u0 respectively. From the convergenc assumption for the sequence of discrete solutions, we thus get: lim m−→+∞T (m) 1 = − Z T 0 Z Ω ¯ρ ¯u ∂tϕ dx dt − Z Ω ρ0(x) u0(x) ϕ(x, 0) dx. Let us now turn to T (m) 2 . From the expression (29) of the ﬂuxes FK and the values uK, reordering the sums, we get: ow turn to T (m) 2 . From the expression (29) of the ﬂuxes FK and the values uK, reordering the sum T (m) 2 = −1 4 N−1 X n=0 δt X K=[ −−→ σσ′]∈M (ρn+1 σ un+1 σ + ρn+1 σ′ un+1 σ′ ) (un+1 σ + un+1 σ′ ) (ϕn σ′ −ϕn σ), which we write T (m) 2 = T (m) 2 + R(m) 2 with: which we write T (m) 2 = T (m) 2 + R(m) 2 with: T (m) 2 = −1 2 N−1 X n=0 δt X K=[ −−→ σσ′]∈M ρn+1 K h (un+1 σ )2 + (un+1 σ′ )2i (ϕn σ′ −ϕn σ). This term reads: This term reads: T (m) 2 = − Z T 0 Z Ω ρ(m) (u(m))2 ðxϕE dx dt, and so lim m−→+∞T (m) 2 = − Z T 0 Z Ω ¯ρ ¯u2 ∂xϕ dx dt. T (m) 2 = − Z T 0 Z Ω ρ(m) (u(m))2 ðxϕE dx dt, and so lim m−→+∞T (m) 2 = − Z T 0 Z Ω ¯ρ ¯u2 ∂xϕ dx dt. T (m) 2 = − Z T 0 Z Ω ρ(m) (u(m))2 ðxϕE dx dt, and so lim m−→+∞T (m) 2 = − Z T 0 Z Ω ¯ρ ¯u2 ∂xϕ dx dt. Then the limit (¯ρ, ¯p, ¯u) satisﬁes the system (31). Thanks to the stability estimate (32), this term tends to zero when m tends to +∞. Momentum balance equation – Let ϕE, ðtϕE and ðxϕE be the interpolate of ϕ on the dual mesh and its discrete time and space derivatives, in the sense of Deﬁnition 3.6, which converge in Lr(Ω× (0, T )), for r ≥1 (including r = +∞), to ϕ, ∂tϕ and ∂xϕ respectively. Let us multiply the discrete momentum balance equation (27c) by δt ϕn σ, and sum the result over n ∈ {0, ..., N −1} and σ ∈Eint. We obtain T (m) 1 + T (m) 2 + T (m) 3 + T (m) 4 = 0 with: T (m) 1 = N−1 X n=0 X σ∈Eint \|Dσ\| ρn+1 Dσ un+1 σ −ρn Dσun σ ϕn σ, T (m) 2 = N−1 X n=0 δt X σ=−−→ K\|L∈Eint h F n+1 L un+1 L −F n+1 K un+1 K i ϕn σ, T (m) 3 = N−1 X n=0 δt X σ=−−→ K\|L∈Eint (pn+1 L −pn+1 K ) ϕn σ, T (m) 4 = N−1 X n=0 δt X σ∈Eint h X K=[σσ′] ν hK (un+1 σ −un+1 σ′ ) i ϕn σ. Thanks to the deﬁnition (29) of the density on the dual mesh ρDσ, reordering the sums, we get for T (m) 1 : Thanks to the deﬁnition (29) of the density on the dual mesh ρDσ, reordering the sums, we get for T (m) 1 : T (m) 1 = − N−1 X n=0 δt X σ=K\|L∈Eint h\|K\| 2 ρn+1 K + \|L\| 2 ρn+1 L i un+1 σ ϕn+1 σ −ϕn σ δt − X σ=K\|L∈Eint h\|K\| 2 ρ0 K + \|L\| 2 ρ0 L i u0 σ ϕ0 σ. T (m) 1 = − N−1 X n=0 δt X σ=K\|L∈Eint h\|K\| 2 ρn+1 K + \|L\| 2 ρn+1 L i un+1 σ ϕn+1 σ −ϕn σ δt − X σ=K\|L∈Eint h\|K\| 2 ρ0 K + \|L\| 2 ρ0 L i u0 σ ϕ0 σ. TITLE WILL BE SET BY THE PUBLISHER 17 Therefore: T (m) 1 = − Z T 0 Z Ω ρ(m) u(m) ðt ϕM(m) dx dt − Z Ω (ρ(m))0(x) (u(m))0(x) ϕM(m)(x, 0) dx. −2ρn+1 K (un+1 σ )2 + (un+1 σ′ )2i (ϕn σ′ −ϕn σ). Then the limit (¯ρ, ¯p, ¯u) satisﬁes the system (31). The remainder term R(m) 2 reads: The remainder term R(m) 2 reads: R(m) 2 = −1 4 N−1 X n=0 δt X K=[ −−→ σσ′]∈M h (ρn+1 σ un+1 σ + ρn+1 σ′ un+1 σ′ )(un+1 σ + un+1 σ′ ) −2ρn+1 K (un+1 σ )2 + (un+1 σ′ )2i (ϕn σ′ −ϕn σ). −2ρn+1 K (un+1 σ )2 + (un+1 σ′ )2i (ϕn σ′ −ϕn σ). Expanding the quantity 2 ρn+1 K ((un+1 σ )2 + (un+1 σ′ )2) thanks to the identity 2(a2 + b2) = (a + b)2 + (a −b)2, we get R(m) 2 = R(m) 2,1 + R(m) 2,2 : Expanding the quantity 2 ρn+1 K ((un+1 σ )2 + (un+1 σ′ )2) thanks to the identity 2(a2 + b2) = (a + b)2 + (a −b)2, we get R(m) 2 = R(m) 2,1 + R(m) 2,2 : R(m) 2,1 = −1 4 N−1 X n=0 δt X K=[ −−→ σσ′]∈M h (ρn+1 σ −ρn+1 K ) un+1 σ + (ρn+1 σ′ −ρn+1 K ) un+1 σ′ (un+1 σ + un+1 σ′ ) i (ϕn σ′ −ϕn σ), R(m) 2,2 = 1 4 N−1 X n=0 δt X K=[ −−→ σσ′]∈M ρn+1 K (un+1 σ −un+1 σ′ )2 (ϕn σ′ −ϕn σ). TITLE WILL BE SET BY THE PUBLISHER 18 First we study R(m) 2,1 . Thanks to the deﬁnition (6) of the upwind value ρn+1 σ , reordering the sum by faces, we get that: First we study R(m) 2,1 . Thanks to the deﬁnition (6) of the upwind value ρn+1 σ , reordering the sum by faces, we get that: \|R(m) 2,1 \| = 1 4 N−1 X n=0 δt X σ∈Eint, σ=L→K, K=[σσ′] (ρn+1 L −ρn+1 K ) un+1 σ (un+1 σ + un+1 σ′ ) (ϕn σ −ϕn σ′) , where the notation L →K means that the ﬂow is going from L to K, or, in other words, that if un+1 σ ≥0 (re un+1 σ ≤0), the cells K and L are chosen such that σ = −−→ L\|K (resp. σ = −−→ K\|L). Since \|ϕn σ −ϕn σ′\| ≤Cϕ \|K Cϕ (\|Dσ\| + \|Dσ′\|), we get: where the notation L →K means that the ﬂow is going from L to K, or, in other words, that if un+1 σ ≥0 (resp. Then the limit (¯ρ, ¯p, ¯u) satisﬁes the system (31). TITLE WILL BE SET BY THE PUBLISHER 19 Let us ﬁnally study T (m) 4 . Reordering the sums, we get: Let us ﬁnally study T (m) 4 . Reordering the sums, we get: Let us ﬁnally study T (m) 4 . Reordering the sums, we get: Let us ﬁnally study T (m) 4 . Reordering the sums, we get: T (m) 4 = N−1 X n=0 δt X K=[ −−→ σσ′]∈M ν(m) hK (un+1 σ −un+1 σ′ ) (ϕn σ −ϕn σ′). The Cauchy-Schwarz inequality yields: The Cauchy-Schwarz inequality yields: \|T (m) 4 \| ≤ hN−1 X n=0 δt X K=[ −−→ σσ′]∈M ν(m) hK (un+1 σ −un+1 σ′ )2i1/2hN−1 X n=0 δt X K=[ −−→ σσ′]∈M ν(m) hK (ϕn σ −ϕn σ′)2i1/2 , and thus, in view of the estimate (24), this term tends to zero when ν(m) tends to zer Conclusion – Gathering the limits of all the terms of the mass and momentum balance equations concludes the proof. □ Remark 3.8 (Sharper bounds and convergence assumptions). The convergence properties and bounds assumed for the solution have been chosen so as to match what may be observed in the theoretical works on compressible Navier-Stokes equations [13, 44, 53]. Note that they are weaker than the assumptions of the Lax-Wendroﬀ theorem for colocated ﬁnite volume schemes on hyperbolic systems, see e.g. [11, Theorem 5.3]. However, examining the proof of Theorem 3.7, we observe that what we really need is that the sequences ρ(m)u(m), ρ(m)(u(m))2, p(m)u(m) converge in the distribution sense to ¯ρ¯u, ¯ρ¯u2 and ¯p¯u respectively, that (ρ(m))γ converge a.e. to ¯ργ, and that the sequence (u(m))m∈N be bounded in L3(Ω× (0, T )). The required second assumption for (ν(m))m∈N is in fact: lim m→+∞ (h(m))2 ν(m) ∥ρ(m)∥L∞(Ω×(0,T )) = 0, lim m→+∞ (h(m))2 ν(m) ∥ρ(m)∥L∞(Ω×(0,T )) = 0, and may be veriﬁed, for instance supposing a relation between δt(m) and h(m) and invoking inverse inequalities, with milder estimates on (ρ(m))m∈N. Then the limit (¯ρ, ¯p, ¯u) satisﬁes the system (31). un+1 σ ≤0), the cells K and L are chosen such that σ = −−→ L\|K (resp. σ = −−→ K\|L). Since \|ϕn σ −ϕn σ′\| ≤Cϕ \|K\| ≤ Cϕ (\|Dσ\| + \|Dσ′\|), we get: \|R(m) 2,1 \| ≤Cϕ 4 N−1 X n=0 δt X σ∈Eint, σ=L→K, K=[σσ′] \|Dσ\| + \|Dσ′\| \|ρn+1 L −ρn+1 K \| \|un+1 σ \| \|un+1 σ + un+1 σ′ \|. Therefore, by the Cauchy-Schwarz inequality, we get: \|R(m) 2,1 \| ≤Cϕ 4 h1/2 N−1 X n=0 δt h X σ=K\|L∈Eint \|un+1 σ \| (ρn+1 L −ρn+1 K )2i1/2 h X σ∈Eint, σ=L→K, K=[σσ′] \|Dσ\| + \|Dσ′\| \|un+1 σ \| un+1 σ + un+1 σ′ 2i1/2 . (33) (33) Since the ratio of the size of two neighboring cells is bounded by the regularity assumption on the mesh (Item (ii) of Deﬁnition 3.5), we get from the estimate (32) on the solution: Since the ratio of the size of two neighboring cells is bounded by the regularity assumption on the mesh (Item (ii) of Deﬁnition 3.5), we get from the estimate (32) on the solution: \|R(m) 2,1 \| ≤C h1/2 ∥u(m)∥ 3/2 L3(Ω×(0,T )), (34) (34) where the real number C is independent of m and therefore R(m) 2,1 tends to zero when m tends to +∞. For R(m) 2,2 , we have, thanks to the estimate (24): where the real number C is independent of m and therefore R(m) 2,1 tends to zero when m tends to +∞. For R(m) 2,2 , we have, thanks to the estimate (24): \|R(m) 2,2 \| ≤Cϕ h2 N−1 X n=0 δt X K∈M ρn+1 K 1 hK (un+1 σ −un+1 σ′ )2 ≤C h2 ν(m) ∥ρ(m)∥L∞(Ω×(0,T )), where C > 0 does not depend on m; therefore, this term also tends to zero when m tends to +∞, since, by assumption, h2/ν(m) tends to zero. where C > 0 does not depend on m; therefore, this term also tends to zero when m tends to +∞, since, by assumption, h2/ν(m) tends to zero. We turn to the term T (m) 3 : T (m) 3 = − N−1 X n=0 δt X K=[ −−→ σσ′]∈M \|K\| pn+1 K ϕn σ′ −ϕn σ hK = − Z T 0 Z Ω p(m)ðxϕE dx dt, and therefore, lim m−→+∞T (m) 3 = − Z T 0 Z Ω ¯p ∂xϕ dx dt. Then the limit (¯ρ, ¯p, ¯u) satisﬁes the system (31). Finally, the bound of (1/ρ(m))m∈N in L∞(Ω× (0, T )) (which, loosely speaking, means that the appearance of void is excluded) is needed to obtain the weak-BV estimate: lim m→+∞h(m) N X n=1 X σ=K\|L∈Eint \|un σ\| (ρn K −ρn L)2 = 0 (35) (35) from the ”weighted weak-BV estimate” (24): from the ”weighted weak-BV estimate” (24): lim m→+∞h(m) N X n=1 X σ=K\|L∈Eint (ρn σ,γ)γ−2 \|un σ\| (ρn K −ρn L)2 = 0, where we recall that ρn σ,γ is equal to either ρn K or ρn L. This assumption is thus useless for γ ≤2. For γ > 2, in the case of a non-vanishing viscosity, Equation (35) may perhaps be derived by using the density itself as test function in the discrete mass balance equation, and invoking a control of the divergence of the velocity (from the momentum balance diﬀusion term), see [12, Proposition 5.5] for such a computation in the steady case. Remark 3.9 (Less sharp bounds and more general meshes). ( p g ) The assumption that the ratio of the size of two neighboring cells is bounded, i.e. Assumption (ii) of Deﬁnition 3.5, is only used to derive (34) from (33), which allows to conclude that the remainder term R(m) 2,1 tends to zero invoking a control of the velocity only in L3(Ω× (0, T )). If we choose to use a uniform bound in L∞(Ω× (0, T )) for the sequence of approximate solutions, we may replace (34) by \|R(m) 2,1 \| ≤C h1/2 ∥u(m)∥ 3/2 L∞(Ω×(0,T )), TITLE WILL BE SET BY THE PUBLISHER 20 and Assumption (ii) is useless. We now turn to the entropy condition. Let us ﬁrst recall that η = ρ Ek +H(ρ) is an entropy of the continuous problem (1), in the sense that if we sum the formal kinetic energy (16) and elastic potential balance (21), we get: ∂tη + ∂x (η + p) u = 0. (36) ∂tη + ∂x (η + p) u = 0. (36) In fact, in order to avoid to invoke unrealistic regularity assumption, such a computation should be done on regularized equations (obtained by adding diﬀusion perturbation terms), and, when making these regularization terms tend to zero, positive measures appear at the left-hand-side of (36), so that we get in the distribution sense: ∂tη + ∂x (η + p) u ≤0. Then the limit (¯ρ, ¯p, ¯u) satisﬁes the system (31). ∂tη + ∂x (η + p) u ≤0. (3 ∂tη + ∂x (η + p) u ≤0. (37) (37) ∂tη + ∂x (η + p) u ≤0. (37) uired to satisfy, for any ϕ ∈C∞ c Ω× [0, T ) , ϕ ≥0: ∂tη + ∂x (η + p) u ≤0. (37) An entropy solution to (1) is thus required to satisfy, for any ϕ ∈C∞ c Ω× [0, T ) , ϕ ≥0: ∂tη + ∂x (η + p) u ≤0. (37) hus required to satisfy, for any ϕ ∈C∞ c Ω× [0, T ) , ϕ ≥0: An entropy solution to (1) is thus required to satisfy, for any ϕ ∈C∞ c Ω× [0, T ) , ϕ ≥0: 1) is thus required to satisfy, for any ϕ ∈C∞ c Ω× [0, T ) , ϕ ≥0: An entropy solution to (1) is thus required to satisfy, for any ϕ ∈C∞ c Ω× [0, T ) , ϕ ≥0: Z T 0 Z Ω η ∂tϕ + (η + p) u ∂xϕ dx dt + Z Ω η0(x) ϕ(x, 0) dx ≥0, (38) (38) where η0 = 1 2 ρ0 u2 0 + H(ρ0). where η0 = 1 2 ρ0 u2 0 + H(ρ0). Theorem 3.10 (Entropy consistency, implicit scheme). Under the assumptions of Theorem 3.7, (¯ρ, ¯p, ¯u) sat- isﬁes the entropy condition (38). Proof. Let ϕ ∈C∞ c Ω× [0, T ) , ϕ ≥0. Then the limit (¯ρ, ¯p, ¯u) satisﬁes the system (31). Using the notations introduced in Deﬁnition 3.6, we multiply the kinetic balance equation (17) by ϕn σ, and the elastic potential balance (22) by ϕn K, sum over the faces and cells respectively, to get X E∈Eint T n+1 σ ϕn σ + X K∈M T n+1 K ϕn K = − X E∈Eint Rn+1 σ ϕn σ − X K∈M Rn+1 K ϕn K, (39) where, for σ = −−→ K\|L, K = [−→ σ′σ] and L = [−−→ σσ′′], T n+1 σ = 1 2 \|Dσ\| δt h ρn+1 Dσ (un+1 σ )2 −ρn Dσ(un σ)2i + 1 2 F n+1 L un+1 σ un+1 σ′′ −1 2 F n+1 K un+1 σ un+1 σ′ + (pn+1 L −pn+1 K ) un+1 σ , X E∈Eint T n+1 σ ϕn σ + X K∈M T n+1 K ϕn K = − X E∈Eint Rn+1 σ ϕn σ − X K∈M Rn+1 K ϕn K, (39) (39) where, for σ = −−→ K\|L, K = [−→ σ′σ] and L = [−−→ σσ′′], where, for σ = −−→ K\|L, K = [−→ σ′σ] and L = [−−→ σσ′′], T n+1 σ = 1 2 \|Dσ\| δt h ρn+1 Dσ (un+1 σ )2 −ρn Dσ(un σ)2i + 1 2 F n+1 L un+1 σ un+1 σ′′ −1 2 F n+1 K un+1 σ un+1 σ′ + (pn+1 L −pn+1 K ) un+1 σ , for K = [−→ σσ′], for K = [−→ σσ′], T n+1 K = \|K\| δt h H(ρn+1 K ) −H(ρn K) i + un+1 σ′ H(ρn+1 σ′ ) −un+1 σ H(ρn+1 σ ) + pn+1 K (un+1 σ′ −un+1 σ ), and the quantities Rn+1 σ and Rn+1 K are given by (the one-dimensional version of) Equation (18) and (23) respectively. The discrete weak form of the entropy balance is obtained by integrating in time (i.e. summing over the time steps) Equation (39). Then the limit (¯ρ, ¯p, ¯u) satisﬁes the system (31). We obtain T (m) 1 + T (m) 2 + T (m) 3 + T (m) 4 + T (m) 5 + R(m) = 0 with: T (m) 1 = 1 2 N−1 X n=0 X σ∈Eint \|Dσ\| h ρn+1 Dσ (un+1 σ )2 −ρn Dσ(un σ)2i ϕn σ, T (m) 1 = 1 2 N−1 X n=0 X σ∈Eint \|Dσ\| h ρn+1 Dσ (un+1 σ )2 −ρn Dσ(un σ)2i ϕn σ, (40a) (40a) T (m) 2 = 1 2 N−1 X n=0 X K∈M \|K\| h H(ρn+1 K ) −H(ρn K) i ϕn K, (40b) T (m) 2 = 1 2 N−1 X n=0 X K∈M \|K\| h H(ρn+1 K ) −H(ρn K) i ϕn K, (40b) TITLE WILL BE SET BY THE PUBLISHER 21 TITLE WILL BE SET BY THE PUBLISHER 21 T (m) 3 = 1 2 N−1 X n=0 δt X σ=−−→ K\|L∈Eint, K=[ −−→ σ′σ], L=[ −−→ σσ′′] h F n+1 L un+1 σ un+1 σ′′ −F n+1 K un+1 σ un+1 σ′ i ϕn σ, (40c) T (m) = N−1 X δt X h un+1H(ρn+1) −un+1H(ρn+1) i ϕn (40d) T (m) 3 = 1 2 N−1 X n=0 δt X σ=−−→ K\|L∈Eint, K=[ −−→ σ′σ], L=[ −−→ σσ′′] h F n+1 L un+1 σ un+1 σ′′ −F n+1 K un+1 σ un+1 σ′ i ϕn σ, (40c) T (m) 4 = N−1 X n=0 δt X K=[ −−→ σσ′]∈M h un+1 σ′ H(ρn+1 σ′ ) −un+1 σ H(ρn+1 σ ) i ϕn K, (40d) T (m) 5 = 1 2 N−1 X n=0 δt h X σ=−−→ K\|L∈Eint (pn+1 L −pn+1 K ) un+1 σ ϕn σ + X K=[ −−→ σσ′]∈M pn+1 K (un+1 σ′ −un+1 σ ) ϕn K i , (40e) (40c) T3 2 X n=0 δt X σ=−−→ K\|L∈Eint, K=[ −−→ σ′σ], L=[ −−→ σσ′′] h FL uσ uσ′′ FK uσ uσ′ i ϕσ, (40c) T (m) 4 = N−1 X n=0 δt X K=[ −−→ σσ′]∈M h un+1 σ′ H(ρn+1 σ′ ) −un+1 σ H(ρn+1 σ ) i ϕn K, (40d) K=[ −−→ σ′σ], L=[ −−→ σσ′′] T (m) 4 = N−1 X n=0 δt X K=[ −−→ σσ′]∈M h un+1 σ′ H(ρn+1 σ′ ) −un+1 σ H(ρn+1 σ ) i ϕn K, (40d) T (m) 5 = 1 2 N−1 X n=0 δt h X σ=−−→ K\|L∈Eint (pn+1 L −pn+1 K ) un+1 σ ϕn σ + X K=[ −−→ σσ′]∈M pn+1 K (un+1 σ′ −un+1 σ ) ϕn K i , (40e) R(m) = 1 N−1 X δt h X Rn σ ϕn σ + X Rn K ϕn K i . Then the limit (¯ρ, ¯p, ¯u) satisﬁes the system (31). (40f) T (m) 4 = N−1 X n=0 δt X K=[ −−→ σσ′]∈M h un+1 σ′ H(ρn+1 σ′ ) −un+1 σ H(ρn+1 σ ) i ϕn K, (40d) (40d) T (m) 5 = 1 2 N−1 X n=0 δt h X σ=−−→ K\|L∈Eint (pn+1 L −pn+1 K ) un+1 σ ϕn σ + X K=[ −−→ σσ′]∈M pn+1 K (un+1 σ′ −un+1 σ ) ϕn K i , (40e) (40e) R(m) = 1 2 N−1 X n=0 δt h X σ=−−→ K\|L∈Eint Rn σ ϕn σ + X K=[ −−→ σσ′]∈M Rn K ϕn K i . (40f) (40f) We ﬁrst study T (m) 1 . Reordering the summations and then using the deﬁnition (29) of the density at the faces, we get: We ﬁrst study T (m) 1 . Reordering the summations and then using the deﬁnition (29) of the density at the faces, we get: T (m) 1 = −1 2 N−1 X n=0 δt X σ∈E \|Dσ\| ρn+1 Dσ (un+1 σ )2 ϕn+1 σ −ϕn σ δt −1 2 X σ∈E \|Dσ\|ρ0 Dσ (u0 σ)2 ϕ0 σ = −1 2 Z T 0 Z Ω ρ(m) (u(m))2 ðtϕE dx dt −1 2 Z Ω (ρ(m))0(x) (u(m))0(x) 2 ϕE(x, 0) dx. By the deﬁnition (56a) of the initial conditions of the scheme, since both ρ0 and u0 are supposed to belong to L∞(Ω), (ρ(m))0 and (u(m))0 converge to ρ0 and u0 respectively in Lr(Ω), for r ≥1. Since, by assumption, the sequence of discrete solutions converges in Lr(Ω× (0, T )) for r ≥1, we can pass to the limit in the previous relation, to get: lim m−→+∞T (m) 1 = −1 2 Z T 0 Z Ω ¯ρ (¯u)2 ∂tϕ dx dt −1 2 Z Ω ρ0(x) u0(x)2 ϕ(x, 0) dx. Then the limit (¯ρ, ¯p, ¯u) satisﬁes the system (31). (41) (41) By a similar computation, we get for T (m) 2 : By a similar computation, we get for T (m) 2 : By a similar computation, we get for T (m) 2 : By a similar computation, we get for T (m) 2 : T (m) 2 = − N−1 X n=0 δt X K∈M \|K\| H(ρn+1 K ) ϕn+1 K −ϕn K δt − X σ∈E \|K\| H(ρ0 K) ϕ0 K = − Z T 0 Z Ω H(ρ(m)) ðtϕM(m) dx dt − Z Ω H (ρ(m))0 (x) ϕM(m)(x, 0) dx, Z 0 Z Ω Z Ω and therefore: lim m−→+∞T (m) 2 = − Z T 0 Z Ω H(¯ρ) ∂tϕ dx dt − Z Ω H(ρ0)(x) ϕ(x, 0) dx. (42) and therefore: lim m−→+∞T (m) 2 = − Z T 0 Z Ω H(¯ρ) ∂tϕ dx dt − Z Ω H(ρ0)(x) ϕ(x, 0) dx. (42) lim m−→+∞T (m) 2 = − Z T 0 Z Ω H(¯ρ) ∂tϕ dx dt − Z Ω H(ρ0)(x) ϕ(x, 0) dx. (42) lim m−→+∞T (m) 2 = − Z T 0 Z Ω H(¯ρ) ∂tϕ dx dt − Z Ω H(ρ0)(x) ϕ(x, 0) dx. (42) (42) TITLE WILL BE SET BY THE PUBLISHER 22 Let us now study the kinetic energy convection term T (m) 3 which reads, after reordering the summations: Let us now study the kinetic energy convection term T (m) 3 which reads, after reordering the summations: T (m) 3 = −1 2 N−1 X n=0 δt X K=[ −−→ σσ′]∈M F n+1 K un+1 σ un+1 σ′ ϕn σ −ϕn σ′ . Using now the deﬁnition of the mass ﬂuxes at the dual edges, we have: Using now the deﬁnition of the mass ﬂuxes at the dual edges, we have: T (m) 3 = −1 4 N−1 X n=0 δt X K=[ −−→ σσ′]∈M ρn+1 σ un+1 σ + ρn+1 σ′ un+1 σ′ ) un+1 σ′ un+1 σ ϕn σ −ϕn σ′ . Then the limit (¯ρ, ¯p, ¯u) satisﬁes the system (31). Reordering the sums, the term R(m) 3,1 reads: Reordering the sums, the term R(m) 3,1 reads: R(m) 3,1 = 1 4 N−1 X n=0 δt X σ∈Eint, σ=L→K, K=[σσ′] εn+1 σ (ρn+1 L −ρn+1 K ) (un+1 σ )2 un+1 σ′ (ϕn σ −ϕn σ′), TITLE WILL BE SET BY THE PUBLISHER 23 TITLE WILL BE SET BY THE PUBLISHER where εn+1 σ = ±1 and the notation L →K means that the ﬂow is going from L to K. Thanks to the Cauchy- Schwarz inequality, we get, by the regularity of ϕ: where εn+1 σ = ±1 and the notation L →K means that the ﬂow is going from L to K. Thanks to the Cauchy- Schwarz inequality, we get, by the regularity of ϕ: \|R(m) 3,1 \| ≤Cϕ h1/2 hN−1 X n=0 δt X σ=K\|L∈Eint \|un+1 σ \| (ρn+1 L −ρn+1 K )2i1/2 hN−1 X n=0 δt X σ∈Eint, σ=L→K, K=[σσ′] \|K\| \|un+1 σ \| (un+1 σ un+1 σ′ )2i1/2 , \|R(m) 3,1 \| ≤Cϕ h1/2 hN−1 X n=0 δt X σ=K\|L∈Eint \|un+1 σ \| (ρn+1 L −ρn+1 K )2i1/2 hN−1 X n=0 δt X σ∈Eint, σ=L→K, K=[σσ′] \|K\| \|un+1 σ \| (un+1 σ un+1 σ′ )2i1/2 , and thus: hus: \|R(m) 3,1 \| ≤Cϕ h1/2 ∥u(m)∥ 5/2 L5(Ω×(0,T )). \|R(m) 3,1 \| ≤Cϕ h1/2 ∥u(m)∥ 5/2 L5(Ω×(0,T )). We now turn to R(m) 3,2 . Thanks to the regularity of ϕ, we get: We now turn to R(m) 3,2 . Thanks to the regularity of ϕ, we get: We now turn to R(m) 3,2 . Thanks to the regularity of ϕ, we get: We now turn to R(m) 3,2 . Thanks to the regularity of ϕ, we get: \|R(m) 3,2 \| ≤Cϕ (h(m))2 ν(m) ∥ρ(m)∥L∞(Ω×(0,T )) ∥u(m)∥ 2 L∞(Ω×(0,T )) X K=[σσ′]∈M ν(m) hK (un+1 σ −un+1 σ′ )2, and thus R(m) 3,2 also tends to zero when m tends to +∞as soon as the ratio (h(m))2/ν(m) tends to zero. As a consequence, we get that T and thus R(m) 3,2 also tends to zero when m tends to +∞as soon as the ratio (h(m))2/ν(m) tends to zero. Then the limit (¯ρ, ¯p, ¯u) satisﬁes the system (31). We now split T (m) 3 = T (m) 3 + R(m) 3 , where We now split T (m) 3 = T (m) 3 + R(m) 3 , where T (m) 3 = −1 4 N−1 X n=0 δt X K=[ −−→ σσ′]∈M ρn+1 K (un+1 σ )3 + (un+1 σ′ )3 ϕn σ −ϕn σ′ = −1 2 Z T 0 Z Ω ρ(m)(u(m))3ðxϕE dx dt, so that lim m−→+∞T (m) 3 = −1 2 Z T 0 Z Ω ¯ρ¯u3∂xϕ dx dt, and R(m) 3 = −1 4 N−1 X n=0 δt X K=[ −−→ σσ′]∈M h (ρn+1 σ un+1 σ + ρn+1 σ′ un+1 σ′ ) un+1 σ un+1 σ′ −ρn+1 K (un+1 σ )3 + (un+1 σ′ )3i (ϕn σ −ϕn σ′). Expanding the quantity (un+1 σ )3 + (un+1 σ′ )3 thanks to the identity a3 + b3 = (a + b)(ab + (a −b)2), we obtain R(m) 3 = R(m) 3,1 + R(m) 3,2 with: Expanding the quantity (un+1 σ )3 + (un+1 σ′ )3 thanks to the identity a3 + b3 = (a + b)(ab + (a −b)2), we obtain R(m) 3 = R(m) 3,1 + R(m) 3,2 with: R(m) 3,1 = −1 4 N−1 X n=0 δt X K=[ −−→ σσ′]∈M h (ρn+1 σ −ρn+1 K ) un+1 σ + (ρn+1 σ′ −ρn+1 K ) un+1 σ′ i un+1 σ un+1 σ′ (ϕn σ −ϕ R(m) 3,2 = 1 4 N X n=0 δt X K=[ −−→ σσ′]∈M ρn+1 K (un+1 σ + un+1 σ′ ) (un+1 σ −un+1 σ′ )2 (ϕn σ −ϕn σ′). R(m) 3,1 = −1 4 N−1 X n=0 δt X K=[ −−→ σσ′]∈M h (ρn+1 σ −ρn+1 K ) un+1 σ + (ρn+1 σ′ −ρn+1 K ) un+1 σ′ i un+1 σ un+1 σ′ (ϕn σ −ϕn σ′), R(m) 3,2 = 1 4 N X n=0 δt X K=[ −−→ σσ′]∈M ρn+1 K (un+1 σ + un+1 σ′ ) (un+1 σ −un+1 σ′ )2 (ϕn σ −ϕn σ′). Then the limit (¯ρ, ¯p, ¯u) satisﬁes the system (31). Therefore, lim m→+∞T (m) 4 = − Z T 0 Z Ω H(¯ρ) ¯u ∂xϕ dx dt. (43) (43) Reordering the sums in the term T (m) 5 , we obtain: Reordering the sums in the term T (m) 5 , we obtain: T (m) 5 = N−1 X n=0 −δt X K=[ −−→ σσ′]∈M pn+1 K (un+1 σ′ ϕn σ′ −un+1 σ ϕn σ) + pn+1 K (un+1 σ −un+1 σ′ ) ϕn K, hence: T (m) 5 = − N−1 X n=0 δt X K=[ −−→ σσ′]∈M \|K\| 2 pn+1 K un+1 σ ϕn K −ϕn σ hK/2 + \|K\| 2 pn+1 K un+1 σ′ ϕn σ′ −ϕn K hK/2 [ ] = − Z T 0 Z Ω p(m) u(m) ðxϕM,E dx dt. = − Z T 0 Z Ω p(m) u(m) ðxϕM,E dx dt. 0 and so, since ðxϕM,E converges to ∂xϕ in Lr(Ω), for r ≥1: lim m−→+∞T (m) 5 = − Z T 0 Z Ω ¯p ¯u ∂xϕ dx lim m−→+∞T (m) 5 = − Z T 0 Z Ω ¯p ¯u ∂xϕ dx dt. (44) (44) From (41)-(44), we get that lim m−→+∞ 5 X i=1 T (m) i = − Z T 0 Z Ω η ∂tϕ + (η + p) u ∂xϕ dx dt − Z Ω η0(x) ϕ(x, 0) dx. In order to complete the proof of Theorem 3.17, there only remains to show that limm→+∞R(m) ≥0. Since we only seek an inequality, the non-negative part of R(m) , i.e. the ﬁrst part in Rn+1 σ and the whole term Rn+1 K , poses no problem, and we only have to study the terms coming from the second part of Rn+1 σ , which reads: (Rdiﬀ)n+1 σ = h X K=[σσ′] ν(m) hK (un+1 σ −un+1 σ′ ) i un+1 σ . For 0 ≤n ≤N −1 and K ∈M, K = [σσ′], let us deﬁne the quantity Qn+1 K by: For 0 ≤n ≤N −1 and K ∈M, K = [σσ′], let us deﬁne the quantity Qn+1 K by: Qn+1 K = ν(m) hK (un+1 σ −un+1 σ′ )2. Then the limit (¯ρ, ¯p, ¯u) satisﬁes the system (31). As a consequence, we get that lim m−→+∞T (m) 3 = −1 2 Z T 0 Z Ω ¯ρ¯u3∂xϕ dx dt, Expressing the mass ﬂuxes as a function of the unknowns in T (m) 4 and reordering the sums, we get: T (m) 4 = − N−1 X n=0 δt X σ=−−→ K\|L∈Eint H(ρn+1 σ ) un+1 σ (ϕn K −ϕn L). rite T (m) 4 = T (m) 4 + R(m) 4 , with, thanks to the deﬁnition of the upwind density (6) at the face: T (m) 4 = − N−1 X n=0 δt X σ=−−→ K\|L∈Eint h \|DK,σ\| H(ρn+1 K ) + \|DL,σ\| H(ρn+1 L ) i un+1 σ ϕn K −ϕn L hσ , R(m) 4 = N−1 X n=0 δt X σ=−−→ K\|L∈Eint \|DL,σ\| h H(ρn+1 L ) −H(ρn+1 K ) i un+1 σ ϕn K −ϕn L hσ . We have: (m) = − Z T 0 Z Ω H(ρ(m)) u(m) ðxϕM(m) dx dt, so lim m−→+∞T (m) 4 = − Z T 0 Z Ω H(¯ρ) ¯u ∂xϕ dx dt. T (m) 4 = − Z T 0 Z Ω H(ρ(m)) u(m) ðxϕM(m) dx dt, so lim m−→+∞T (m) 4 = − Z T 0 Z Ω H(¯ρ) ¯u ∂xϕ dx dt. T (m) 4 = − Z T 0 Z Ω H(ρ(m)) u(m) ðxϕM(m) dx dt, so lim m−→+∞T (m) 4 = − Z T 0 Z Ω H(¯ρ) ¯u ∂xϕ dx dt. By the regularity of ϕ, we get: By the regularity of ϕ, we get: \|R(m) 4 \| ≤Cϕ h(m) N−1 X n=0 δt X σ=K\|L∈Eint H(ρn+1 K ) −H(ρn+1 L ) \|un+1 σ \|. \|R(m) 4 \| ≤Cϕ h(m) N−1 X n=0 δt X σ=K\|L∈Eint H(ρn+1 K ) −H(ρn+1 L ) \|un+1 σ \|. TITLE WILL BE SET BY THE PUBLISHER 24 Since both sequences (ρ(m))m∈N and (1/ρ(m))m∈N are supposed to be uniformly bounded, we have H(ρn+1 K ) − H(ρn+1 L ) ≤C \|ρn+1 K −ρn+1 L \| with a constant real number C, and therefore R(m) 4 tends to zero as h(m). Therefore, Since both sequences (ρ(m))m∈N and (1/ρ(m))m∈N are supposed to be uniformly bounded, we have H(ρn+1 K ) − H(ρn+1 L ) ≤C \|ρn+1 K −ρn+1 L \| with a constant real number C, and therefore R(m) 4 tends to zero as h(m). Then the limit (¯ρ, ¯p, ¯u) satisﬁes the system (31). We have Qn+1 K ≥0, and, reordering the summation, we get that Q(m) = N−1 X n=0 δt h X σ∈Eint ϕn σ (Rdiﬀ)n+1 σ − X K∈M ϕn K Qn+1 K i = N−1 X n=0 δt X K=[ −−→ σσ′]∈M ν hK (un+1 σ −un+1 σ′ ) un+1 σ (ϕσ −ϕK) −un+1 σ′ (ϕσ′ −ϕK) 25 TITLE WILL BE SET BY THE PUBLISHER By the Cauchy-Schwarz inequality and the regularity of ϕ, we thus get: By the Cauchy-Schwarz inequality and the regularity of ϕ, we thus get: By the Cauchy-Schwarz inequality and the regularity of ϕ, we thus get: By the Cauchy-Schwarz inequality and the regularity of ϕ, we thus get: Q(m) ≤Cϕ hN−1 X n=0 δt X K=[ −−→ σσ′]∈M ν(m) hK (un+1 σ −un+1 σ′ )2i1/2 hN−1 X n=0 δt X K=[ −−→ σσ′]∈M ν(m) \|DK,σ\| (un+1 σ )2 + \|DK,σ′\| (un+1 σ′ )2i1/2 . hN−1 X n=0 δt X K=[ −−→ σσ′]∈M ν(m) \|DK,σ\| (un+1 σ )2 + \|DK,σ′\| (un+1 σ′ )2i1/2 . From estimate (24), we thus get that From estimate (24), we thus get that Q(m) ≤C (ν(m))1/2, Q(m) ≤C (ν(m))1/2, where C > 0 only depends on ϕ, on C0 and on the assumed bounds on the solution. Since ν(m) tends to 0 with m, we then obtain that limm→+∞R(m) ≥0, which concludes the proof. □ where C > 0 only depends on ϕ, on C0 and on the assumed bounds on the solution. Since ν(m) tends to 0 with m, we then obtain that limm→+∞R(m) ≥0, which concludes the proof. □ 3.2. The pressure correction scheme for the barotropic equations The implicit scheme which we studied in the previous section is easy to write, but diﬃcult to implement in practice, because of the large nonlinear systems to be solved at the algebraic level. Pressure correction methods are based on the idea that one may compute the velocity and the pressure in a sequential way, thus yielding a more practical scheme. The algorithm presented in this section is implemented in the open-source software component library for ﬂuid ﬂows simulation CALIF3S [5], developed at IRSN on the basis of the software components library PELICANS [55]; in this context, it is routinely used for industrial applications. 3.2.1. The scheme In the algorithm given below, the velocity is predicted by solving the momentum balance equation with a known pressure. This latter is obtained from the beginning-of-step pressure through a “renormalization” step, in order to be able to perform the stability analysis (stability of the scheme and satisfaction of the entropy condition). Note that the renormalization proposed here is diﬀerent than that proposed in [22] in the context of variable density incompressible ﬂows or in [14] in the context of compressible barotropic ﬂows. Indeed, in these latter works, this step requires the resolution of a discrete elliptic problem for the pressure, while, here, we only scale the pressure gradient by a simple weight. Then, the velocity is corrected and the other variables are advanced in time. As for the implicit scheme, a discrete kinetic energy balance can be derived, provided that the mass balance over the dual cells (8) holds; since the mass balance is not yet solved when performing the prediction step, this leads us to perform a time shift of the density at this stage. The algorithm reads, for 0 ≤n ≤N −1: Pressure gradient renormalization step: Pressure gradient renormalization step: ∀σ ∈E, (g ∇p)n+1 σ = s ρn Dσ ρn−1 Dσ (∇p)n σ. E, (g ∇p)n+1 σ = s ρn Dσ ρn−1 Dσ (∇p)n σ. (45a) (45a) Prediction step – Solve for ˜un+1: For 1 ≤i ≤d, ∀σ ∈E(i) S , \|Dσ\| δt (ρn Dσ ˜un+1 σ,i −ρn−1 Dσ un σ,i) + X ǫ∈¯E(Dσ) F n σ,ǫ˜un+1 ǫ,i −\|Dσ\| (∆M˜u)n+1 σ,i + \|Dσ\| (g ∇p)n+1 σ,i = 0. (45 (45b) 26 TITLE WILL BE SET BY THE PUBLISHER 26 Correction step – Solve for ρn+1, pn+1 and un+1: Correction step – Solve for ρn+1, pn+1 and un+1: For 1 ≤i ≤d, ∀σ ∈E(i) S , \|Dσ\| δt ρn Dσ (un+1 σ,i −˜un+1 σ,i ) + \|Dσ\| (∇p)n+1 σ,i −(g ∇p)n+1 σ,i = 0, (45c) ∀K ∈M, \|K\| δt (ρn+1 K −ρn K) + X σ∈E(K) F n+1 K,σ = 0, with F n+1 K,σ = \|σ\| ρn+1 σ un+1 K,σ , (45d) ∀K ∈M, pn+1 K = (ρn+1 K )γ. (45e) For 1 ≤i ≤d, ∀σ ∈E(i) S , \|Dσ\| δt ρn Dσ (un+1 σ,i −˜un+1 σ,i ) + \|Dσ\| (∇p)n+1 σ,i −(g ∇p)n+1 σ,i = 0, (45c) (45c) (45d) ∀K ∈M, pn+1 K = (ρn+1 K )γ. ∀K ∈M, pn+1 K = (ρn+1 K )γ. (45e) Recall that the notation ρn+1 σ in (45d) stands for the upwind choice of ρ deﬁned by (6), while ρn Dσ (resp. ρn−1 Dσ ) in (45b) is the convex combination of ρn K and ρn L (resp. ρn−1 K and ρn−1 L ) deﬁned by (7). The initialization of the scheme is performed as follows. First, ρ−1 and u0 are given by the average of the initial conditions ρ0 and u0 on the primal and dual cells respectively: ∀K ∈M, ρ−1 K = 1 \|K\| Z K ρ0(x) dx, for 1 ≤i ≤d, ∀σ ∈E(i) S , u0 σ,i = 1 \|Dσ\| Z Dσ (u0(x))i dx. (46) ∀K ∈M, ρ−1 K = 1 \|K\| Z K ρ0(x) dx, (46) (46) for 1 ≤i ≤d, ∀σ ∈E(i) S , u0 σ,i = 1 \|Dσ\| Z Dσ (u0(x))i dx. (46) Then, we compute ρ0 by solving the mass balance equation (45d). Finally, the initial pressure p0 is computed from the initial density ρ0 by the equation of state: ∀K ∈M, p0 K = (ρ0 K)γ. This procedure allows to perform the ﬁrst prediction step with (ρ−1 Dσ )σ∈E, (ρ0 Dσ)σ∈E and the dual mass ﬂuxes satisfying the mass balance. 3.2.2. Estimates As for the implicit scheme, we begin with an estimate on the velocity which is a discrete equivalent of the kinetic energy balance. Lemma 3.11 (Discrete kinetic energy balance, pressure correction scheme). A solution to the system (45) satisﬁes the following equality, for 1 ≤i ≤d, σ ∈E(i) S and 0 ≤n ≤N −1: Lemma 3.11 (Discrete kinetic energy balance, pressure correction scheme). A solution to the system (45) satisﬁes the following equality, for 1 ≤i ≤d, σ ∈E(i) S and 0 ≤n ≤N −1: 1 2 \|Dσ\| δt h ρn Dσ(un+1 σ,i )2 −ρn−1 Dσ (un σ,i)2i + 1 2 X ǫ=Dσ\|Dσ′ F n σ,ǫ ˜un+1 σ,i ˜un+1 σ′,i + \|Dσ\| (∇p)n+1 σ,i un+1 σ,i = −Rn+1 σ,i −P n+1 σ,i , (47) (47) where Rn+1 σ,i = \|Dσ\| 2 δt ρn−1 Dσ ˜un+1 σ,i −un σ,i 2 + h X ǫ=Dσ\|Dσ′ ν hd−2 ǫ (˜un+1 σ,i −˜un+1 σ′,i ) i ˜un+1 σ,i , P n+1 σ,i = \|Dσ\| δt 2 ρn Dσ h(∇p)n+1 σ,i 2 − (g ∇p)n+1 σ,i 2i . (48) (48) TITLE WILL BE SET BY THE PUBLISHER 27 Proof. Let us multiply the velocity prediction equation (45b) by the corresponding velocity unknown ˜un+1 σ,i , and use the equality (88) of Lemma A.2, on the dual mesh and with P = Dσ. We obtain: Proof. Let us multiply the velocity prediction equation (45b) by the corresponding velocity unknown ˜un+1 σ,i , and use the equality (88) of Lemma A.2, on the dual mesh and with P = Dσ. We obtain: 1 2 \|Dσ\| δt h ρn Dσ(˜un+1 σ,i )2 −ρn−1 Dσ (un σ,i)2i + 1 2 X ǫ=Dσ\|Dσ′ F n σ,ǫ ˜un+1 σ,i ˜un+1 σ′,i 1 2 \|Dσ\| δt h ρn Dσ(˜un+1 σ,i )2 −ρn−1 Dσ (un σ,i)2i + 1 2 X ǫ=Dσ\|Dσ′ F n σ,ǫ ˜un+1 σ,i ˜un+1 σ′,i + 1 2 \|Dσ\| δt ρn−1 Dσ ˜un+1 σ,i −un σ,i 2 −\|Dσ\|(∆M˜u)n+1 σ,i ˜un+1 σ,i + \|Dσ\| (g ∇p)n+1 σ,i ˜un+1 σ,i = 0. (49) + 1 2 \|Dσ\| δt ρn−1 Dσ ˜un+1 σ,i −un σ,i 2 −\|Dσ\|(∆M˜u)n+1 σ,i ˜un+1 σ,i + \|Dσ\| (g ∇p)n+1 σ,i ˜un+1 σ,i = 0. 3.2.2. Estimates (49) (49) ing the velocity correction equation (45c) by (\|Dσ\| δt ρn Dσ) 1 2 , we obtain: Dividing the velocity correction equation (45c) by (\|Dσ\| δt ρn Dσ) 1 2 , we obtain: h\|Dσ\| δt ρn Dσ i1/2 un+1 σ,i + h\|Dσ\| δt ρn Dσ i1/2 (∇p)n+1 σ,i = h\|Dσ\| δt ρn Dσ i1/2 ˜un+1 σ,i + h\|Dσ\| δt ρn Dσ i1/2 (g ∇p)n+1 σ,i . Squaring this relation, dividing by two and summing it with (49) yields the result, using the deﬁnition (10) o (∆M˜u)n+1. □ The discrete potential balance is again valid for the pressure correction algorithm, thanks to the fact that the mass balance (45d) is satisﬁed. The proof is identical to that of Lemma 3.2 given for the implicit scheme. Lemma 3.12 (Discrete potential balance, pressure correction scheme). A solution to the system (45) satisﬁes the discrete potential balance (22), with Rn+1 K deﬁned by (23). The discrete potential balance is again valid for the pressure correction algorithm, thanks t the mass balance (45d) is satisﬁed. The proof is identical to that of Lemma 3.2 given for the im Lemma 3.12 (Discrete potential balance, pressure correction scheme). A solution to the system (45) satisﬁes the discrete potential balance (22), with Rn+1 K deﬁned by (23). Let us now turn to the entropy inequality. Let us now turn to the entropy inequality. Proposition 3.13 (Global discrete entropy inequality, existence of a solution). Assume that the initial condition ρ0 is positive. Then there exists a solution (un, ρn) 0≤n≤N and (˜un) 1≤n≤N to the scheme (45), and, for 1 ≤n ≤N, ρn > 0 and the following inequality holds: 1 2 d X i=1 X σ∈E(i) S \|Dσ\| ρn−1 Dσ (un σ,i)2 + X K∈M \|K\| H(ρn K) + Rn ≤C0, (50) (50) where C0 ∈R+ only depends on the initial conditions and on the density ﬁeld ρ0 computed at the initialization of the algorithm. 3.2.2. Estimates We obtain a ”local in time” version of Equation (50), which reads: T n+1 −T n + Rn+1 + Pn+1 = 0, (52) T n+1 −T n + Rn+1 + Pn+1 = 0, (52) TITLE WILL BE SET BY THE PUBLISHER 28 where: where: T n+1 = X K∈M \|K\| H(ρn+1 K ) + 1 2 d X i=1 X σ∈E(i) S \|Dσ\| ρn Dσ (un+1 σ,i )2, and: Rn+1 = X 1≤i≤d X σ∈E(i) S Rn+1 σ,i , Pn+1 = X 1≤i≤d X σ∈E(i) S ∩Eint P n+1 σ,i , where: T n+1 = X K∈M \|K\| H(ρn+1 K ) + 1 2 d X i=1 X σ∈E(i) S \|Dσ\| ρn Dσ (un+1 σ,i )2, d T n+1 = X K∈M \|K\| H(ρn+1 K ) + 1 2 d X i=1 X σ∈E(i) S \|Dσ\| ρn Dσ (un+1 σ,i )2, K∈M i σ∈E( ) S and: Rn+1 = X 1≤i≤d X σ∈E(i) S Rn+1 σ,i , Pn+1 = X 1≤i≤d X σ∈E(i) S ∩Eint P n+1 σ,i , and: and: and: Rn+1 = X 1≤i≤d X σ∈E(i) S Rn+1 σ,i , Pn+1 = X 1≤i≤d X σ∈E(i) S ∩Eint P n+1 σ,i , with Rn+1 σ,i , and P n+1 σ,i given by Equation (48). The term Pn+1 thus reads: with Rn+1 σ,i , and P n+1 σ,i given by Equation (48). The term Pn+1 thus reads: Pn+1 = δt2 2 X σ∈Eint \|Dσ\| ρn Dσ h \|(∇p)n+1 σ \|2 −\|(g ∇p)n+1 σ \|2i = δt2 2 X σ∈Eint \|Dσ\| h\|(∇p)n+1 σ \|2 ρn Dσ −\|(∇p)n σ\|2 ρn−1 Dσ i . (53) (53) Then, summing (52) over the time steps yields the estimate (50) with: Then, summing (52) over the time steps yields the estimate (50) with: C0 = X K∈M \|K\| H(ρ0 K) + 1 2 X 1≤i≤d X σ∈E(i) S \|Dσ\| ρ−1 Dσ (u0 σ,i)2 + δt2 2 X σ∈Eint \|Dσ\| ρ−1 Dσ \|(∇p)0 σ\|2. □ Remark 3.14. As in the implicit case, the inequality (50) thus provides an estimate on the unknowns (see Remark 3.4). Remark 3.14. As in the implicit case, the inequality (50) thus provides an estimate on the unknowns (see Remark 3.4). Remark 3.15 (Regularity assumptions for the initial conditions). For a given mesh, the quantity denoted above by C0 is bounded whenever ρ0 is positive and belongs to L1(Ω) and u0 belongs to L1(Ω)d. 3.2.2. Estimates The remainder term Rn is non-negative, and gathers some estimates of the space and time translates of the unknowns: Rn = d X i=1 n X k=1 h1 2 X σ∈E(i) S \|Dσ\| ρk−2 Dσ (˜uk σ,i −uk−1 σ,i )2 + δt X ǫ=Dσ\|Dσ′ ∈¯E(i) S ν hd−2 σ (˜uk σ,i −˜uk σ′,i)2i + γ 2 n X k=1 δt X σ=K\|L∈Eint \|σ\| (ρk σ,γ)γ−2 \|uk K,σ\| (ρk K −ρk L)2 + δt2 2 X σ∈Eint \|Dσ\| ρn−1 Dσ \|(∇p)n σ\|2, (51) with ρk σ,γ equal to either ρk K or ρk L and such that (ρk σ,γ)γ−2 = min (ρk K)γ−2, (ρk L)γ−2 . with ρk σ,γ equal to either ρk K or ρk L and such that (ρk σ,γ)γ−2 = min (ρk K)γ−2, (ρk L)γ−2 . Proof. The essential arguments of the proof of this proposition are given in [14, Theorem 3.8] with slightly diﬀerent notations, so we brieﬂy recall here how to obtain the estimate (50), for the sake of completeness. We sum the kinetic energy balance equation (47) over the faces, and the elastic potential balance (22) over the cells, and ﬁnally sum the two obtained relations. We obtain a ”local in time” version of Equation (50), which reads: Proof. The essential arguments of the proof of this proposition are given in [14, Theorem 3.8] with slightly diﬀerent notations, so we brieﬂy recall here how to obtain the estimate (50), for the sake of completeness. We sum the kinetic energy balance equation (47) over the faces, and the elastic potential balance (22) over the cells, and ﬁnally sum the two obtained relations. 3.2.2. Estimates When dealing with a sequence of discretizations to pass to the limit in the scheme, we need to assume that C0 is controlled independently of the mesh and time step, which necessitates (i) that the initial kinetic energy is bounded, (ii) that H(ρ0 K) is bounded in L1(Ω), and (iii) that the last term involving the discrete pressure gradient does not blow-up. Assumption (ii) (and, of course, (i)) may be obtained by supposing that both u0 and ρ0 belongs to L∞(Ω) and L∞(Ω)d respectively and that δt/h is bounded (possibly by a number much larger than 1); indeed, ρ0 is then obtained in this case by a single time step of a (discrete) transport equation with a velocity ﬁeld the divergence of which is controlled by 1/h, and so ρ0 is controlled in L∞(Ω). Assumption (iii) may then be inferred by the same assumption on the ratio δt/h, together with the hypothesis that the data ρ0 (and so ρ−1) is bounded away from zero. Indeed, since ρ0 is bounded, so is p0 and we get: X σ∈Eint \|Dσ\| δt2 ρ−1 Dσ \|(∇p)0 σ\|2 = X σ=K\|L∈Eint δt2 \|σ\|2 ρ−1 Dσ \|Dσ\| (p0 K −p0 L)2 ≤C ∥1 ρ−1 ∥ L∞(Ω) ∥p0∥ 2 L∞(Ω) X σ∈Eint h2 \|σ\|2 \|Dσ\| sum is bounded. We shall work under these assumptions for the passage to the limit in the schem The expression (53) in the proof of Proposition 3.13, where Pn+1 is set under the form of a diﬀerence of the same two quantities taken at two consecutive time steps, shows the interest of the pressure gradient renormal- ization step. We prove in addition in the following lemma that, under stability conditions, this remainder term tends to zero in a discrete distribution sense. Lemma 3.16 (Pressure remainder terms). Let (M(m), δt(m))m∈N be a sequence of meshes and time steps, such that h(m) and δt(m) tend to zero as m tends to inﬁnity, and satisfying the CFL-like condition: ∀m ∈N, δt(m) h(m) ≤C, with h(m) = min σ∈E(m) int \|Dσ\| \|σ\| , TITLE WILL BE SET BY THE PUBLISHER 29 and where C is a positive real number which can be greater than 1. Let (ρ(m))m∈N and (p(m))m∈N be (part of) the associated sequence of discrete solutions, satisfying equations (45). 3.2.2. Estimates We assume that the sequence (p(m))m∈N is uniformly bounded in L∞(Ω× (0, T )) and in the discrete L1(0, T ; BV (Ω)) norm: ∀m ∈N, ∥p(m)∥T ,x,BV = N (m) X n=0 δt X σ=K\|L∈E(m) int \|σ\| (p(m))n L −(p(m))n K ≤C. (54) (54) We furthermore suppose that (ρ(m))m∈N and (1/ρ(m))m∈N are bounded in L∞(Ω×(0, T )). Let ϕ ∈C∞ c (Ω×[0, T )), and, for m ∈N, 0 ≤n ≤N (m) and σ ∈E(m) int , let ϕn σ = ϕ(xσ, tn), with xσ the mass center of σ. Let us deﬁne the quantity P n+1 σ , for 0 ≤n ≤N −1 and σ ∈Eint, as: P n+1 σ = \|Dσ\| δt ρn Dσ h \|(∇p)n+1 σ \|2 −\|(g ∇p)n+1 σ \|2i . (55) (55) Then lim m→+∞ hn−1 X n=0 δt X σ∈Eint P n+1 σ ϕn σ i = 0. Proof. By deﬁnition of P n+1 σ , we get that N−1 X n=0 δt X σ=K\|L∈Eint P n+1 σ ϕn σ = N−1 X n=0 δt2 X σ=K\|L∈Eint \|σ\|2 \|Dσ\| h 1 ρn Dσ (pn+1 K −pn+1 L )2 − 1 ρn−1 Dσ (pn K −pn L)2i ϕn σ. N−1 X n=0 δt X σ=K\|L∈Eint P n+1 σ ϕn σ = N−1 X n=0 δt2 X σ=K\|L∈Eint \|σ\|2 \|Dσ\| h 1 ρn Dσ (pn+1 K −pn+1 L )2 − 1 ρn−1 Dσ (pn K −pn L)2i ϕn σ. A discrete integration by parts yields: A discrete integration by parts yields: A discrete integration by parts yields: \| N−1 X n=0 δt X σ=K\|L∈Eint P n+1 σ ϕn σ\| ≤δt2   X σ=K\|L∈Eint 1 ρ−1 Dσ \|σ\|2 \|Dσ\|\|p0 K −p0 L\|2 \|ϕ0 σ\| + N−1 X n=0 X σ=K\|L∈Eint 1 ρn Dσ \|σ\|2 \|Dσ\|(pn+1 K −pn+1 L )2\|ϕn+1 σ −ϕn σ\|  . + N−1 X n=0 X σ=K\|L∈Eint 1 ρn Dσ \|σ\|2 \|Dσ\|(pn+1 K −pn+1 L )2\|ϕn+1 σ −ϕn σ\|  . Prediction step – Solve for ˜un+1: ∀σ = −−→ K\|L ∈Eint, ∀σ = −−→ K\|L ∈Eint, \|Dσ\| δt (ρn Dσ ˜un+1 σ −ρn−1 Dσ un σ) + F n L ˜un+1 L −F n K ˜un+1 K −\|Dσ\| (∆M˜u)n+1 σ + ( eδp)n+1 σ = 0, (56c) (56c) Correction step – Solve for ρn+1, pn+1 and un+1: Correction step – Solve for ρn+1, pn+1 and un+1: Correction step – Solve for ρn+1, pn+1 and un+1: Correction step – Solve for ρn+1, pn+1 and un+1: ∀σ = −−→ K\|L ∈Eint, \|Dσ\| δt ρn Dσ (un+1 σ −˜un+1 σ ) + (pn+1 L −pn+1 K ) −( eδp)n+1 σ = 0, (56d) ∈ int, \|Dσ\| δt ρn Dσ (un+1 σ −˜un+1 σ ) + (pn+1 L −pn+1 K ) −( eδp)n+1 σ = 0, (56d) (56d) δt ∀K = [−→ σσ′] ∈M, \|K\| δt (ρn+1 K −ρn K) + F n+1 σ′ −F n+1 σ = 0, (56e) ∀K ∈M, pn+1 K = (ρn+1 K )γ. (56f) = [σσ′] ∈M, \|K\| δt (ρn+1 K −ρn K) + F n+1 σ′ −F n+1 σ = 0, \|K\| δt (ρn+1 K −ρn K) + F n+1 σ′ −F n+1 σ = 0, \|K\| δt (ρn+1 K −ρn K) + F n+1 σ′ −F n+1 σ = 0, (56e) (56e) ∀K ∈M, pn+1 K = (ρn+1 K )γ. (56f) Theorem 3.17 (Consistency of the pressure correction scheme). Let Ωbe an open bounded interval of R. We suppose that ρ0 ∈L∞(Ω), 1/ρ0 ∈L∞(Ω) and u0 ∈L∞(Ω). Let (M(m), δt(m), ν(m))m∈N be a regular sequence of discretizations in the sense of Deﬁnition 3.5, and let (ρ(m), p(m), u(m), ˜u(m))m∈N be the corresponding sequence of solutions. We suppose that this sequence con- verges in Lp(Ω× (0, T ))4, for 1 ≤p < +∞, to (¯ρ, ¯p, ¯u, ¯˜u) ∈L∞(Ω× (0, T ))4. We suppose in addition that both sequences (ρ(m))m∈N and (1/ρ(m))m∈N are uniformly bounded in L∞(Ω× (0, T )). Th ¯ ¯˜ d th t i l t (¯ ¯ ¯) ti ﬁ th t (31) Theorem 3.17 (Consistency of the pressure correction scheme). Let Ωbe an open bounded interval of R. We suppose that ρ0 ∈L∞(Ω), 1/ρ0 ∈L∞(Ω) and u0 ∈L∞(Ω). Let (M(m), δt(m), ν(m))m∈N be a regular sequence of discretizations in the sense of Deﬁnition 3.5, and let (ρ(m), p(m), u(m), ˜u(m))m∈N be the corresponding sequence of solutions. 3.2.3. Passing to the limit in the scheme Using the notations of Section 3.1.3, the pressure correction scheme in one space dimension reads: Initialization – Compute ρ−1, u0, solve for ρ0 and compute p0: ∀K ∈M, ρ−1 K = 1 \|K\| Z K ρ0(x) dx, ∀σ ∈Eint, u0 σ = 1 \|Dσ\| Z Dσ u0(x) dx, ∀K = [−→ σσ′] ∈M, \|K\| δt (ρ0 K −ρ−1 K ) + F 0 σ′ −F 0 σ = 0, ∀K ∈M, p0 K = (ρ0 K)γ. (56a) (56a) Pressure gradient renormalization step: Pressure gradient renormalization step: essure gradient renormalization step: ∀σ = −−→ K\|L ∈Eint, ( eδp)n+1 σ = s ρn Dσ ρn−1 Dσ (pn L −pn K). (56b) (56b) Prediction step – Solve for ˜un+1: 3.2.2. Estimates Using the fact that, for 0 ≤n ≤N and σ = K\|L ∈Eint, (pn K −pn L)2 ≤2 ∥p∥L∞(Ω×(0,T )) \|pn K −pn L\|, we get, thanks to the regularity of ϕ, that Using the fact that, for 0 ≤n ≤N and σ = K\|L ∈Eint, (pn K −pn L)2 ≤2 ∥p∥L∞(Ω×(0,T )) \|pn K −pn L\|, we get, thanks to the regularity of ϕ, that \| N−1 X n=0 δt X σ=K\|L∈Eint P n+1 σ ϕn σ\| ≤Cϕ δt2 h ∥p∥L∞(Ω×(0,T )) h ∥p0∥BV (Ω) + ∥p∥T ,x,BV i , which concludes the proof. □ which concludes the proof. which concludes the proof. □ which concludes the proof. TITLE WILL BE SET BY THE PUBLISHER 30 3.2.3. Passing to the limit in the scheme 3.2.3. Passing to the limit in the scheme Theorem 3.17 (Consistency of the pressure correction scheme). Let Ωbe an open bounded interval of R. We suppose that ρ0 ∈L∞(Ω), 1/ρ0 ∈L∞(Ω) and u0 ∈L∞(Ω). Let (M(m), δt(m), ν(m))m∈N be a regular sequence of discretizations in the sense of Deﬁnition 3.5, and let (ρ(m), p(m), u(m), ˜u(m))m∈N be the corresponding sequence of solutions. We suppose that this sequence con- verges in Lp(Ω× (0, T ))4, for 1 ≤p < +∞, to (¯ρ, ¯p, ¯u, ¯˜u) ∈L∞(Ω× (0, T ))4. We suppose in addition that both sequences (ρ(m))m∈N and (1/ρ(m))m∈N are uniformly bounded in L∞(Ω× (0, T )). Then ¯u = ¯˜u and the triplet (¯ρ, ¯p, ¯u) satisﬁes the system (31). Letting m tend to +∞in this equation yields ¯u = ¯˜u. Letting m tend to +∞in this equation yields ¯u = ¯˜u. As for the implicit scheme, with the assumed convergence for the sequence of solutions, the limit satisﬁes the equation of state. The passage to the limit in the mass balance equation is the same as in the implicit case, and we only need to address here the momentum balance equation. Let ϕ ∈C∞ c (Ω× [0, T )), and let its interpolate ϕE and its discrete derivatives be deﬁned by Deﬁnition 3.6. Summing the velocity prediction and correction equations, multiplying the result by δt ϕn σ and then summing over the faces and time steps, we get T (m) 1 + T (m) 2 + T (m) 3 + T (m) 4 = 0, with: T (m) 1 = N−1 X n=0 X σ∈Eint \|Dσ\| ρn Dσ ˜un+1 σ −ρn−1 Dσ un σ ϕn σ, T (m) 2 = N−1 X n=0 δt X σ=−−→ K\|L∈Eint h F n L ˜un+1 L −F n K ˜un+1 K i ϕn σ, T (m) 3 = N−1 X n=0 δt X σ=−−→ K\|L∈Eint (pn+1 L −pn+1 K ) ϕn σ, T (m) 4 = N−1 X n=0 δt X σ∈Eint h X K=[σσ′] ν hK (˜un+1 σ −˜un+1 σ′ ) i ϕn σ T (m) 1 = N−1 X n=0 X σ∈Eint \|Dσ\| ρn Dσ ˜un+1 σ −ρn−1 Dσ un σ ϕn σ, T (m) 2 = N−1 X n=0 δt X σ=−−→ K\|L∈Eint h F n L ˜un+1 L −F n K ˜un+1 K i ϕn σ, T (m) 3 = N−1 X n=0 δt X σ=−−→ K\|L∈Eint (pn+1 L −pn+1 K ) ϕn σ, T (m) 4 = N−1 X n=0 δt X σ∈Eint h X K=[σσ′] ν hK (˜un+1 σ −˜un+1 σ′ ) i ϕn σ. Prediction step – Solve for ˜un+1: We suppose that this sequence con- verges in Lp(Ω× (0, T ))4, for 1 ≤p < +∞, to (¯ρ, ¯p, ¯u, ¯˜u) ∈L∞(Ω× (0, T ))4. We suppose in addition that both sequences (ρ(m))m∈N and (1/ρ(m))m∈N are uniformly bounded in L∞(Ω× (0, T )). ¯ ( ) ( ) Then ¯u = ¯˜u and the triplet (¯ρ, ¯p, ¯u) satisﬁes the system (31). TITLE WILL BE SET BY THE PUBLISHER 31 TITLE WILL BE SET BY THE PUBLISHER TITLE WILL BE SET BY THE PUBLISHER 31 Proof. Let m ∈N be given. Dropping for short the superscript (m), the estimate of Proposition 3.13 yields: n X k=1 δt X σ∈Eint \|Dσ\| ρk−1 Dσ (˜uk σ −uk−1 σ )2 ≤C δt, (57) (57) where, by the assumption on the initial data, the real number C is independent of m (see Remark ∥˜u(m) −u(m)(., . −δt)∥ 2 L2(Ω×(0,T )) ≤C δt(m) ∥ 1 ρ(m) ∥ L∞(Ω×(0,T )) . Letting m tend to +∞in this equation yields ¯u = ¯˜u. Thanks to the deﬁnition (29) of the density on the dual mesh ρDσ, reordering the sums, we get for T (m) 1 : Thanks to the deﬁnition (29) of the density on the dual mesh ρDσ, reordering the sums, we get for T (m) 1 : T (m) 1 = − N−1 X n=0 δt X σ=K\|L∈Eint h\|K\| 2 ρn K + \|L\| 2 ρn L i ˜un+1 σ ϕn+1 σ −ϕn σ δt − X σ=K\|L∈Eint h\|K\| 2 ρ−1 K + \|L\| 2 ρ−1 L i u0 σ ϕ0 σ = − Z T 0 Z Ω ρ(m) ˜u(m) ðt ϕM(m) dx dt − Z Ω (ρ(m))−1(x) (u(m))0(x) ϕM(m)(x, 0) dx. From the deﬁnition (56a) of the initial conditions, the sequences (ρ(m))−1 and u(m))0 converge in Lr(Ω), for r ≥1, to ρ0 and u0 respectively. Thanks to the convergence assumption for the sequence of discrete solutions, and noting that the sequence ρ(m)(·, · −δt) m∈N converges to ¯ρ as (ρ(m))m∈N, we get: From the deﬁnition (56a) of the initial conditions, the sequences (ρ(m))−1 and u(m))0 converge in Lr(Ω), for r ≥1, to ρ0 and u0 respectively. Thanks to the convergence assumption for the sequence of discrete solutions, and noting that the sequence ρ(m)(·, · −δt) m∈N converges to ¯ρ as (ρ(m))m∈N, we get: lim m−→+∞T (m) 1 = − Z T 0 Z Ω ¯ρ ¯u ∂tϕ dx dt − Z Ω ρ0(x) u0(x) ϕ(x, 0) dx. 32 TITLE WILL BE SET BY THE PUBLISHER TITLE WILL BE SET BY THE PUBLISHER TITLE WILL BE SET BY THE PUBLISHER 32 Let us now turn to T (m) 2 . From the expression (29) of the ﬂuxes FK and the values uK, reordering the sums, we get: T (m) 2 = −1 4 N−1 X n=0 δt X K=[ −−→ σσ′]∈M (ρn σun σ + ρn σ′ un σ′) (˜un+1 σ + ˜un+1 σ′ ) (ϕn σ′ −ϕn σ), which we write T (m) 2 = T (m) 2 + R(m) 2 with: T (m) 2 = −1 2 N−1 X n=0 δt X K=[ −−→ σσ′]∈M ρn K h un σ ˜un+1 σ + un σ′ ˜un+1 σ′ i (ϕn σ′ −ϕn σ). Letting m tend to +∞in this equation yields ¯u = ¯˜u. (59) = − Z T 0 Z Ω ρ(m)(·, · −δt) u(m)(·, · −δt) ˜u(m) ðxϕE dx dt, (60) T (m) 2 = −1 4 N−1 X n=0 δt X K=[ −−→ σσ′]∈M (ρn σun σ + ρn σ′ un σ′) (˜un+1 σ + ˜un+1 σ′ ) (ϕn σ′ −ϕn σ), which we write T (m) 2 = T (m) 2 + R(m) 2 with: which we write T (m) 2 = T (m) 2 + R(m) 2 with: which we write T (m) 2 = T (m) 2 + R(m) 2 with: T (m) 2 = −1 2 N−1 X n=0 δt X K=[ −−→ σσ′]∈M ρn K h un σ ˜un+1 σ + un σ′ ˜un+1 σ′ i (ϕn σ′ −ϕn σ). (59) = − Z T 0 Z Ω ρ(m)(·, · −δt) u(m)(·, · −δt) ˜u(m) ðxϕE dx dt, (60) T (m) 2 = −1 2 N−1 X n=0 δt X K=[ −−→ σσ′]∈M ρn K h un σ ˜un+1 σ + un σ′ ˜un+1 σ′ i (ϕn σ′ −ϕn σ). (59) (59) = − Z T 0 Z Ω ρ(m)(·, · −δt) u(m)(·, · −δt) ˜u(m) ðxϕE dx dt, (60) (60) and therefore, lim m−→+∞T (m) 2 = − Z T 0 Z Ω ¯ρ ¯u2 ∂xϕ dx dt. The remainder term R(m) 2 reads: R(m) 2 = −1 4 N−1 X n=0 δt X K=[ −−→ σσ′]∈M h (ρn σun σ + ρn σ′un σ′)(˜un+1 σ + ˜un+1 σ′ ) −2ρn K (un σ ˜un+1 σ + un σ′ ˜un+1 σ′ ) i (ϕn σ′ −ϕn σ). Letting m tend to +∞in this equation yields ¯u = ¯˜u. TITLE WILL BE SET BY THE PUBLISHER 33 TITLE WILL BE SET BY THE PUBLISHER 33 TITLE WILL BE SET BY THE PUBLISHER 33 Therefore, by the Cauchy-Schwarz inequality, we get: Therefore, by the Cauchy-Schwarz inequality, we get: Therefore, by the Cauchy-Schwarz inequality, we get: Therefore, by the Cauchy-Schwarz inequality, we get: \|R(m) 2,1 \| ≤Cϕ 4 (h(m))1/2 hN−1 X n=0 δt X σ=K\|L∈Eint \|un σ\| (ρn L −ρn K)2i1/2 hN−1 X n=0 δt X σ∈Eint, σ=L→K, K=[σσ′] \|Dσ\| + \|Dσ′\| \|un σ\| ˜un+1 σ + ˜un+1 σ′ 2i1/2 . Since the ratio of the size of two neighboring meshes is bounded by the regularity assumption on the mesh, we get from the estimate (50) on the solution: Since the ratio of the size of two neighboring meshes is bounded by the regularity assumption on the mesh, we get from the estimate (50) on the solution: \|R(m) 2,1 \| ≤C (h(m))1/2 h ∥u(m)∥L2(Ω×(0,T )) + ∥˜u(m)∥ 2 L4(Ω×(0,T )) i , (61) (61) where C ∈R+ does not depend on m, and so R(m) 2,1 tends to zero when m tends to +∞. For R(m) 2,2 , we have, by the Cauchy-Schwarz inequality: where C ∈R+ does not depend on m, and so R(m) 2,1 tends to zero when m tends to +∞. For R(m) 2,2 , we have, by the Cauchy-Schwarz inequality: \|R(m) 2,2 \| ≤Cϕ N−1 X n=0 δt X K=[ −−→ σσ′]∈M \|K\| ρn+1 K \|un σ + un σ′\| (˜un+1 σ −˜un+1 σ′ ) ≤Cϕ h(m) (ν(m))1/2 ∥ρ(m)∥L∞(Ω×(0,T )) ∥u(m)∥L2(Ω×(0,T )) hN−1 X n=0 δt X K=[σσ′]∈M ν(m) hK (˜un+1 σ −˜un+1 σ′ )2i1/2 , [ ]∈M ≤Cϕ h(m) (ν(m))1/2 ∥ρ(m)∥L∞(Ω×(0,T )) ∥u(m)∥L2(Ω×(0,T )) hN−1 X n=0 δt X K=[σσ′]∈M ν(m) hK (˜un+1 σ −˜un+1 σ′ )2i1/2 , and thus, thanks to the estimate (50): \|R(m) 2,2 \| ≤C h(m) (ν(m))1/2 ∥ρ(m)∥L∞(Ω×(0,T )) ∥u(m)∥L2(Ω×(0,T )), e C ∈R+ does not depend on m. Therefore, this term also tends to zero when m tends to +∞. where C ∈R+ does not depend on m. Therefore, this term also tends to zero when m tends to +∞. Letting m tend to +∞in this equation yields ¯u = ¯˜u. Expanding the quantity 2ρn K (un σ ˜un+1 σ +un σ′ ˜un+1 σ′ ) thanks to the identity 2(ab+cd) = (a+c)(b+d)+(a−c)(b−d), we get R(m) 2 = R(m) 2,1 + R(m) 2,2 : Expanding the quantity 2ρn K (un σ ˜un+1 σ +un σ′ ˜un+1 σ′ ) thanks to the identity 2(ab+cd) = (a+c)(b+d)+(a−c)(b−d) we get R(m) 2 = R(m) 2,1 + R(m) 2,2 : R(m) 2,1 = −1 4 N−1 X n=0 δt X K [ −−→ σσ′]∈M (ρn σ −ρn K) un σ + (ρn σ′ −ρn K) un σ′ (˜un+1 σ + ˜un+1 σ′ ) (ϕn σ′ −ϕn σ), R(m) 2,1 = −1 4 N−1 X n=0 δt X K=[ −−→ σσ′]∈M (ρn σ −ρn K) un σ + (ρn σ′ −ρn K) un σ′ (˜un+1 σ + ˜un+1 σ′ ) (ϕn σ′ −ϕn σ), R(m) 2,2 = 1 4 N−1 X n=0 δt X K=[ −−→ σσ′]∈M ρn K (un σ −un σ′) (˜un+1 σ −˜un+1 σ′ ) (ϕn σ′ −ϕn σ). First we study R(m) 2,1 . Thanks to the deﬁnition of the upwind approximation, reordering the sum by faces, we get: First we study R(m) 2,1 . Thanks to the deﬁnition of the upwind approximation, reordering the sum by faces, we get: R(m) 2,1 = 1 4 N−1 X n=0 δt X σ∈Eint, σ=L→K, K=[σσ′] εn σ (ρn L −ρn K) un σ (˜un+1 σ + ˜un+1 σ′ ) (ϕn σ −ϕn σ′), εn σ (ρn L −ρn K) un σ (˜un+1 σ + ˜un+1 σ′ ) (ϕn σ −ϕn σ′), where we recall that the notation σ = L →K means that the face σ separates K and L and the ﬂow goes from L to K at the time level n, and where εn σ = ±1. Since \|ϕn σ −ϕn σ′\| ≤Cϕ \|K\| ≤Cϕ (\|Dσ\| + \|Dσ′\|), we get: \|R(m) 2,1 \| ≤Cϕ 4 N−1 X n=0 δt \|R(m) 2,1 \| ≤Cϕ 4 N−1 X n=0 δt X σ∈Eint, σ=L→K, K=[σσ′] \|Dσ\| + \|Dσ′\| \|ρn L −ρn K\| \|un σ\| \|˜un+1 σ + ˜un+1 σ′ \|. \|Dσ\| + \|Dσ′\| \|ρn L −ρn K\| \|un σ\| \|˜un+1 σ + ˜un+1 σ′ \|. Letting m tend to +∞in this equation yields ¯u = ¯˜u. Next, we turn to the term T (m) 3 : Next, we turn to the term T (m) 3 : Next, we turn to the term T (m) 3 : T (m) 3 = − N−1 X n=0 δt X K=[ −−→ σσ′]∈M \|K\| pn+1 K ϕn σ′ −ϕn σ hK = − Z T 0 Z Ω p(m)ðxϕE dx dt, and therefore, lim m−→+∞T (m) 3 = − Z T 0 Z Ω ¯p ∂xϕ dx dt. lim m−→+∞T (m) 3 = − Z T 0 Z Ω ¯p ∂xϕ dx dt. Let us ﬁnally study T (m) 4 . Reordering the sums, we get: T (m) 4 = N−1 X n=0 δt X K=[ −−→ σσ′]∈M ν(m) hK (˜un+1 σ −˜un+1 σ′ ) (ϕn σ −ϕn σ′). The Cauchy-Schwarz inequality yields: \|T (m) 4 \| ≤ hN−1 X n=0 δt X K=[ −−→ σσ′]∈M ν(m) hK (˜un+1 σ −˜un+1 σ′ )2i1/2hN−1 X n=0 δt X K=[ −−→ σσ′]∈M ν(m) hK (ϕn σ −ϕn σ′)2i1/2 , TITLE WILL BE SET BY THE PUBLISHER 34 □ and thus, again in view of the estimate (50), this term tends to zero when ν(m) tends to zero. and thus, again in view of the estimate (50), this term tends to zero when ν(m) tends to zero. Remark 3.18 (On the ”non appearance of void assumption”). ( ) The assumption that (1/ρ(m))m∈N is bounded in L∞(Ω× (0, T )) (which, loosely speaking, means that the appearance of void is excluded) is used twice in the proof of Theorem 3.17. We use it for the ﬁrst time to obtain ¯u = ¯˜u. Here, the hypothesis may be circumvented by replacing this conclusion by ¯ρ¯u = ¯ρ¯˜u (or, in other words, ¯u = ¯˜u wherever ¯ρ ̸= 0), which is easily obtained from Inequality (57) below. The second time is to obtain, as in the implicit case, the ”non–weighted” estimate of the density space translates (35) for γ ≥2, see Remark 3.8. Remark 3.19 (Less sharp bounds and more general meshes). ( ) As in the implicit case, the assumption that the ratio of the size of two neighboring cells is bounded, i.e. Assumption (ii) of Deﬁnition 3.5, is only used for the remainder associated with the the convection term in the momentum balance. Letting m tend to +∞in this equation yields ¯u = ¯˜u. It may be avoided if we suppose that the sequence of solution is uniformly bounded, replacing (61) by \|R(m) 2,1 \| ≤C (h(m))1/2 ∥u(m)∥ 1/2 L∞(Ω×(0,T )) ∥˜u(m)∥L∞(Ω×(0,T )). \|R(m) 2,1 \| ≤C (h(m))1/2 ∥u(m)∥ 1/2 L∞(Ω×(0,T )) ∥˜u(m)∥L∞(Ω×(0,T )). We now turn to the entropy condition. For any piecewise constant function q on primal cells, we deﬁne its L1(0, T ; BV (Ω)) norm by: ∥q∥T ,x,BV = N X n=0 δt X σ=K\|L∈Eint \|qn L −qn K\|. (62) (62) With this notation, we are now in position to state the following resul With this notation, we are now in position to state the following result. With this notation, we are now in position to state the following result. Theorem 3.20 (Entropy consistency, pressure correction scheme). Under the assumptions of Theorem 3.17, we furthermore assume that: Theorem 3.20 (Entropy consistency, pressure correction scheme). Under the assumptions of Theorem 3.17, we furthermore assume that: - the sequence of regular meshes satisﬁes: - the sequence of regular meshes satisﬁes: ∀m ∈N, δt(m) h(m) ≤C, with h(m) = min σ∈E(m) int \|hσ\|, and where C is a positive real number which can be greater than 1, and where C is a positive real number which can be greater than 1, and where C is a positive real number which can be greater than 1, - the sequence (p(m))m∈N is uniformly bounded in the discrete L1(0, T ; BV (Ω)) norm deﬁned by (62). Then the limit (¯ρ, ¯p, ¯u) satisﬁes the entropy condition (38). Proof. Let ϕ ∈C∞ c Ω× [0, T ) , ϕ ≥0. Letting m tend to +∞in this equation yields ¯u = ¯˜u. Again using the notations of Deﬁnition 3.6, we multiply the kinetic balance equation (47) by ϕn σ, and the elastic potential balance (22) by ϕn K, sum over the faces and cells respectively and over the time steps, to get: X σ∈Eint T n+1 σ ϕn σ + X K∈M T n+1 K ϕn K = − X σ∈Eint Rn+1 σ ϕn σ − X K∈M Rn+1 K ϕn K − X σ∈Eint P n+1 σ ϕn σ, (63) where, for σ = −−→ K\|L, K = [−→ σ′σ] and L = [−−→ σσ′′], T n+1 σ = 1 2 \|Dσ\| δt h ρn Dσ(un+1 σ )2 −ρn−1 Dσ (un σ)2i + 1 2 F n+1 L ˜un+1 σ ˜un+1 σ′′ −1 2 F n+1 K ˜un+1 σ ˜un+1 σ′ + (pn+1 L −pn+1 K ) un+1 σ , for K = [−→ σσ′], nt T n+1 σ ϕn σ + X K∈M T n+1 K ϕn K = − X σ∈Eint Rn+1 σ ϕn σ − X K∈M Rn+1 K ϕn K − X σ∈Eint P n+1 σ ϕn σ, (63) X σ∈Eint T n+1 σ ϕn σ + X K∈M T n+1 K ϕn K = − X σ∈Eint Rn+1 σ ϕn σ − X K∈M Rn+1 K ϕn K − X σ∈Eint P n+1 σ ϕn σ, (63) where, for σ = −−→ K\|L, K = [−→ σ′σ] and L = [−−→ σσ′′], T n+1 1 \|Dσ\|h n ( n+1)2 n−1( n)2i + 1 F n+1˜n+1 ˜n+1 1 F n+1˜n+1 ˜n+1 + ( n+1 n+1) n+1 (63) T n+1 σ = 1 2 \|Dσ\| δt h ρn Dσ(un+1 σ )2 −ρn−1 Dσ (un σ)2i + 1 2 F n+1 L ˜un+1 σ ˜un+1 σ′′ −1 2 F n+1 K ˜un+1 σ ˜un+1 σ′ + (pn+1 L −pn+1 K ) un+1 σ , → for K = [−→ σσ′], for K = [−→ σσ′], T n+1 K = \|K\| δt h H(ρn+1 K ) −H(ρn K) i + un+1 σ′ H(ρn+1 σ′ ) −un+1 σ H(ρn+1 σ ) + pn+1 K (un+1 σ′ −un+1 σ ), T n+1 K = \|K\| δt h H(ρn+1 K ) −H(ρn K) i + un+1 σ′ H(ρn+1 σ′ ) −un+1 σ H(ρn+1 σ ) + pn+1 K (un+1 σ′ −un+1 σ ), TITLE WILL BE SET BY THE PUBLISHER 3 ITLE WILL BE SET BY THE PUBLISHER 3 TITLE WILL BE SET BY THE PUBLISHER TITLE WILL BE SET BY THE PUBLISHER 35 the quantities Rn+1 σ and P n+1 σ are given by (the one-dimensional version of) Equation (48), and Rn+1 K is given by (the one-dimensional version of) Equation (23). Letting m tend to +∞in this equation yields ¯u = ¯˜u. The discrete weak form of the entropy balance is obtained by integrating in time (i.e. summing over the time steps) Equation (63). We obtain T (m) 1 + T (m) 2 + T (m) 3 + T (m) 4 + T (m) 5 + R(m) + P (m) = 0 where T (m) 1 , T (m) 2 , T (m) 4 , T (m) 5 and R(m) are identical to their namesakes in the implicit case (see proof of Theorem 3.10, deﬁned by (40a), (40b), (40d), (40e) and (40f) respectively, and T (m) 3 = 1 2 N−1 X n=0 δt X σ=−−→ K\|L∈Eint, K=[ −−→ σ′σ], L=[ −−→ σσ′′] h F n+1 L ˜un+1 σ ˜un+1 σ′′ −F n+1 K ˜un+1 σ ˜un+1 σ′ i ϕn σ, (64a) P (m) = N−1 X δt X P n+1 ϕn (64b) h F n+1 L ˜un+1 σ ˜un+1 σ′′ −F n+1 K ˜un+1 σ ˜un+1 σ′ i ϕn σ, (64a) (64a) P (m) = N−1 X n=0 δt X σ∈Eint P n+1 σ ϕn σ. P (m) = N−1 X n=0 δt X σ∈Eint P n+1 σ ϕn σ. (64b) (64b) The terms T (m) 1 , T (m) 2 , T (m) 4 , T (m) 5 and R(m) were studied in the proof of Theorem 3.10. Let us then study the kinetic energy convection term T (m) 3 which reads, after reordering the summations: The terms T (m) 1 , T (m) 2 , T (m) 4 , T (m) 5 and R(m) were studied in the proof of Theorem 3.10. Let us then study the kinetic energy convection term T (m) 3 which reads, after reordering the summations: T (m) 3 = −1 2 N−1 X n=0 δt X K=[ −−→ σσ′]∈M F n K ˜un+1 σ ˜un+1 σ′ (ϕn σ′ −ϕn σ). Letting m tend to +∞in this equation yields ¯u = ¯˜u. Furthermore, Lemma 3.16 implies that limm→+∞P (m) = 0, which concludes the proof of the theorem. □ In the proof of Theorem 3.10, we obtained that limm→+∞R(m) ≥0. Furthermore, Lemma 3.16 implies tha limm→+∞P (m) = 0, which concludes the proof of the theorem. □ Letting m tend to +∞in this equation yields ¯u = ¯˜u. Using the identity 2(ab −cd) = (a −c)(b + d) + (a + c)(b −d), we split this ﬁrst part of R(m) 3 into TITLE WILL BE SET BY THE PUBLISHER 36 TITLE WILL BE SET BY THE PUBLISHER 36 TITLE WILL BE SET BY THE PUBLISHER 36 R(m) 31 = −1 8 N−1 X n=0 δt X K=[ −−→ σσ′]∈M un σ ˜un+1 σ (ρn σ −ρn K) (˜un+1 σ′ + ˜un+1 σ ) (ϕn σ′ −ϕn σ), R(m) 32 = −1 8 N−1 X n=0 δt X K=[ −−→ σσ′]∈M un σ ˜un+1 σ (ρn σ + ρn K) (˜un+1 σ′ −˜un+1 σ ) (ϕn σ′ −ϕn σ). Thanks to the regularity of ϕ, the Cauchy-Schwarz inequality yields: Thanks to the regularity of ϕ, the Cauchy-Schwarz inequality yields: \|R(m) 31 \| ≤Cϕ h1/2hN−1 X n=0 δt X K=[ −−→ σσ′]∈M \|un σ\| (ρn σ −ρn K)2 i1/2hN−1 X n=0 δt X K=[ −−→ σσ′]∈M \|K\| \|un σ\| ˜un+1 σ (˜un+1 σ′ + ˜un+1 σ ) 2i1/2 , and thus, invoking the estimate (50), and thus, invoking the estimate (50), \|R(m) 31 \| ≤C (h(m))1/2 ∥u(m)∥L2(Ω×(0,T )) + ∥˜u(m)∥ 4 L8(Ω×(0,T )) . Similarly, we get: \|R(m) 32 \| ≤Cϕ h ν1/2 hN−1 X n=0 δt X K=[ −−→ σσ′]∈M ν hK (˜un+1 σ′ −˜un+1 σ )2i1/2 hN−1 X n=0 δt X K=[ −−→ σσ′]∈M \|K\| un σ ˜un+1 σ (ρn σ + ρn K) 2i1/2 , and thus: \|R(m) 32 \| ≤C h(m) (ν(m))1/2 ∥u(m)∥ 3 L6(Ω×(0,T )) + ∥˜u(m)∥ 3 L6(Ω×(0,T )) + ∥ρ(m)∥ 3 L6(Ω×(0,T )) . \|R(m) 32 \| ≤C h(m) (ν(m))1/2 ∥u(m)∥ 3 L6(Ω×(0,T )) + ∥˜u(m)∥ 3 L6(Ω×(0,T )) + ∥ρ(m)∥ 3 L6(Ω×(0,T )) . Therefore, under the assumptions of the theorem, we have Therefore, under the assumptions of the theorem, we have lim m−→+∞T (m) 3 = −1 2 Z T 0 Z Ω ¯ρ ¯u3 ∂xϕ dx dt. (65) (65) Together with the results which were obtained in the proof of Theorem 3.10, this yields lim m−→+∞ 5 X i=1 T (m) i + R(m)) ≥− Z T 0 Z Ω η ∂tϕ + (η + p) u ∂xϕ dx dt − Z Ω η0(x) ϕ(x, 0) dx. In the proof of Theorem 3.10, we obtained that limm→+∞R(m) ≥0. Letting m tend to +∞in this equation yields ¯u = ¯˜u. We write T m 3 = T (m) 3 + R(m) 3 , where T (m) 3 = −1 N−1 X δt X \|K\| ρn K un(˜un+1)2 + un ′(˜un+1 ′ )2 ϕn σ′ −ϕn σ We write T m 3 = T (m) 3 + R(m) 3 , where We write T m 3 = T (m) 3 + R(m) 3 , where T (m) 3 = −1 2 N−1 X n=0 δt X K=[ −−→ σσ′]∈M \|K\| 2 ρn K un σ(˜un+1 σ )2 + un σ′(˜un+1 σ′ )2 ϕn σ′ −ϕn σ hK = −1 2 Z T 0 Z Ω ρ(m)(x, t −δt) u(m)(x, t −δt) (˜u(m)(x, t))2 ðxϕE dx dt, T (m) 3 = −1 2 N−1 X n=0 δt X K=[ −−→ σσ′]∈M \|K\| 2 ρn K un σ(˜un+1 σ )2 + un σ′(˜un+1 σ′ )2 ϕn σ′ −ϕn σ hK so that lim m−→+∞T (m) 3 = −1 2 Z T 0 Z Ω ¯ρ ¯u3 ∂xϕ dx dt. The remainder term R(m) 3 reads: R(m) 3 = −1 4 N−1 X n=0 δt X K=[ −−→ σσ′]∈M h (ρn σun σ + ρn σ′un σ′) ˜un+1 σ′ ˜un+1 σ −ρn K un σ (˜un+1 σ )2 + un σ′ (˜un+1 σ′ )2i (ϕn σ′ −ϕn σ). Reordering the terms in the sum, we get: Reordering the terms in the sum, we get: R(m) 3 = −1 4 N−1 X n=0 δt X K=[ −−→ σσ′]∈M h (ρn σ ˜un+1 σ′ −ρn K ˜un+1 σ ) un σ ˜un+1 σ \| {z } D1 + (ρn σ′ ˜un+1 σ −ρn K ˜un+1 σ′ ) un σ′ ˜un+1 σ′ \| {z } D2 i (ϕn σ′ −ϕn σ). D2 Let us consider the term involving D1, and skip the exposition of the treatment of the term with D2, which is similar. Using the identity 2(ab −cd) = (a −c)(b + d) + (a + c)(b −d), we split this ﬁrst part of R(m) 3 into Let us consider the term involving D1, and skip the exposition of the treatment of the term with D2, which is similar. 4.1. Internal energy, kinetic energy and total energy In these latter models, the natural energy balance equation is the internal energy equation (67). In addition, discretizing (67) instead of the total energy balance (2c) presents two advantages: - ﬁrst, it avoids the space discretization of the total energy, which is rather unnatural for staggered schemes since the degrees of freedom for the velocity and the scalar variables are not colocated, - ﬁrst, it avoids the space discretization of the total energy, which is rather unnatural for stagge since the degrees of freedom for the velocity and the scalar variables are not colocated - ﬁrst, it avoids the space discretization of the total energy, which is rather unnatural for staggered schemes since the degrees of freedom for the velocity and the scalar variables are not colocated, f ( ) f f - second, a suitable discretization of (67) may yield, ”by construction” of the scheme, the positivity of the internal energy [18]. - second, a suitable discretization of (67) may yield, ”by construction” of the scheme, the positivity of the internal energy [18]. However, in the inviscid case and for solutions with shocks, Equation (67) (with τ = 0) is not equivalent to the conservative total energy balance (2c) (with τ = 0); more precisely speaking, at the locations of shocks, positive measures should replace, at the right-hand side of Equation (67), the term τ(u) : ∇u which is formally the product of vanishing quantities (for a Newtonian ﬂuid, the viscosity) and inﬁnite derivatives of the velocity. Discretizing (67) instead of (2c) may thus yield a scheme which does not compute the correct weak discontin- uous solutions; in particular, the numerical solutions may present (smeared) shocks which do not satisfy the Rankine-Hugoniot conditions associated with (2c). The essential result of this section is to provide a solution to circumvent this problem. To this purpose, we closely mimic the above performed formal computation: - Starting from the discrete momentum balance equation, we derive a discrete kinetic energy balance (in fact, this computation is already performed in the previous sections, since we use for the full Euler equations the same discrete momentum balance as in the barotropic case). In this relation, residual terms which do no tend to zero with space and time step appear (they are the discrete manifestations of the above mentioned measures). 4.1. Internal energy, kinetic energy and total energy Let us suppose that the solution to the Navier–Stokes equations (2) is regular. As already mentioned, taking the inner product of the momentum balance equation (2b) by u and using the mass balance equation, we obtain the so-called kinetic energy balance equation: 1 2∂t(ρ \|u\|2) + 1 2div(ρ \|u\|2u) + ∇p · u = div(τ(u)) · u. (66) (66) ing this relation from the total energy balance, we obtain the internal energy balance equation: total energy balance, we obtain the internal energy balance equation: (67) ∂t(ρe) + div(ρeu) + p div(u) = τ(u) : ∇u. (67) ∂t(ρe) + div(ρeu) + p div(u) = τ(u) : ∇u. Since, Since, - from thermodynamical arguments, τ(u) : ∇u ≥0, - thanks to the mass balance equation, the ﬁrst two terms in the left-hand side of (67) may be recast as a transport operator: ∂t(ρe) + div(ρeu) = ρ [∂te + u · ∇e], - thanks to the mass balance equation, the ﬁrst two terms in the left-h transport operator: ∂t(ρe) + div(ρeu) = ρ [∂te + u · ∇e], - thanks to the mass balance equation, the ﬁrst two terms in the left-hand side of (67) may be recast as a transport operator: ∂t(ρe) + div(ρeu) = ρ [∂te + u · ∇e], - thanks to the mass balance equation, the ﬁrst two terms in the left-hand side of (67) may be recast t t t ∂( ) di ( ) [∂ ∇] p p t(ρ ) + (ρ ) ρ [ t + ], - and, ﬁnally, from the equation of state, the pressure vanishes when e = 0, this equation implies that, if e ≥0 at t = 0 and with suitable boundary conditions, then e remains non-negative at all times. p (ρ ) (ρ ) ρ [ ], ally, from the equation of state, the pressure vanishes when e = 0, - and, ﬁnally, from the equation of state, the pressure vanishes when e = 0, this equation implies that, if e ≥0 at t = 0 and with suitable boundary conditions, then e remains non-negative at all times. Our aim here is to build a scheme stable and accurate at all Mach numbers, and, in particular, which boils down to a usual scheme for incompressible ﬂows (or, more generally speaking, for the asymptotic model of vanishing Mach number ﬂows [45]) when the Mach number tends to zero. 4. The pressure correction scheme for the full Euler equations We now turn to the development and study of a similar pressure correction scheme for the full Euler equa- tions, that is for the system (2) assuming τ(u) = 0. Numerical schemes for the Euler equations have been widely studied, and are very often based on Riemann solvers on the system consisting of the mass balance, the momentum balance, and the total energy balance. We start this section by explaining why we use the internal energy balance rather the the total energy in the correction scheme, and the precautions that must be taken in order to compute correct shocks in this way. TITLE WILL BE SET BY THE PUBLISHER 37 TITLE WILL BE SET BY THE PUBLISHER 38 the second scheme cures this problem, at the price of the introduction of an additional elliptic problem which must be solved at the beginning of each time step to determine a tentative pressure. Here, we present a variant of these schemes, that preserves the positivity of the internal energy thanks to a renormalization step which only consists in a weighting of the discrete pressure gradient and does not require any elliptic solve (and which is thus much less costly). As the correction scheme for barotropic ﬂows, it is implemented in the industrial open-source code CALIF3S [5]. Let us mention also that fully explicit versions have been studied [30,31]. the second scheme cures this problem, at the price of the introduction of an additional elliptic problem which must be solved at the beginning of each time step to determine a tentative pressure. Here, we present a variant of these schemes, that preserves the positivity of the internal energy thanks to a renormalization step which only consists in a weighting of the discrete pressure gradient and does not require any elliptic solve (and which is thus much less costly). As the correction scheme for barotropic ﬂows, it is implemented in the industrial open-source code CALIF3S [5]. Let us mention also that fully explicit versions have been studied [30,31]. Pressure gradient renormalization step: +1 = s ρn Dσ ρn−1 Dσ (∇p)n σ. (68a) ∀σ ∈E, (g ∇p)n+1 σ = s ρn Dσ ρn−1 Dσ (∇p)n σ. (68a) 4.1. Internal energy, kinetic energy and total energy - Starting from the discrete momentum balance equation, we derive a discrete kinetic energy balance (in fact, this computation is already performed in the previous sections, since we use for the full Euler equations the same discrete momentum balance as in the barotropic case). In this relation, residual terms which do no tend to zero with space and time step appear (they are the discrete manifestations of the above mentioned measures). ) hese residual terms are then compensated by corrective terms in the internal energy balance. - These residual terms are then compensated by corrective terms in the internal energy bal We provide a theoretical justiﬁcation of this process by showing that, in the 1D case, if the scheme is stable and converges to a limit (in a sense to be deﬁned), this limit satisﬁes a weak form of (2c) which implies the correct Rankine-Hugoniot conditions. Then, we perform numerical tests which substantiate this analysis. A fully implicit scheme was studied in [26] along with two pressure correction schemes: the ﬁrst correction scheme is appealing for its (relative) simplicity, but does not seem to warrant the sign of the internal energy (so that the unconditional stability induced by the above mentioned conservation of the total energy property is lost); TITLE WILL BE SET BY THE PUBLISHER Prediction step – Solve for ˜un+1: Prediction step – Solve for ˜un+1: For 1 ≤i ≤d, ∀σ ∈E(i) S , \|Dσ\| δt (ρn Dσ ˜un+1 σ,i −ρn−1 Dσ un σ,i) + X ǫ∈¯E(Dσ) F n σ,ǫ˜un+1 ǫ,i −\|Dσ\| (∆M˜u)n+1 σ,i + \|Dσ\| (g ∇p)n+1 σ,i = 0. (68b) For 1 ≤i ≤d, ∀σ ∈E(i) S , ≤ ≤ , S , \|Dσ\| δt (ρn Dσ ˜un+1 σ,i −ρn−1 Dσ un σ,i) + X ǫ∈¯E(Dσ) F n σ,ǫ˜un+1 ǫ,i −\|Dσ\| (∆M˜u)n+1 σ,i + \|Dσ\| (g ∇p)n+1 σ,i = 0. (68b) \|Dσ\| δt (ρn Dσ ˜un+1 σ,i −ρn−1 Dσ un σ,i) + X ǫ∈¯E(Dσ) F n σ,ǫ˜un+1 ǫ,i −\|Dσ\| (∆M˜u)n+1 σ,i + \|Dσ\| (g ∇p)n+1 σ,i = 0. (68 (68b) Correction step – Solve for ρn+1, pn+1, en+1 and un+1: Correction step – Solve for ρn+1, pn+1, en+1 and un+1: 4.2. The scheme We propose in this section a pressure correction scheme, which, as in the barotropic case, features a renor- malization step for the pressure gradient. As previously mentioned, we add a corrective term in the internal energy equation; we are able to show that this corrective term is non negative, which ensures the positivity of the internal energy and the existence of a solution to the scheme. With the notations that were introduced in Section 3.2.1, the algorithm reads, for 0 ≤n ≤N −1: Pressure gradient renormalization step: Correction step – Solve for ρn+1, pn+1, en+1 and un+1: Finally, the initial pressure p0 is computed from the initial density ρ0 by the equation of state: ∀K ∈M, p0 K = (γ −1) ρ0 K e0 K. As in the barotropic case, the objective of this procedure is to perform the ﬁrst prediction step with (ρ−1 Dσ )σ∈E, (ρ0 Dσ)σ∈E and the dual mass ﬂuxes satisfying the mass balance. Then, we compute ρ0 by solving the mass balance equation (68d). Finally, the initial pressure p0 is computed from the initial density ρ0 by the equation of state: ∀K ∈M, p0 K = (γ −1) ρ0 K e0 K. As in the barotropic case, the objective of this procedure is to perform the ﬁrst prediction step with (ρ−1 Dσ )σ∈E, (ρ0 Dσ)σ∈E and the dual mass ﬂuxes satisfying the mass balance. There only remains to deﬁne the corrective terms Sn+1 K in the internal energy balance (68e), with the aim to recover a consistent discretization of the total energy balance. The ﬁrst idea to do this could be just to sum the (discrete) kinetic energy balance with the internal energy balance: it is indeed possible for a colocated discretization. But here, we face the fact that the kinetic energy balance is associated with the dual mesh, while the internal energy balance is discretized on the primal one. The way to circumvent this diﬃculty is to remark that we do not really need a discrete total energy balance; in fact, we only need to recover (a weak form of) this equation when the mesh and time steps tend to zero. To this purpose, we choose the quantities (Sn+1 K ) in such a way as to somewhat compensate the terms (Rn+1 σ,i ) deﬁned by (48) appearing at the right-hand-side of the discrete kinetic energy identity (47): ∀K ∈M, Sn+1 K = d X i=1 Sn+1 K,i , ∀K ∈M, Sn+1 K = d X i=1 Sn+1 K,i , with: with: with: Sn+1 K,i = 1 2 ρn−1 K X σ∈E(K)∩E(i) S \|DK,σ\| δt ˜un+1 σ,i −un σ,i 2 + X ǫ∈¯E(i) S , ǫ∩¯ K̸=∅, ǫ=Dσ\|Dσ′ αK,ǫ ν hd−2 ǫ (˜un+1 σ,i −˜un+1 σ′,i )2. (71) (71) The coeﬃcient αK,ǫ is ﬁxed to 1 if the face ǫ is included in K, and this is the only situation to consider for the RT and CR discretizations. Correction step – Solve for ρn+1, pn+1, en+1 and un+1: Correction step – Solve for ρn+1, pn+1, en+1 and un+1: For 1 ≤i ≤d, ∀σ ∈E(i) S , \|Dσ\| δt ρn Dσ (un+1 σ,i −˜un+1 σ,i ) + \|Dσ\| (∇p)n+1 σ,i −(g ∇p)n+1 σ,i = 0, (68c) ∀K ∈M, \|K\| δt (ρn+1 K −ρn K) + X σ∈E(K) F n+1 K,σ = 0, (68d) ∀K ∈M, \|K\| δt (ρn+1 K en+1 K −ρn Ken K) + X σ∈E(K) F n+1 K,σ en+1 σ +\|K\| pn+1 K (divu)n+1 K = Sn+1 K , (68e) \|Dσ\| δt ρn Dσ (un+1 σ,i −˜un+1 σ,i ) + \|Dσ\| (∇p)n+1 σ,i −(g ∇p)n+1 σ,i = 0, (68c) (68c) \|K\| δt (ρn+1 K −ρn K) + X σ∈E(K) F n+1 K,σ = 0, (68d) (68d) ∀K ∈M, \|K\| δt (ρn+1 K en+1 K −ρn Ken K) + X σ∈E(K) F n+1 K,σ en+1 σ ∀K ∈M, \|K\| δt (ρn+1 K en+1 K −ρn Ken K) + X σ∈E(K) F n+1 K,σ en+1 σ +\|K\| pn+1 K (divu)n+1 K = Sn+1 K , (68e) (68e) ∀K ∈M, pn+1 K = (γ −1) ρn+1 K en+1 K . +1 = (γ −1) ρn+1 K en+1 K . (68f) ∀K ∈M, pn+1 K = (γ −1) ρn+1 K en+1 K . (68f) (68f) where we make an upwind choice for e (again a crucial choice to ensure the positivity of the internal energy): where we make an upwind choice for e (again a crucial choice to ensure the positivity of the internal energy): for σ = K\|L ∈Eint, en+1 σ = en+1 K if F n+1 K,σ ≥0, en+1 L otherwise. (69) (69) 39 TITLE WILL BE SET BY THE PUBLISHER The initialization of the scheme is performed in a way similar to the barotropic case. First, ρ−1, e0 and u0 are given by the average of the initial conditions ρ0, e0 and u0, i.e. by (46) and the following relation: The initialization of the scheme is performed in a way similar to the barotropic case. First, ρ−1, e0 and u0 are given by the average of the initial conditions ρ0, e0 and u0, i.e. by (46) and the following relation: ∀K ∈M, e0 K = 1 \|K\| Z K e0(x) dx. (70) (70) Then, we compute ρ0 by solving the mass balance equation (68d). Correction step – Solve for ρn+1, pn+1, en+1 and un+1: For the MAC scheme, some dual faces are included in the primal cells, but some lie on their boundary; for such a dual face ǫ, we denote by Nǫ the set of cells M such that ¯ M ∩ǫ ̸= ∅(the cardinal of this set being always 4), and compute αK,ǫ by: αK,ǫ = \|K\| P M∈Nǫ \|M\|. αK,ǫ = \|K\| P M∈Nǫ \|M\|. For a uniform grid, this formula yields αK,ǫ = 1/4. For a uniform grid, this formula yields αK,ǫ = 1/4. The expression of the terms(Sn+1 K )K∈M is justiﬁed by the passage to the limit in the scheme (for a one- dimensional problem) performed in Section 4.3. However, its expression may be anticipated, thanks to the following remarks. First, we note that: X K∈M Sn+1 K − d X i=1 X σ∈E(i) S Rn+1 σ,i = 0. (72) (72) Indeed, for K ∈M and σ = K\|L, the ﬁrst part of Sn+1 K,i , thanks to the expression (7) of the density at the face ρn Dσ, results from a dispatching of the ﬁrst part of the kinetic energy balance residual Rn+1 σ,i over the two cells Indeed, for K ∈M and σ = K\|L, the ﬁrst part of Sn+1 K,i , thanks to the expression (7) of the density at the face ρn Dσ, results from a dispatching of the ﬁrst part of the kinetic energy balance residual Rn+1 σ,i over the two cells TITLE WILL BE SET BY THE PUBLISHER 40 adjacent to σ: adjacent to σ: 1 2 \|Dσ\| δt ρn−1 Dσ ˜un+1 σ,i −un σ,i 2 = 1 2 \|DK,σ\| δt ρn−1 K ˜un+1 σ,i −un σ,i 2 \| {z } aﬀected to K + 1 2 \|DL,σ\| δt ρn−1 L ˜un+1 σ,i −un σ,i 2 \| {z } aﬀected to L . For the second part of the remainder, a standard reordering of the sum yields: d X i=1 X σ∈E(i) S h X ǫ=Dσ\|Dσ′ ν hd−2 ǫ (˜un+1 σ,i −˜un+1 σ′,i ) i ˜un+1 σ,i = d X i=1 X ǫ=Dσ\|Dσ′ ∈¯E(i) S ν hd−2 ǫ (˜un+1 σ,i −˜un+1 σ′,i )2. One may wonder why we do not use in Sn+1 K the expression of this term as it is written in the remainder Rn+1 σ,i , i.e., in other words, use the numerical diﬀusion multiplied by u instead of the dissipation. Correction step – Solve for ρn+1, pn+1, en+1 and un+1: A ﬁrst answer is that we mimic what happens at the continuous level: the term which appears in the kinetic energy balance is div τ(u) · u and the corresponding term in the internal energy balance is the dissipation τ(u) : ∇u. A more involved argument is that the expression in Sn+1 K provides a positive source term to the internal energy balance, and we may hope that the diﬀerence between the numerical diﬀusion multiplied by u and the associated dissipation tends to zero (because the numerical diﬀusion tends to zero) in the sense of distributions. To have an intuition of this fact, let us consider the toy elliptic problem, posed over Ω: v −ν∆v = f, where ν is a positive parameter and f ∈L2(Ω). Assuming homogeneous Dirichlet boundary conditions, we obtain by standard variational arguments ∥v∥L2(Ω) + ν1/2∥∇v∥L2(Ω)d ≤C, with C only depending on Ωand f. We thus get, with ϕ ∈C∞ c (Ω): Z Ω h ν(∆v)v + ν\|∇v\|2i ϕ dx = ν Z Ω div(v∇v)ϕ dx = −ν Z Ω v∇v · ∇ϕ dx, and so, ﬁnally, by the Cauchy-Schwarz inequality: Z Ω h ν(∆v)v + ν\|∇v\|2i ϕ dx ≤C ∥∇ϕ∥L∞(Ω)d ν1/2, so this term tends to zero if so does ν. A discrete analogue of this simple computation is used to pass to the limit in the scheme in the next section (with a control on the unknown assumed and not proven). so this term tends to zero if so does ν. A discrete analogue of this simple computation is used to pass to the limit in the scheme in the next section (with a control on the unknown assumed and not proven). Note that the term Sn+1 K is non-negative. Consequently, adapting the proof of [18, Theorem 4.1] to cope with this additional term, we obtain that the scheme admits at least one solution, which satisﬁes p ≥0, ρ ≥0 and e ≥0. In addition, Equation (72) shows that the scheme conserves the integral of the total energy over the computational domain. Theorem 4.1 (Existence and stability). Assume that for all K ∈M, e0 K > 0 and ρ−1 K > 0. Correction step – Solve for ρn+1, pn+1, en+1 and un+1: Then there exists a solution to the scheme (68), which furthermore satisﬁes ρ0 K > 0 and, for 1 ≤n ≤N and K ∈M, en K > 0, ρn K > 0, and the following discrete analogue of the total energy balance: X K∈M \|K\| en K + 1 2 d X i=1 X σ∈E(i) S \|Dσ\| ρn−1 Dσ (un σ,i)2 + Rn ≤ X K∈M \|K\| e0 K + 1 2 d X i=1 X σ∈E(i) S \|Dσ\| ρ−1 Dσ (u0 σ,i)2 + R0, 41 TITLE WILL BE SET BY THE PUBLISHER 41 where: Rn = δt2 X σ∈Eint \|Dσ\| ρn−1 Dσ \|(∇p)n σ\|2 = δt2 X σ=K\|L∈Eint \|σ\|2 \|Dσ\| ρn−1 Dσ (pn K −pn L)2. 4.3. Passing to the limit in the scheme (1D case) We now undertake the passage to the limit in the scheme (68) in the one-dimensional case. Using the notations (28)–(30), the scheme may be rewritten in “one-dimensional notations” as follows: tialization – Compute ρ−1, u0 and ρ0 by (56a) and e0 and p0 by: Initialization – Compute ρ−1, u0 and ρ0 by (56a) and e0 and p0 by: ∀K ∈M, e0 K = 1 \|K\| Z K e0(x) dx, ∀K ∈M, p0 K = (γ −1) ρ0 K e0 K. (73a) Pressure gradient renormalization step Pressure gradient renormalization step Eint, ( eδp)n+1 σ = s ρn Dσ ρn−1 Dσ (pn K −pn L) . (73b) ∀σ = K\|L ∈Eint, ( eδp)n+1 σ = s ρn Dσ ρn−1 Dσ (pn K −pn L) . (73b) Prediction step – Solve for ˜un+1: ∀σ = −−→ K\|L ∈Eint, \|Dσ\| δt (ρn D ˜un+1 σ −ρn−1 D un σ) + F n L ˜un+1 L −F n K ˜un+1 K −\|Dσ\| (∆M˜u)n+1 σ + ( eδp)n+1 σ = 0. (73c) Prediction step – Solve for ˜un+1: ∀σ = −−→ K\|L ∈Eint, \|Dσ\| δt (ρn Dσ ˜un+1 σ −ρn−1 Dσ un σ) + F n L ˜un+1 L −F n K ˜un+1 K −\|Dσ\| (∆M˜u)n+1 σ + ( eδp)n+1 σ = 0. (73c) Correction step – Solve for ρn+1, pn+1, en+1 and un+1: Correction step – Solve for ρn+1, pn+1, en+1 and un+1: Correction step – Solve for ρn+1, pn+1, en+1 and un+1: ∀σ = −−→ K\|L ∈Eint, \|Dσ\| δt ρn Dσ(un+1 σ −˜un+1 σ ) + (pn+1 L −pn+1 K ) −( eδp)n+1 σ = 0, (73d) ∀K = [−→ σσ′] ∈M, \|K\| δt (ρn+1 K −ρn K) + F n+1 σ′ −F n+1 σ = 0, (73e) ∀K = [−→ σσ′] ∈M, \|K\| δt (ρn+1 K en+1 K −ρn Ken K) + F n+1 σ′ en+1 σ′ −F n+1 σ en+1 σ (73f) K = [−→ σσ′] ∈M, \|K\| δt (ρn+1 K −ρn K) + F n+1 σ′ −F n+1 σ = 0, (73e) ∀K = [−→ σσ′] ∈M, \|K\| δt (ρn+1 K en+1 K −ρn Ken K) + F n+1 σ′ en+1 σ′ −F n+1 σ en+1 σ +pn+1 K (un+1 σ′ −un+1 σ ) = Sn+1 K , (73f) (73f) ∀K ∈M, pn+1 K = (γ −1) ρn+1 K en+1 K . ∀K ∈M, pn+1 K = (γ −1) ρn+1 K en+1 K . 4.3. Passing to the limit in the scheme (1D case) ∀K ∈M, pn+1 K = (γ −1) ρn+1 K en+1 K . (73g) pn+1 K = (γ −1) ρn+1 K en+1 K . (73g) (73g) The corrective term Sn+1 K reads: The corrective term Sn+1 K reads: ∀K = [σσ′], Sn+1 K = \|K\| 4 δt ρn−1 K (˜un+1 σ −un σ)2 + (˜un+1 σ′ −un σ′)2 + ν hK (˜un+1 σ −˜un+1 σ′ )2. (74) (74) For discrete functions q and v deﬁned on the primal and dual mesh, respectively, we deﬁne a discrete L1((0, T ); BV (Ω)) norm by: For discrete functions q and v deﬁned on the primal and dual mesh, respectively, we deﬁne a discrete L1((0, T ); BV (Ω)) norm by: ∥q∥T ,x,BV = N X n=0 δt X σ=K\|L∈Eint \|qn L −qn K\|, ∥v∥T ,x,BV = N X n=0 δt X ǫ=Dσ\|Dσ′∈¯Eint \|vn σ′ −vn σ\|, TITLE WILL BE SET BY THE PUBLISHER 42 TITLE WILL BE SET BY THE PUBLISHER 42 and a discrete L1(Ω; BV ((0, T ))) norm by: and a discrete L1(Ω; BV ((0, T ))) norm by: ∥q∥T ,t,BV = X K∈M \|K\| N−1 X n=0 \|qn+1 K −qn K\|, ∥v∥T ,t,BV = X σ∈E \|Dσ\| N−1 X n=0 \|vn+1 σ −vn σ\|. For the proof of the consistency of the scheme (i.e. the proof of the theorem 4.3 below), we need to introduce the following stability assumptions on a sequence (ρ(m), p(m), e(m), ˜u(m), u(m))m∈N of discrete solutions: For the proof of the consistency of the scheme (i.e. 4.3. Passing to the limit in the scheme (1D case) the proof of the theorem 4.3 below), we need to introduce the following stability assumptions on a sequence (ρ(m), p(m), e(m), ˜u(m), u(m))m∈N of discrete solutions: \|(ρ(m))n K\| + \|(p(m))n K\| + \|(e(m))n K\| ≤C, ∀K ∈M(m), for 0 ≤n ≤N (m), ∀m ∈N, (75a) 1 \|(ρ(m))n K\| ≤C, ∀K ∈M(m), for 0 ≤n ≤N (m), ∀m ∈N, (75b) \|(u(m))n\| + \|(˜u(m))n\| ≤C ∀σ ∈E(m) for 0 ≤n ≤N (m) ∀m ∈N (75c) \|(ρ(m))n K\| + \|(p(m))n K\| + \|(e(m))n K\| ≤C, ∀K ∈M(m), for 0 ≤n ≤N (m), ∀m ∈N, (75 \|(ρ(m))n K\| + \|(p(m))n K\| + \|(e(m))n K\| ≤C, ∀K ∈M(m), for 0 ≤n ≤N (m), ∀m ∈N, (75a) 1 \|(ρ(m))n K\| ≤C, ∀K ∈M(m), for 0 ≤n ≤N (m), ∀m ∈N, (75b) \|(u(m))n σ\| + \|(˜u(m))n σ\| ≤C, ∀σ ∈E(m), for 0 ≤n ≤N (m), ∀m ∈N, (75c) ∥ρ(m)∥T ,x,BV + ∥e(m)∥T ,x,BV + ∥p(m)∥T ,x,BV + ∥˜u(m)∥T ,x,BV ≤C, ∀m ∈N, (75d) ∥ρ(m)∥T ,t,BV + ∥u(m)∥T ,t,BV ≤C, ∀m ∈N. (75e) (75a) (75b) \|(u )σ\| + \|(u )σ\| ≤C, ∀σ ∈E , for 0 ≤n ≤N , ∀m ∈N, (75 ∥ρ(m)∥T ,x,BV + ∥e(m)∥T ,x,BV + ∥p(m)∥T ,x,BV + ∥˜u(m)∥T ,x,BV ≤C, ∀m ∈N, (75 Note that we do not suppose any control on time discrete derivatives of ˜u(m) (i.e. on ∥˜u(m)∥T ,t,BV ) and on the discrete space derivatives of u(m) (i.e. on ∥u(m)∥T ,x,BV ). Note also that, by deﬁnition of the initial conditions of the scheme, these inequalities imply that the functions ρ0, e0 and u0 belong to L∞(Ω) ∩BV (Ω). As in the barotropic case, we are not able to prove (75) for the solutions of the scheme; however, such inequalities are satisﬁed by the ”interpolation” (for instance, by taking the cell average) of the solution to a Riemann problem, and are also observed in computations. The following deﬁnition gathers the assumptions on the discretization which are needed in the proof of Theorem 4.3 below. Deﬁnition 4.2 (Regular sequence of discretizations). Deﬁnition 4.2 (Regular sequence of discretizations). 4.3. Passing to the limit in the scheme (1D case) (77d) (77d) p = (γ −1)ρ e, E = 1 2u2 + e, E0 = 1 2u2 0 + e0. As in the barotropic case, these relations are not suﬃcient to deﬁne a weak solution to the problem, since they do not imply anything about the boundary conditions, but they allow to derive the Rankine-Hugoniot conditions. As in the barotropic case, these relations are not suﬃcient to deﬁne a weak solution to the problem, since they do not imply anything about the boundary conditions, but they allow to derive the Rankine-Hugoniot conditions. in position to state the following consistency result. We are now in position to state the following consistency result. We are now in position to state the following consistency result. Theorem 4.3. Let Ωbe an open bounded interval of R. Let (M(m), δt(m), ν(m))m∈N be a regular sequence of discretizations in the sense of Deﬁnition 4.2. Let (ρ(m), p(m), e(m), ˜u(m), u(m))m∈N be the corresponding sequence of solutions. We suppose that this sequence satisﬁes the bounds (75) and converges in Lp(Ω× (0, T ))5, for 1 ≤p < +∞, to (¯ρ, ¯p, ¯e, ¯˜u, ¯u) ∈L∞(Ω× (0, T ))5. Then ¯˜u = ¯u and (¯ρ, ¯p, ¯e, ¯u) satisﬁes the system (77). Then ¯˜u = ¯u and (¯ρ, ¯p, ¯e, ¯u) satisﬁes the system (77). Then ¯˜u = ¯u and (¯ρ, ¯p, ¯e, ¯u) satisﬁes the system (77). Proof. Let us ﬁrst check that ¯˜u = ¯u. We ﬁrst note that thanks to assumptions (75a) and (75b), the pressure prediction step (73b) yields that\|( eδp)n+1 σ \| ≤C \|pn K −pn L\|. Therefore, from the expression of the correction step for the velocity (73d), we have, again using assumption (75b): ∥u(m) −˜u(m)∥L1(Ω×(0,T )) ≤Cδt ∥p(m)∥T ,x,BV which, passing to the limit when m →+∞, yields the result. 4.3. Passing to the limit in the scheme (1D case) ( ) ( ) ( ) ( g q ) We deﬁne a regular sequence of discretizations (M(m), δt(m), ν(m))m∈N as a sequence of meshes, time steps and numerical diﬀusion coeﬃcients which are assumed to satisfy the following conditions: (i) both the time step δt(m) and the size h(m) of the mesh M(m) tend to zero as m →+∞ (i) both the time step δt(m) and the size h(m) of the mesh M(m) tend to zero as m →+∞; (ii) there exists C ∈R+ (not necessarily lower than 1) such that the following CFL-like condition (i) both the time step δt(m) and the size h(m) of the mesh M(m) tend to zero as m →+∞; (ii) there exists C ∈R+ (not necessarily lower than 1) such that the following CFL-like condition holds: ( ) p ; (ii) there exists C ∈R+ (not necessarily lower than 1) such that the following CFL-like condition holds: ∀m ∈N, δt(m) h(m) ≤C, with h(m) = min σ=K\|L∈E(m) int 1 2 (hK + hL); (76) (76) (iii) there exists C ∈R+ such that the sequence of numerical diﬀusion coeﬃcients (ν(m))m∈N satisﬁes lim m→+∞ ν(m) h(m) ≤C. (iii) there exists C ∈R+ such that the sequence of numerical diﬀusion coeﬃcients (ν(m))m∈N satisﬁes lim m→+∞ ν(m) h(m) ≤C. TITLE WILL BE SET BY THE PUBLISHER 43 TITLE WILL BE SET BY THE PUBLISHER 43 A weak solution to the continuous problem satisﬁes, for any ϕ ∈C∞ c [0, T ) × Ω : A weak solution to the continuous problem satisﬁes, for any ϕ ∈C∞ c [0, T ) × Ω : − Z Ω×(0,T ) h ρ ∂tϕ + ρ u ∂xϕ i dx dt − Z Ω ρ0(x) ϕ(x, 0) dx = 0, (77a) − Z Ω×(0,T ) h ρ u ∂tϕ + (ρ u2 + p) ∂xϕ i dx dt − Z Ω ρ0(x) u0(x) ϕ(x, 0) dx = 0, (77b) − Z Ω×(0,T ) h ρ E ∂tϕ + (ρ E + p) u ∂xϕ i dx dt − Z Ω ρ0(x) E0(x) ϕ(x, 0) dx = 0, (77c) p = (γ −1)ρ e, E = 1 2u2 + e, E0 = 1 2u2 0 + e0. (77d) (77a) p = (γ −1)ρ e, E = 1 2u2 + e, E0 = 1 2u2 0 + e0. which, passing to the limit when m →+∞, yields the result. and where P n+1 σ is deﬁned by (48), and Sn+1 K by (74). The study of the terms T (m) 1 , T (m) 3 and T (m) 5 has already been performed in the proof of Theorem 3.20. The study of the term eT (m) 2 (resp. eT (m) 4 ) is similar to that of the term T (m) 2 (resp. T (m) 4 ) of the proof of Theorem 3.20, replacing the elastic potential H(ρ) by the internal energy e. Finally, we have to deal with the term eR(m). Here, the situation is diﬀerent from Theorem 3.20 since we have to prove that this term tends to zero whereas for the entropy inequality in the barotropic case, we only had to prove that it is non-negative at the limit of vanishing time and space steps. We split eR(m) into three terms: eR(m) = R(m) δt + R(m) ∆ + P (m) and give the expression of each of these three terms hereafter. The ﬁrst term reads: R(m) δt = −1 2 N−1 X n=0 X σ∈E \|Dσ\| ρn−1 Dσ (˜un+1 σ −un σ)2 ϕn σ + 1 2 N−1 X n=0 X K∈M X σ∈E(K) \|DK,σ\| ρn−1 K (˜un+1 σ −un σ)2 ϕn K. Thanks to the deﬁnition of the density on the faces, we get: R(m) δt = 1 2 N−1 X n=0 X K∈M X σ∈E(K) \|DK,σ\| ρn−1 K (˜un+1 σ −un σ)2 (ϕn K −ϕn σ), and therefore, thanks to the regularity of ϕ: and therefore, thanks to the regularity of ϕ: \|R(m) δt \| ≤Cϕ h N−1 X n=0 X K∈M X σ∈E(K) \|DK,σ\| ρn−1 K (˜un+1 σ −un σ)2. Using the assumed uniform bound in L∞(Ω× (0, T )) for the sequence (ρ(m))m∈N, we obtain that the remainder R(m) δt satisﬁes \|R(m) δt \| ≤C h(m) R(m) δt,1 + R(m) δt,2 , where C is a real positive number, independent of m and R(m) δt,1 = N−1 X n=0 X σ∈Eint \|Dσ\| (un+1 σ −un σ)2, R(m) δt,2 = N−1 X n=0 X σ∈Eint \|Dσ\| (un+1 σ −˜un+1 σ )2. which, passing to the limit when m →+∞, yields the result. Summing the two obtained relations, we get T (m) 1 + eT (m) 2 + T (m) 3 + eT (m) 4 + T (m) 5 = R(m), where the terms T (m) 1 , T (m) 3 , and T (m) 5 are deﬁned by (40a), (40b), (40c) in the proof of Theorem 3.20, and eT (m) 2 = N−1 X n=0 δt X K∈M \|K\| δt ρn+1 K en+1 K −ρn Ken K ϕn K, (78) eT (m) 4 = N−1 X n=0 δt X K=[ −−→ σσ′]∈M F n+1 σ′ en+1 σ′ −F n+1 σ en+1 σ ϕn K, (79) X K∈M \|K\| δt ρn+1 K en+1 K −ρn Ken K ϕn K, (78) (78) eT (m) 4 = N 1 X n=0 δt X K=[ −−→ σσ′]∈M F n+1 σ′ en+1 σ′ −F n+1 σ en+1 σ ϕn K, (79) (79) 44 TITLE WILL BE SET BY THE PUBLISHER 44 eR(m) = − N−1 X n=0 δt X σ∈E ( eRn+1 σ −P n+1 σ ) ϕn σ + N−1 X n=0 δt X K∈M Sn+1 K ϕn K (80) (80) where, in the latter relation, the remainder term reads, for σ = −−→ K\|L ∈Eint with K = [−→ σ′σ] and L = [−−→ σσ′′]: eRn+1 σ = 1 2 \|Dσ\| δt ρn−1 Dσ ˜un+1 σ −un σ 2 + ˜un+1 σ h ν h (˜un+1 σ −˜un+1 σ′ ) + ν h (˜un+1 σ −˜un+1 σ′′ ) i , where, in the latter relation, the remainder term reads, for σ = −−→ K\|L ∈Eint with K = [−→ σ′σ] and L = [−−→ σσ′′]: where, in the latter relation, the remainder term reads, for σ = −−→ K\|L ∈Eint with K = [−→ σ′σ] and L = [−−→ σσ′′]: where, in the latter relation, the remainder term reads, for σ = −−→ K\|L the latter relation, the remainder term reads, for σ = K\|L ∈Eint with K = [σ σ] and L = [σσ ] eRn+1 σ = 1 2 \|Dσ\| δt ρn−1 Dσ ˜un+1 σ −un σ 2 + ˜un+1 σ h ν hK (˜un+1 σ −˜un+1 σ′ ) + ν hL (˜un+1 σ −˜un+1 σ′′ ) i , eRn+1 σ = 1 2 \|Dσ\| δt ρn−1 Dσ ˜un+1 σ −un σ 2 + ˜un+1 σ h ν hK (˜un+1 σ −˜un+1 σ′ ) + ν hL (˜un+1 σ −˜un+1 σ′′ ) i , and where P n+1 σ is deﬁned by (48), and Sn+1 K by (74). which, passing to the limit when m →+∞, yields the result. We now observe that the stability assumptions (75) and the regularity assumptions for the discretization of Deﬁnition 4.2 are stronger than the hypotheses made in the barotropic case (see Deﬁnition 3.5, Remark 3.19 and the assumptions of Theorem 3.20). In particular, combining L∞, space BV estimates and the assumption (iii) of Deﬁnition 4.2 on the numerical diﬀusion coeﬃcient, we can prove the same control on the space translates of ρ and ˜u as provided by the remainder terms (51) of the inequality (50). Consequently, the passage to the limit in the scheme for the mass and momentum balance equations is the same as in the barotropic case. We thus only need to prove that (¯ρ, ¯p, ¯e, ¯u) satisﬁes (77c). Let us ﬁrst multiply the one dimensional version of the discrete kinetic energy equation (47) by δt ϕn σ and sum over the faces and the time steps. Similarly, we multiply the discrete internal energy equation (73f) by δt ϕn K, and sum over the primal cells and the time steps. which, passing to the limit when m →+∞, yields the result. We thus get, thanks to the regularity of ϕ: We thus get, thanks to the regularity of ϕ: \|R(m) ∆\| ≤ν Cϕ ∥˜u(m)∥L∞(Ω×(0,T )) ∥˜u(m)∥T ,x,BV , which yields the desired estimate. Finally, P (m) reads: which yields the desired estimate. Finally, P (m) reads: P (m) = N−1 X n=0 δt X σ∈E P n+1 σ ϕn σ, P (m) = N−1 X n=0 δt X σ∈E P n+1 σ ϕn σ, P (m) = N−1 X n=0 δt X σ∈E P n+1 σ ϕn σ, and the fact that this term tends to zero is stated in Lemma 3.16. This concludes the proof. □ and the fact that this term tends to zero is stated in Lemma 3.16. This concludes the proof. □ □ which, passing to the limit when m →+∞, yields the result. The ﬁrst of this term satisﬁes \|R(m) δt,1\| ≤2 ∥u(m)∥L∞(Ω×(0,T )) ∥u(m)∥T ,t,BV , and, thanks once again to the expression of the velocity correction step (73d), we have for the second one: \|R(m) δt,2\| ≤C ∥ 1 ρ(m) ∥ L∞(Ω×(0,T )) ∥u(m)∥L∞(Ω×(0,T )) + ∥˜u(m)∥L∞(Ω×(0,T )) ∥p(m)∥T ,x,BV , \|R(m) δt,2\| ≤C ∥ 1 ρ(m) ∥ L∞(Ω×(0,T )) ∥u(m)∥L∞(Ω×(0,T )) + ∥˜u(m)∥L∞(Ω×(0,T )) ∥p(m)∥T ,x,BV , TITLE WILL BE SET BY THE PUBLISHER 45 TITLE WILL BE SET BY THE PUBLISHER 45 where C is again a real positive number, independent of m. Hence R(m) δt tends to zero as m tends to +∞. Let us now turn to R(m) ∆, which reads: where C is again a real positive number, independent of m. Hence R(m) δt tends to zero as m tends to +∞. Let us now turn to R(m) ∆, which reads: R(m) ∆ = − N−1 X n=0 δt X σ=−−→ K\|L∈Eint, K=[ −−→ σ′σ], L=[ −−→ σσ′′] ˜un+1 σ h ν hK (˜un+1 σ −˜un+1 σ′ ) + ν hL (˜un+1 σ −˜un+1 σ′′ ) i ϕn σ R(m) ∆ = − N−1 X n=0 δt X σ=−−→ K\|L∈Eint, K=[ −−→ σ′σ], L=[ −−→ σσ′′] ˜un+1 σ h ν hK (˜un+1 σ −˜un+1 σ′ ) + ν hL (˜un+1 σ −˜un+1 σ′′ ) i ϕn σ + N−1 X n=0 δt X K=[σσ′]∈M ν hK (˜un+1 σ −˜un+1 σ′ )2 ϕn K. + N−1 X n=0 δt X K=[σσ′]∈M ν hK (˜un+1 σ −˜un+1 σ′ )2 ϕn K. As explained in Section 4.2, the general idea is now to recast this term as a discrete version of the integral over space and time of a quantity of the form −u ∂xu ∂xϕ scaled by a numerical viscosity vanishing with the space step; then, the supposed controls on the solution imply that the term tends to zero. Reordering the sums, we get: R(m) ∆ = N−1 X n=0 δt X K=[ −−→ σσ′]∈M ν hK (˜un+1 σ −˜un+1 σ′ ) (˜un+1 σ′ ϕn σ′ −˜un+1 σ ϕn σ) + ν hK (˜un+1 σ′ −˜un+1 σ )2ϕn K, and thus: and thus: R(m) ∆ = N−1 X n=0 δt X K=[ −−→ σσ′]∈M ν hK (˜un+1 σ −˜un+1 σ′ ) ˜un+1 σ (ϕn K −ϕn σ) + ˜un+1 σ′ (ϕn σ′ −ϕn K) . 4.4. Numerical tests In this section, we assess the behaviour of the scheme on a one dimensional Riemann problem. We choose initial conditions such that the structure of the solution consists in two shock waves, separated by the contact discontinuity, with suﬃciently strong shocks to allow an easy discrimination of correct numerical solutions. These initial conditions are those proposed in [63, chapter 4], for the test referred to as Test 5: left state:   ρleft uleft pleft  =   5.99924 19.5975 460.894   right state:   ρright uright pright  =   5.99242 −6.19633 46.0950   The problem is posed over Ω= (−0.5, 0.5), and the discontinuity is initially located at x = 0 The problem is posed over Ω= (−0.5, 0.5), and the discontinuity is initially located at x = 0. problem is posed over Ω= (−0.5, 0.5), and the discontinuity is initially located at x = 0. At the boundaries, since, in this test, the ﬂow is entering the domain, the solution is prescribed (which, in fact, is unimportant, the solution being constant at any time in a suﬃciently large neighborhood of these boundaries). Previous numerical experiments addressing barotropic ﬂows [27] showed that, at least for one dimensional computations with schemes similar to the one under study here, it was not necessary to upwind the At the boundaries, since, in this test, the ﬂow is entering the domain, the solution is prescribed (which, in fact, is unimportant, the solution being constant at any time in a suﬃciently large neighborhood of these boundaries). Previous numerical experiments addressing barotropic ﬂows [27] showed that, at least for one dimensional computations with schemes similar to the one under study here, it was not necessary to upwind the TITLE WILL BE SET BY THE PUBLISHER 46 convection term in the momentum balance equation; consequently, we only employ a centered approximation of the velocity at the dual faces. The computations are performed with the open-source software CALIF3S [5]. The computations are performed with the open-source software CALIF3S [5]. The density ﬁelds obtained with h = 1/2000 (or a number of cells n = 2000) at t = 0.035, with and without assembling the corrective source term in the internal energy balance (SK)K∈M, together with the analytical solution, are shown on Figure 2. 4.4. Numerical tests The density and the pressure obtained, still with and without corrective terms, for various meshes, are plotted on Figures 3 and 4 respectively. For these computations, we take δt = h/20, which yields a cﬂnumber, with respect to the material velocity only, close to one. The ﬁrst conclusion is that both schemes seem to converge, but the corrective term is necessary to obtain the right solution. In this case, for instance, we obtain the correct intermediate state for the pressure and velocity up to four digits in the essential part of the corresponding zone: (analytical) intermediate state: p∗ u∗ = 1691.65 8.68977 for x ∈(0.028, 0.428) numerical results: p ∈(1691.6, 1691.8) u ∈(8.689, 8.690) for x ∈(0.032, 0.417) p∗ u∗ = 1691.65 8.68977 for x ∈(0.028, 0.428) p ∈(1691.6, 1691.8) u ∈(8.689, 8.690) for x ∈(0.032, 0.417) p∗ u∗ = 1691.65 8.68977 for x ∈(0.028, 0.428) p ∈(1691.6, 1691.8) u ∈(8.689, 8.690) for x ∈(0.032, 0.417) (analytical) intermediate state: p∗ u∗ = 1691.65 8.68977 for x ∈(0.028, 0.428) numerical results: p ∈(1691.6, 1691.8) u ∈(8.689, 8.690) for x ∈(0.032, 0.417) (analytical) intermediate state: numerical results: Without a corrective term, one can check that the obtained solution is not a weak solution to the Euler system: indeed, the Rankine-Hugoniot condition applied to the total energy balance, with the states obtained numerically, yields a right shock speed slightly greater than the analytical solution one, while the same shock speed obtained numerically is clearly lower. We also observe that the scheme is rather diﬀusive especially for contact discontinuities for which the beneﬁcial compressive eﬀect of the shocks does not apply. More accurate variants may certainly be derived, using for instance MUSCL-like techniques; this work is underway [17]. 0 5 10 15 20 25 30 35 40 -0.4 -0.2 0 0.2 0.4 x with without exact Figure 2. Test 5 of [63, chapter 4] - Density obtained with n = 2000 cells, with and without corrective source terms, and analytical solution. Figure 2. Test 5 of [63, chapter 4] - Density obtained with n = 2000 cells, with and without corrective source terms, and analytical solution. TITLE WILL BE SET BY THE PUBLISHER 47 0 5 10 15 20 25 30 35 40 -0.4 -0.2 0 0.2 0.4 x n=500 n=1000 n=2000 0 5 10 15 20 25 30 35 40 -0.4 -0.2 0 0.2 0.4 x n=500 n=1000 n=2000 Figure 3. Test 5 of [63, chapter 4] - Density obtained with various meshes, with (left) and without (right) corrective source terms. 0 5 10 15 20 25 30 35 40 -0.4 -0.2 0 0.2 0.4 x n=500 n=1000 n=2000 0 5 10 15 20 25 30 35 40 -0.4 -0.2 0 0.2 0.4 x n=500 n=1000 n=2000 0 5 10 15 -0.4 -0.2 0 0.2 0.4 x n=500 n=1000 n=2000 0 5 10 15 -0.4 -0.2 0 0.2 0.4 x n=500 n=1000 n=2000 Figure 3. Test 5 of [63, chapter 4] - Density obtained with various meshes, with (left) and without (right) corrective source terms. 0 200 400 600 800 1000 1200 1400 1600 1800 -0.4 -0.2 0 0.2 0.4 x n=500 n=1000 n=2000 0 200 400 600 800 1000 1200 1400 1600 1800 -0.4 -0.2 0 0.2 0.4 x n=500 n=1000 n=2000 Figure 4. Test 5 of [63, chapter 4] - pressure obtained with various meshes, with (left) and without (right) corrective source terms. Figure 3. Test 5 of [63, chapter 4] - Density obtained with various meshes, with (left) and without (right) corrective source terms. numerical results: 0 200 400 600 800 1000 1200 1400 1600 1800 -0.4 -0.2 0 0.2 0.4 x n=500 n=1000 n=2000 0 200 400 600 800 1000 1200 1400 1600 1800 -0.4 -0.2 0 0.2 0.4 x n=500 n=1000 n=2000 0 200 400 600 800 1000 1200 1400 1600 1800 -0.4 -0.2 0 0.2 0.4 x n=500 n=1000 n=2000 Figure 4. Test 5 of [63, chapter 4] - pressure obtained with various meshes, with (left) and without (right) corrective source terms. 0 200 400 600 800 1000 1200 1400 1600 1800 -0.4 -0.2 0 0.2 0.4 x n=500 n=1000 n=2000 Figure 4. Test 5 of [63, chapter 4] - pressure obtained with various meshes, with (left) and without (right) corrective source terms. In order to check the consistency of the scheme, we give in the table below the L1(Ω)-norm of the diﬀerence between the numerical and the exact solution (denoted (ρex, pex, uex)) at t = 0.035, for various grids and still for δt = h/20. h ∥ρ −ρex∥L1(Ω) ∥p −pex∥L1(Ω) ∥u −uex∥L1(Ω) 1/250 0.0662 1.235 0.00911 1/500 0.0452 0.619 0.00437 1/1000 0.0313 0.365 0.00232 1/2000 0.0215 0.170 0.00125 1/4000 0.0148 0.0849 0.000625 1/8000 0.0102 0.0357 0.000358 We observe a convergence rate of approximatively 1 for the variables which are continuous at the contact discontinuity (p and u) and 1/2 for the other ones (in the table, only ρ). Since, as explained above, the error essentially lies at the jumps of the solution, it means that a shock is captured in approximatively the same In order to check the consistency of the scheme, we give in the table below the L1(Ω)-norm of the diﬀerence between the numerical and the exact solution (denoted (ρex, pex, uex)) at t = 0.035, for various grids and still for δt = h/20. In order to check the consistency of the scheme, we give in the table below the L1(Ω)-norm of the diﬀerence between the numerical and the exact solution (denoted (ρex, pex, uex)) at t = 0.035, for various grids and still for δt = h/20. 5. Conclusion, perspectives In this work, we studied staggered implicit and semi-implicit schemes which had been found earlier to be very eﬃcient for viscous compressible ﬂows [14–16]. We were able to show here that they are also well adapted to the shallow water equations or barotropic Euler equations. We also showed that, with a careful discretization of the internal energy equation, they are again an eﬃcient choice for the full Euler equations. There are several open questions under study at the present time or that will be in the near future: There are several open questions under study at the present time or that will be in - A proof of the consistency of the scheme in the multidimensional case is underway: there is a real diﬃculty in going from the 1D case that we studied here in sections 3.1.3, 3.2.3 and 4.3 to the multidimensional case, due to the intricate deﬁnition of the nonlinear convection term in the momentum balance equation, which makes complex the passage to the limit in this term. This diﬃculty has been adressed both for the ﬂuxes deﬁned on unstructured meshes, with the Crouzeix-Raviart and Rannacher-Turek ﬁnite element unknowns [42], and in the MAC case [29]. [ ] [ ] - The consistency of the scheme with the entropy condition in the case of the full Euler equation is an open question, although the numerical results suggest that this is true. We shall continue to investigate this important issue. - The consistency of the scheme with the entropy condition in the case of the full Euler equation is an open question, although the numerical results suggest that this is true. We shall continue to investigate this important issue. - Comparisons are currently being performed, for the proposed pressure correction scheme, between the present staggered discretization and a colocated version (density, pressure and velocity unknowns all in the cells), for the Euler equations [62]. - Comparisons are currently being performed, for the proposed pressure correction scheme, between the present staggered discretization and a colocated version (density, pressure and velocity unknowns all in the cells), for the Euler equations [62]. ) [ ] - An explicit version of this scheme has been studied both for the shallow water and Euler equations [30]. For this time discretization, higher order schemes using a MUSCL technique [56] have been studied and implemented [17]. 5. Conclusion, perspectives ) [ ] - An explicit version of this scheme has been studied both for the shallow water and Euler equations [30]. For this time discretization, higher order schemes using a MUSCL technique [56] have been studied and implemented [17]. [ ] - Finally, setting ρ to a constant in the pressure correction scheme yields the usual projection scheme for the incompressible Euler (or Navier-Stokes) equations. A natural question is therefore to know whether the scheme is indeed asymptotic preserving: does the approximate solution tend to the incompressible solution as the Mach number tends to 0? This question should be addressed in the near future. numerical results: h ∥ρ −ρex∥L1(Ω) ∥p −pex∥L1(Ω) ∥u −uex∥L1(Ω) 1/250 0.0662 1.235 0.00911 1/500 0.0452 0.619 0.00437 1/1000 0.0313 0.365 0.00232 1/2000 0.0215 0.170 0.00125 1/4000 0.0148 0.0849 0.000625 1/8000 0.0102 0.0357 0.000358 h ∥ρ −ρex∥L1(Ω) ∥p −pex∥L1(Ω) ∥u −uex∥L1(Ω) 1/250 0.0662 1.235 0.00911 1/500 0.0452 0.619 0.00437 1/1000 0.0313 0.365 0.00232 1/2000 0.0215 0.170 0.00125 1/4000 0.0148 0.0849 0.000625 1/8000 0.0102 0.0357 0.000358 We observe a convergence rate of approximatively 1 for the variables which are continuous at the contact discontinuity (p and u) and 1/2 for the other ones (in the table, only ρ). Since, as explained above, the error essentially lies at the jumps of the solution, it means that a shock is captured in approximatively the same We observe a convergence rate of approximatively 1 for the variables which are continuous at the contact discontinuity (p and u) and 1/2 for the other ones (in the table, only ρ). Since, as explained above, the error essentially lies at the jumps of the solution, it means that a shock is captured in approximatively the same We observe a convergence rate of approximatively 1 for the variables which are continuous at the contact discontinuity (p and u) and 1/2 for the other ones (in the table, only ρ). Since, as explained above, the error essentially lies at the jumps of the solution, it means that a shock is captured in approximatively the same 48 TITLE WILL BE SET BY THE PUBLISHER number of cells, for any of the meshes used in this test; by a simple computation, this implies that the corrective term (SK)K∈M does not tend to zero. number of cells, for any of the meshes used in this test; by a simple computation, this implies that the corrective term (SK)K∈M does not tend to zero. Appendix A. Some results associated with finite volume convection operators Let ψ be a regular function from (0, +∞) to R; then: ψ′(ρ) C(ρ) = ψ′(ρ) ∂t(ρ) + ψ′(ρ) u · ∇ρ + ψ′(ρ) ρ divu = ∂t(ψ(ρ)) + u · ∇ψ(ρ) + ρ ψ′(ρ) divu; adding and subtracting ψ(ρ) divu yields: adding and subtracting ψ(ρ) divu yields: ψ′(ρ) C(ρ) = ∂t ψ(ρ) + div ψ(ρ)u + ρψ′(ρ) −ψ(ρ) divu. (83) (83) This computation is of course completely formal and only valid for regular functions ρ and u. The following lemma states a discrete analogue to (83), and its proof follows the formal computation which we just described. This computation is of course completely formal and only valid for regular functions ρ and u. The following lemma states a discrete analogue to (83), and its proof follows the formal computation which we just described. Lemma A.1. Let P be a polygonal (resp. polyhedral) bounded set of R2 (resp. R3), and let E(P) be the set of its edges (resp. faces). Let ψ be a continuously diﬀerentiable function deﬁned over (0, +∞). Let ρ∗ P > 0, ρP > 0, δt > 0; consider three families (ρη)η∈E(P ) ⊂R+ \ {0}, (Vη)η∈E(P ) ⊂R and (Fη)η∈E(P ) ⊂R such that ∀η ∈E(P), Fη = ρη Vη, ∀η ∈E(P), Fη = ρη Vη, and deﬁne: and deﬁne: RP,δt = h\|P\| δt (ρP −ρ∗ P ) + X η∈E(P ) Fη i ψ′(ρP ) − h\|P\| δt ψ(ρP ) −ψ(ρ∗ P ) + X η∈E(P ) ψ(ρη) Vη + ρP ψ′(ρP ) −ψ(ρP ) X η∈E(P ) Vη i (84) Then Then (i) If ψ is convex and ρη = ρP whenever Vη > 0, then RP,δt ≥0. (ii) If ψ is twice continuously diﬀerentiable then (i) If ψ is convex and ρη = ρP whenever Vη > 0, then RP,δt ≥0. (i) If ψ is convex and ρη = ρP whenever Vη > 0, then RP,δt ≥0. (ii) If ψ is twice continuously diﬀerentiable then RP,δt = 1 2 \|P\| δt ψ′′(ρP ) (ρP −ρ∗ P )2 −1 2 X η∈E(P ) Vη ψ′′(ρη) (ρη −ρP )2, with ρP ∈[min(ρP , ρ∗ P ), max(ρP , ρ∗ P )] and ∀η ∈E(P), ρη ∈[min(ρη, ρP ), max(ρη, ρP )]. Proof. We have: with ρP ∈[min(ρP , ρ∗ P ), max(ρP , ρ∗ P )] and ∀η ∈E(P), ρη ∈[min(ρη, ρP ), max(ρη, ρP )]. Proof. Appendix A. Some results associated with finite volume convection operators We gather in this section some results concerning the ﬁnite volume discretization of the two convectio operators which appear in the Navier–Stokes equations: the convection operator C appearing in the mass balance, which reads, at the continuous level, ρ 7→C(ρ) = ∂tρ + div(ρu), (81) (81) where u stands for a given velocity ﬁeld (which is not assumed to satisfy any divergence constraint), h i C i i h d b l hi h d i h i where u stands for a given velocity ﬁeld (which is not assumed to satisfy any divergence constraint), - the convection operator Cρ appearing in the momentum and energy balances, which reads, in the contin- where u stands for a given velocity ﬁeld (which is not assumed to satisfy any divergence constraint), - the convection operator Cρ appearing in the momentum and energy balances, which reads, in the contin- uous setting, g y ( y y g ), - the convection operator Cρ appearing in the momentum and energy balances, which reads, in the contin- uous setting, z 7→Cρ(z) = ∂t(ρz) + div(ρzu), (82) z 7→Cρ(z) = ∂t(ρz) + div(ρzu), (82) where ρ (resp. u) stands for a given scalar (resp. vector) ﬁeld; we wish to obtain some property of Cρ under the assumption that ρ and u satisfy a mass balance equation, i.e. ∂tρ + div(ρu) = 0. Multiplying these operators by functions depending on the unknown is a classical technique to obtain convection operators acting over diﬀerent variables, possibly with residual terms: one may think, for instance, to the theory of renormalized solutions or entropy solutions for the operator C, or, in ﬂuid mechanics, to the derivation of the TITLE WILL BE SET BY THE PUBLISHER 49 so-called kinetic energy transport identity for the operator Cρ, with z standing for a component of the velocity. The results provided in this section are the discrete analogs of these properties. so-called kinetic energy transport identity for the operator Cρ, with z standing for a component of the velocity. The results provided in this section are the discrete analogs of these properties. We begin with a property of the convection operator C deﬁned by (81); at the continuous level, this property may be formally obtained as follows. + X η∈E(P ) [ρηψ′(ρP ) −ψ(ρη)] Vη, Appendix A. Some results associated with finite volume convection operators = ρ ∂tψ(z) + u · ∇ψ(z) = ∂t ρ ψ(z) + div ρ ψ(z) u . Taking for z a component of the velocity ﬁeld, this relation is the central argument used to derive the kinetic energy balance. The following lemma states a discrete counterpart of this identity. Taking for z a component of the velocity ﬁeld, this relation is the central argument used to derive the kinetic energy balance. The following lemma states a discrete counterpart of this identity. Lemma A.2. Let P be a polygonal (resp. polyhedral) bounded set of R2 (resp. R3) and let E(P) be the set of its edges (resp. faces). Let ρ∗ P > 0, ρP > 0, δt > 0, and (Fη)η∈E(P ) ⊂R be such that \|P\| δt (ρP −ρ∗ P ) + X η∈E(P ) Fη = 0. (85) (85) Let ψ be a continuously diﬀerentiable function deﬁned over (0, +∞). For u∗ P ∈R, uP ∈R and (uη)η∈E(P ) ⊂R let us deﬁne: Let ψ be a continuously diﬀerentiable function deﬁned over (0, +∞). For u∗ P ∈R, uP ∈R and (uη)η∈E(P ) ⊂R let us deﬁne: RP,δt = h\|P\| δt ρP uP −ρ∗ P u∗ P + X η∈E(P ) Fη uη i ψ′(uP ) − h\|P\| δt ρP ψ(uP ) −ρ∗ P ψ(u∗ P ) + X η∈E(P ) Fη ψ(uη) i . (86) RP,δt = h\|P\| δt ρP uP −ρ∗ P u∗ P + X η∈E(P ) Fη uη i ψ′(uP ) − h\|P\| δt ρP ψ(uP ) −ρ∗ P ψ(u∗ P ) + X η∈E(P ) Fη ψ(uη) i . (86) (86) Then: Then: Then: (i) If ψ is convex and uη = uP whenever Fη > 0, then RP,δt ≥0. (ii) If ψ is twice continuously diﬀerentiable, then (i) If ψ is convex and uη = uP whenever Fη > 0, then RP,δt ≥0. (ii) If ψ is twice continuously diﬀerentiable, then RP,δt = 1 2 \|P\| δt ρ∗ P ψ′′(uP )(uP −u∗ P )2 −1 2 X η∈E(P ) Fη ψ′′(uη) (uη −uP )2, (87) (87) with, uP ∈[min(uP , u∗ P ), max(uP , u∗ P )] and, ∀η ∈E(P), uη ∈[min(uη, uP ), max(uη, uP )]. Appendix A. Some results associated with finite volume convection operators We have: h\|P\| δt (ρP −ρ∗ P ) + X η∈E(P ) Fη i ψ′(ρP ) = \|P\| δt (ρP −ρ∗ P ) ψ′(ρP ) + X η∈E(P ) ψ(ρη) Vη + X η∈E(P ) [ρηψ′(ρP ) −ψ(ρη)] Vη, so the remainder term RP,δt reads RP,δt = \|P\| δt rP + P η∈E(P ) Vη rη, with: rP = (ρP −ρ∗ P ) ψ′(ρP ) − ψ(ρP ) −ψ(ρ∗ P ) , rη = ρηψ′(ρP ) −ψ(ρη) − ρP ψ′(ρP ) −ψ(ρP ) . so the remainder term RP,δt reads RP,δt = \|P\| δt rP + P η∈E(P ) Vη rη, with: rP = (ρP −ρ∗ P ) ψ′(ρP ) − ψ(ρP ) −ψ(ρ∗ P ) , rη = ρηψ′(ρP ) −ψ(ρη) − ρP ψ′(ρP ) −ψ(ρP ) . so the remainder term RP,δt reads RP,δt = \|P\| δt rP + P η∈E(P ) Vη rη, with: δt η ( ) rP = (ρP −ρ∗ P ) ψ′(ρP ) − ψ(ρP ) −ψ(ρ∗ P ) , rη = ρηψ′(ρP ) −ψ(ρη) − ρP ψ′(ρP ) −ψ(ρP ) . TITLE WILL BE SET BY THE PUBLISHER 50 If the function ψ is convex, rP is non-negative while rη is non-positive (and vanishes if ρη = ρP ). If ψ is twice continuously diﬀerentiable, a Taylor expansion gives that: (ρP −ρ∗ P )ψ′(ρP ) = ψ(ρP ) −ψ(ρ∗ P ) + 1 2ψ ′′(ρP )(ρP −ρ∗ P )2, ρηψ′(ρP ) −ψ(ρη) = ρP ψ′(ρP ) −ψ(ρP ) −1 2ψ′′(ρη)(ρη −ρP )2, with ρP ∈[min(ρP , ρ∗ P ), max(ρP , ρ∗ P )] and for any η ∈E(P), ρη ∈[min(ρη, ρP ), max(ρη, ρP )]; hence the result. □ with ρP ∈[min(ρP , ρ∗ P ), max(ρP , ρ∗ P )] and for any η ∈E(P), ρη ∈[min(ρη, ρP ), max(ρη, ρP )]; hence the result. □ □ We now turn to the momentum convection operator Cρ deﬁned by (82); formally, using twice the assumption ∂tρ + div(ρu) = 0 yields: ψ′(z) Cρ(z) = ψ′(z) ∂t(ρ z) + div(ρ z u) = ψ′(z)ρ ∂tz + u · ∇z = ρ ∂tψ(z) + u · ∇ψ(z) = ∂t ρ ψ(z) + div ρ ψ(z) u . Appendix A. Some results associated with finite volume convection operators (iii) As a consequence of (ii), for ψ deﬁned by ψ(s) = s2/2, and ∀η ∈E(P), uη such that uη = with, uP ∈[min(uP , u∗ P ), max(uP , u∗ P )] and, ∀η ∈E(P), uη ∈[min(uη, uP ), max(uη, uP )]. (iii) As a consequence of (ii), for ψ deﬁned by ψ(s) = s2/2, and ∀η ∈E(P), uη such that uη = (uP + uPη)/2 (this is simply obtained by deﬁning uPη = 2 uη −uP ), we get the following identity: , [ ( , P ), ( , P )] , η ( ), η [ ( η, ), ( η, )] (iii) As a consequence of (ii), for ψ deﬁned by ψ(s) = s2/2, and ∀η ∈E(P), uη such that uη = (uP + uPη)/2 (this is simply obtained by deﬁning uPη = 2 uη −uP ), we get the following identity: h\|P\| δt ρP uP −ρ∗ P u∗ P + X η∈E(P ) Fη uη i uP = 1 2 \|P\| δt ρP u2 P −ρ∗ P (u∗ P )2 + 1 2 X η∈E(P ) Fη uP uPη + ˜RP,δt, (88) (88) 51 TITLE WILL BE SET BY THE PUBLISHER with with ˜RP,δt = 1 2 \|P\| δt ρ∗ P (uP −u∗ P )2, with ˜RP,δt = 1 2 \|P\| δt ρ∗ P (uP −u∗ P )2, Proof. Let TP be deﬁned by: Proof. Let TP be deﬁned by: TP = h\|P\| δt (ρP uP −ρ∗ P u∗ P ) + X η∈E(P ) Fη uη i ψ′(uP ). Using Equation (85), we obtain: TP = h\|P\| δt ρ∗ P (uP −u∗ P ) + X η∈E(P ) Fη(uη −uP ) i ψ′(uP ). We now deﬁne the remainder terms rP and (rη)η∈E(P ) by: rP = (uP −u∗ P ) ψ′(uP ) − ψ(uP ) −ψ(u∗ P ) , rη = (uP −uη) ψ′(uP ) − ψ(uP ) −ψ(uη) rP = (uP −u∗ P ) ψ′(uP ) − ψ(uP ) −ψ(u∗ P ) , rη = (uP −uη) ψ′(uP ) − ψ(uP ) −ψ(uη) . rP = (uP −u∗ P ) ψ′(uP ) − ψ(uP ) −ψ(u∗ P ) , rη = (uP −uη) ψ′(uP ) − ψ(uP ) −ψ(uη) . Appendix A. Some results associated with finite volume convection operators With these notations, we get: With these notations, we get: TP = \|P\| δt ρ∗ P ψ(uP ) −ψ(u∗ P ) + X η∈E(P ) Fη ψ(uη) −ψ(uP ) + \|P\| δt ρ∗ P rP − X η∈E(P ) Fη rη. Using Equation (85) once again, we have: Using Equation (85) once again, we have: TP = \|P\| δt ρP ψ(uP ) −ρ∗ P ψ(u∗ P ) + X η∈E(P ) Fη ψ(uη) + \|P\| δt ρ∗ P rP − X η∈E(P ) Fη rη, and thus: and thus: RP,δt = \|P\| δt ρ∗ P rP − X η∈E(P ) Fη rη. If ψ is convex, the remainder terms rP and (rη)η∈E(P ) are non-negative, and if uη = uP , rη = 0; hence, if we suppose that uη = uP when Fη ≥0, then RP,δt ≥0. 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https://openalex.org/W2755458164	https://zenodo.org/record/1438362/files/ArcaSedda_Kocsis_Brandt18_arxiv.pdf	English	null	Gamma-ray and X-ray emission from the Galactic centre: hints on the nuclear star cluster formation history	Monthly Notices of the Royal Astronomical Society	2,018	cc-by	19,971	Mon. Not. R. Astron. Soc. 000, 1–?? (2015) Printed 29 May 2018 (MN LATEX style ﬁle v2.2) Mon. Not. R. Astron. Soc. 000, 1–?? (2015) Printed 29 May 2018 (MN LATEX style ﬁle v2.2) ABSTRACT The Milky Way centre exhibits an intense ﬂux in the gamma and X-ray bands, whose origin is partly ascribed to the possible presence of a large population of millisecond pulsars (MSPs) and cataclysmic variables (CVs), respectively. However, the number of sources required to generate such an excess is much larger than what is expected from in situ star formation and evolution, opening a series of questions about the formation history of the Galactic nucleus. In this paper we make use of direct N- body simulations to investigate whether these sources could have been brought to the Galactic centre by a population of star clusters that underwent orbital decay and formed the Galactic nuclear star cluster (NSC). Our results suggest that the gamma ray emission is compatible with a population of MSPs that were mass segregated in their parent clusters, while the X-ray emission is consistent with a population of CVs born via dynamical interactions in dense star clusters. Combining observations with our modelling, we explore how the observed γ ray ﬂux can be related to diﬀerent NSC formation scenarios. Finally, we show that the high-energy emission coming from the galactic central regions can be used to detect black holes heavier than 105 M⊙in nearby dwarf galaxies. arXiv:1709.03119v3 [astro- Key words: Galaxy: centre, gamma-rays: galaxies, X-rays: galaxies, cataclysmic variables, pulsars: general, dark matter. Revised to arXiv:1709.03119v3 [astro-ph.GA] 1 T ⋆e-mail: m.arcasedda@ari.uni-heidelberg.de Gamma-ray and X-ray emission from the Galactic centre: hints on the nuclear star cluster formation history 3119v3 [astro-ph.GA] 26 May 2018 Xiv:1709.03119v3 [astro-ph.GA] 26 May 201 R 1 Manuel Arca-Sedda1⋆and Bence Kocsis2 and Timothy D. Brandt3,4 1Zentrum f¨ur Astronomie der Universit¨at Heidelberg, Astronomisches Rechen-Institut, M¨onchhofstr. 12-14, 69120 Heidelberg 2Institute of Physics, E¨otv¨os University, P´azm´any P. s. 1/A, Budapest, 1117, Hungary 3Institute for Advanced Study, Einstein Dr., Princeton, NJ, 08540 USA 4University of California, Santa Barbara, Santa Barbara, CA, 93106, USA Manuel Arca-Sedda1⋆and Bence Kocsis2 and Timothy D. Brandt3,4 1Zentrum f¨ur Astronomie der Universit¨at Heidelberg, Astronomisches Rechen-Institut, M¨onchhofstr. 12-14, 69120 Heidelberg 2Institute of Physics, E¨otv¨os University, P´azm´any P. s. 1/A, Budapest, 1117, Hungary 3Institute for Advanced Study, Einstein Dr., Princeton, NJ, 08540 USA 4University of California, Santa Barbara, Santa Barbara, CA, 93106, USA Revised to Revised to Manuel Arca-Sedda1⋆and Bence Kocsis2 and Timothy D. Brandt3,4 1Zentrum f¨ur Astronomie der Universit¨at Heidelberg, Astronomisches Rechen-Institut, M¨onchhofstr. 12-14, 69120 Heidelberg 2Institute of Physics, E¨otv¨os University, P´azm´any P. s. 1/A, Budapest, 1117, Hungary 3Institute for Advanced Study, Einstein Dr., Princeton, NJ, 08540 USA 4University of California, Santa Barbara, Santa Barbara, CA, 93106, USA 1 INTRODUCTION (2016) propose that the emission is likely due to intermediate polars (IPs), a type of CV with longer orbital periods and non-synchronized orbits compared to polars (Evidence for Intermediate Polars as the Origin of the Galactic Center Hard X-ray Emission pat; Pretorius et al. 2013). The authors note that the cen- tral X-ray emission proﬁle is quite similar to the luminosity proﬁle of the Galactic NSC, thus suggesting a stellar origin for the X-rays. Understanding the nature of the gamma-ray excess and its possible connection with the X-ray excess may shed light on the extreme processes that take place in the vicinity of an SMBH. dense systems (Brandt & Kocsis 2015). Indeed, known as “recycled pulsars”, MSPs form primarily in binary systems. The high stellar encounter rates in dense systems, facilitates to decrease the binary separation, until the neutron star’s companion transfers material and angular momentum, re- ducing the neutron star’s magnetic ﬁeld and increasing its spin rate (Michel 1987; Bhattacharya & van den Heuvel 1991). During this phase, lasting ∼107 −109 yr, the system is observable as a low mass X-ray binary (LMXB, Ivanova et al. 2008). After the mass transfer stops, the MSP phase will live beyond ∼1010 yr. Given their long life time, MSPs constitute a promis- ing source for the Galactic γ ray ﬂux, although many ob- jections have been put forth against this scenario. Haggard et al. (2017) argued that if the observed gamma-ray ﬂux is only due to MSPs, they should have observed ∼1000 LMXBs within 10deg from the Galactic centre (∼1.4 kpc @ 8 kpc), but only ∼40 −80 LMXBs have been observed there with INTEGRAL (Lavigne et al. 1998). The MSP scenario thus requires that most LMXB activity, and hence MSP creation, ceased long ago. If the MSP population is old, the present- day emission depends strongly on the gamma-ray eﬃciency, which varies as a function of spin-down power (O’Leary et al. 2016). The observed excess is inconsistent with MSPs as- suming a constant gamma-ray eﬃciency (Hooper & Linden 2016). However, recently Fragione et al. (2017) showed that the observed emission is consistent with the expected MSP emission accounting for spin down eﬀects. Finally, Hooper & Linden (2016) claimed that the MSP luminosity func- tion requires several very bright (and individually resolv- able) MSPs around the Galactic center in order to explain the excess. 1 INTRODUCTION young pulsars born in the star-forming nuclear star cluster (NSC) (O’Leary et al. 2015, 2016); injection of cosmic-ray protons (Carlson & Profumo 2014); or cosmic ray outbursts (Petrovi´c et al. 2014). While a diﬀuse background would be expected from annihilation in a smooth dark matter proﬁle (Bartels et al. 2016; Mishra-Sharma et al. 2017), observa- tions instead indicate an unresolved population of gamma ray point sources, consistent with the hypothesis that MSPs play a signiﬁcant role in the development of gamma ray emis- sion (Daylan et al. 2016; Fermi-LAT Collaboration 2017). The Fermi satellite’s discovery of strong excess emission in the Milky Way Galaxy centre opened a series of questions about the physical origin of this phenomenon. The excess is characterised by a nearly spherical morphology, and extends up to ∼1 −3 kpc from the Galaxy’s supermassive black hole (SMBH). One possible explanation for such a strong signal is the annihilation of ∼30 GeV dark matter particles (Hooper & Goodenough 2011). In this case, the Galactic centre would provide the ﬁrst evidence of dark matter parti- cles beyond the Standard Model interacting with the electro- magnetic sector. However, such exotic explanations presume that astrophysical processes cannot account for the observed emission. Possible alternatives to dark matter annihilation, among others, are millisecond pulsars (MSPs), rapidly ro- tating neutron stars observed throughout the galaxy and characterised by a gamma ray spectrum similar to that ob- served for the excess (Abazajian 2011); highly magnetized Fermi’s resolution is insuﬃcient to study the morphol- ogy of the excess over the inner few pc. The excess is con- sistent with a compact, unresolved (≲10 pc) source or set of sources, plus much more extended emission (Abazajian et al. 2014; Brandt & Kocsis 2015), or with a steep cusp toward Sgr A* (Daylan et al. 2016). The morphology of this inner region is accessible in X-rays. Recent observations with the NuSTAR satellite show a complex X-ray structure in the inner 10 pc from Sgr A, characterised by a nearly spheri- cal structure and emitting ﬁlaments. The source of emission may be an unresolved population of cataclysmic variables Arca-Sedda, M. and Kocsis, B. and Brandt, T. D. 2 (CVs, see Mukai (2017) for a review on the argument) with mass ∼0.9 M⊙(Mori et al. 2015), a population of MSPs (Perez et al. 2015), or a more heterogeneous population of MSPs, CVs and X-ray binaries. Hailey et al. 1.2 Formation of the nuclear star cluster The distribution of MSPs and CVs in the Galactic center carries important information about the formation history of the nuclear star cluster (NSC). The NSC is a massive star cluster surrounding Sgr A which is characterised by a very compact size (half-mass radius ∼4.2 pc) and a to- tal mass of 2.5 × 107 M⊙(Sch¨odel et al. 2014; Gallego- Cano et al. 2017; Sch¨odel et al. 2017). In what is called the “dry-merger” scenario, NSCs are assembled by the se- quence of mergers of dense star clusters that spiral toward the galaxy centre due to dynamical friction (Tremaine et al. 1975; Tremaine 1976b,a; Capuzzo-Dolcetta 1993; Antonini et al. 2012; Antonini 2013; Gnedin et al. 2014; Arca-Sedda & Capuzzo-Dolcetta 2014a,c; Arca-Sedda et al. 2015; Arca- Sedda & Capuzzo-Dolcetta 2016, 2017b). During this pro- cess, GCs bring their stellar content to the Galactic centre altering the stellar population therein (Antonini 2014; Arca- Sedda & Capuzzo-Dolcetta 2017b). The “dry” scenario con- trasts with a “wet” formation in which gas was brought to the Galactic centre where stars formed in-situ (Milosavljevi´c 1.1 Controversy of the MSP origin of the Fermi excess 1 INTRODUCTION In this paper, we use direct N-body simulations to ex- amine the role of infalling globular clusters (GCs) in shaping the observed gamma-ray and hard X-ray proﬁles. GCs eﬃ- ciently form MSPs, CVs, and X-ray binaries due to the high likelihood of close dynamical encounters. Using semianalytic arguments and comparisons to extant GCs, Brandt & Kocsis (2015) showed that the observed gamma-ray ﬂux is consis- tent with the emission of MSPs that were delivered to the Galactic centre by inspiralling GCs. Similarly, we point out that infalling clusters would also deposit their CV popula- tions around the Galactic centre. Indeed, CVs are expected to form via dynamical encounters in dense stellar environ- ments (Ivanova et al. 2006; Belloni et al. 2016; Dieball et al. 2017), and their lifetimes are estimated to lie in the range 108-1011 yr (Kolb & Stehle 1996; Mukai 2017). CVs with period shorter than 2.2 hr have a lifetime > 109 yr1. Recent observations in the far UV have outlined the presence of a population of both CVs and MSPs mass-segregated into the core of the NGC 6397 GC (Dieball et al. 2017). This poses interesting questions about the formation and evolution of such objects, conﬁrming at the same time their presence in the inner regions of GCs. With the ongoing development of hybrid strategies to infer the actual number of MSPs from Fermi data (Bartels et al. 2016; Bhakta et al. 2017) and with future radio ob- servations with the square kilometer array (SKA) it may be possible to detect MSPs directly and map out their distribu- tion in the MW inner 10 pc (Dewdney et al. 2009; Macquart & Kanekar 2015; Brandt & Kocsis 2015; Calore et al. 2016), as recently suggested by Abbate et al. (2017). Dynamically formed MSPs and CVs carry information on the GC infall history of the Galactic center. Using our simulations, we examine the formation of the central Milky Way nuclear star cluster, and predict the distribution of MSPs and CVs. We use this information to investigate the implications for gamma ray and hard X-ray emission pro- ﬁles. 1 i.e. those below the so-called “period gap”, and constitute ∼30% of the CV population (Kolb & Stehle 1996), although it is diﬃcult to determine the actual fraction due to observational se- lection eﬀects (Mukai 2017) or to the complex modelling required (Podsiadlowski et al. 2003; Goliasch & Nelson 2015). 2 After the original submission of this paper, a paper appeared on the predicted gamma-ray emission of the Galactic bulge for a delivered population of MSPs accounting for the spin-down eﬀect (Fragione et al. 2017). Their calculation leads to a γ ﬂux an order of magnitude larger than observed in the case in which the MSP spindown eﬀect is taken into account. 2004; Nayakshin et al. 2009; Antonini 2013; Aharon & Perets 2015). 2004; Nayakshin et al. 2009; Antonini 2013; Aharon & Perets 2015). with γ = 0.5 the negative inner density slope, Mg = 1.6 × 109 M⊙the total galaxy mass and rg = 110 pc its length scale. The truncation radius rcut = 150 pc allowed us to model self-consistently the inner region of the Galaxy. In this paper we use state-of-the-art N-body simula- tions to investigate whether a “dry” origin of the Galaxy’s nuclear star cluster (NSC) can account for the Galactic cen- tre’s gamma-ray and X-ray emission observed by Fermi and NuSTAR, and examine implications for MSP and CV source candidates.2 We also determine the number of stellar mass BHs delivered to this region by GCs. This choice of parameters is chosen to roughly represent the observed cumulative mass proﬁle and velocity dispersion (see Arca-Sedda et al. 2015 for more details). The outer cut at 150 pc is set by computational limitations while keeping the necessary high resolution of the inner regions. This ap- proach allows us to create a self-consistent model that repro- duces the whole region inside rcut, whose evolution is driven by two-body relaxation processes. In other words, cutting the density proﬁle with the exponential cut in Equation (2) ensures a correct representation of the dynamics inside rcut avoiding spurious relaxation processes related to the absence of particles outside of this region. The paper is organized as follows: in Section 2 we de- scribe our numerical setup and our models; in Section 3 we discuss the outcomes of the simulations and in Section 5 we draw the conclusions of this work. 1.1 Controversy of the MSP origin of the Fermi excess In the MSP interpretation of the Fermi excess, the observed gamma ray ﬂux requires at least 103 MSPs (Bednarek & Sobczak 2013), a number that seems exceedingly large with respect to our current knowledge of in situ MSP formation mechanisms in the Galactic centre. The MSPs could have formed in a dense stellar environment, such as a GC, and have been delivered to the central region by the inspiral of Gamma-ray and X-ray emission from the Galactic centre Gamma-ray and X-ray emission from the Galactic centre 3 This model represents the inner regions of a galaxy much smaller than the Milky Way, but provides a view on its galactic nucleus before the formation of its NSC. Indeed, while we know the current morphology and mass distribu- tion of the Galactic NSC, the larger-scale properties of the Milky Way’s nucleus (and its properties before NSC forma- tion) remain uncertain. Table 1. Star clusters properties and mass deposited ID MGC MGC(< 10 pc)/MGC (%) 106 M⊙ S1 S2 S3 1 2.29 54.6 54.6 66.4 2 0.92 42.8 44.8 42.9 3 1.14 15.3 38.6 31.3 4 0.91 47.4 55.3 79.2 5 0.40 0.02 33.4 0 6 0.40 0 37.9 0 7 0.46 0 0 0 8 0.45 1.1 0 1.0 9 0.20 0 40.8 0 10 0.45 0 57.1 0 11 0.20 0 41.8 0 Col. 1: GC identiﬁcation number. Col. 2: GC mass. Col 3-6: per- centage of the GC mass deposited within 10 pc from the SMBH for models S1, S2, and S3. Table 1. Star clusters properties and mass deposited The formation of an NSC leads to a signiﬁcant enhance- ment of the central density slope (Merritt 2006; Antonini et al. 2012; Arca-Sedda et al. 2015). Before this took place, the stellar distribution in the Galactic centre could have been diﬀerent from today. The minimum negative radial density exponent required to achieve a self-consistent dis- tribution around a SMBH is γ = 0.5, which is the choice assumed here (Merritt 2006, 2013a). Col. 1: GC identiﬁcation number. Col. 2: GC mass. Col 3-6: per- centage of the GC mass deposited within 10 pc from the SMBH for models S1, S2, and S3. Our galaxy model is a truncated Dehnen sphere (Dehnen 1993; Arca-Sedda et al. 2015): ρD(r) = (3 −γ)Mg 4πr3g r rg −γ 1 + r rg −4+γ 1 cosh(r/rcut), (1) Table 2. Masses and sizes of the NSCs formed in the simulations. Table 2. Masses and sizes of the NSCs formed in the simulations. model MNSC rNSC 106 M⊙ pc S1 4.7 4.2 S2 6.0 2.6 S3 5.1 2.0 Col. 1: model name. Col. 2: NSC mass. Col. 3: NSC eﬀective radius. The ﬁnal NSC angular momentum is a fraction of the sum of all the merging GCs’ angular momenta, which are partly erased by the action of dynamical friction. For ini- tially co-planar orbits, GCs momenta are parallel and have the same sign, thus leading to a rotating NSC. If GCs move on random orientation orbits, the mergers lead to a partial cancellation of the angular momentum and produce a slowly rotating NSC.3 Col. 1: model name. Col. 2: NSC mass. Col. 3: NSC eﬀective radius. According to the observed properties of the Galactic NSC, we assumed MMWNSC = 2.5 × 107 M⊙and rMWNSC = 4.2 pc (Sch¨odel et al. 2014, 2017). The recent discovery of a population of massive young clusters orbiting in a disc conﬁguration (Nguyen et al. 2014) also motivates the S2 model, which allows us to investi- gate the possibility of NSC formation by GCs formed in the Galactic disk. Finally, comparing models S1 and S3 allows us to highlight the role played by the central SMBH in shap- ing the nuclear cluster’s properties and its stellar content. We refer the reader to Arca-Sedda et al. (2015) for further details on the GCs’ initial conditions and orbital properties. As dynamical friction drags the star clusters toward the Galactic centre, they lose stars from their outskirts due to tidally-enhanced evaporation. Table 1 shows the fraction of GC mass deposited within the inner 10 pc for the three models investigated. According to Table 1, we ﬁnd that ∼ 20 −30% of the mass of the most massive clusters (1-4) is deposited into the NSC. After the NSC formation, nearly 60 −70% of the to- tal GC mass is deposited in the innermost 20 pc, 20% is dispersed between 20-60 pc and the remaining mass is dis- persed around the Galactic centre, up to 100 pc and be- yond. Since the numerical costs limited our simulations to within the innermost 200 pc region of the Galactic bulge, we cannot model the mass deposited from massive GCs falling in from larger distances. 2 NUMERICAL MODEL The central SMBH is modelled with a point-like particle with mass MBH = 2.6 × 106 M⊙, in agreement with the observational estimates in Henize 2-10 (Reines et al. 2011; Reines & Deller 2012; Reines et al. 2016). Note that this value is quite similar to the Sgr A* mass, 3.6+0.2 −0.4 × 106 M⊙ (Ghez et al. 2008; Gillessen et al. 2009; Sch¨odel et al. 2009), making the Henize 2-10 nucleus models capable of testing the dry-merger origin for the Milky Way’s NSC. In order to investigate how the GCs’ initial conditions can aﬀect the gamma-ray emission proﬁle, we used the set of direct N-body simulations presented in Arca-Sedda et al. (2015), which was used to model the long-term evolution of the galaxy Henize 2-10. This dwarf starburst galaxy is an excellent observational target to study NSC formation, as it contains 11 massive star clusters with masses in the range (0.2−2)×106 M⊙(Nguyen et al. 2014), orbiting at distances ≲200 pc around an SMBH with mass 2.6× 106 M⊙(Reines et al. 2011). In the rest of the paper we do not distinguish young massive star clusters and globular clusters and refer to both as GCs. These N-body simulations conﬁrmed that the star clusters likely segregate to the Galactic centre rapidly on very short timescales, ∼100 Myr, leading to the formation of a dense NSC with mass ≃(4 −6) × 106 M⊙depending on the star clusters’ initial conditions (see Table 2) (Arca- Sedda et al. 2015). We list the masses of the assumed GCs in Table 1. We analysed three diﬀerent initial conditions for the geometry of the distribution of the globular cluster and the presence of the SMBH: (i) model S1, in which the number density distribution of clusters were assumed to follow the distribution of the background galaxy, (ii) model S2, corresponding to the assumption that the clusters are all initially located in the same plane and co- rotate, and (iii) model S3, where clusters have the same initial con- ditions as in model S1, but in this case the galaxy does not contain any central SMBH. Models S1 and S2 represent two limiting cases. While in S1 the star clusters are distributed in phase space accord- ing to the Galactic background, in S2 they are distributed over the same plane and have distances from the central Arca-Sedda, M. and Kocsis, B. and Brandt, T. D. Table 2. Masses and sizes of the NSCs formed in the simulations. However, our simulations indicate that a signiﬁcant fraction of the emission produced by MSPs comes from an extended region in the Galactic bulge, rather then from the central NSC, in agreement with observations (Abazajian et al. 2014; Daylan et al. 2016). We ﬁnd that a well detectable central NSC forms in all of the simulations within ∼15 −80 Myr mainly due to the merger of the four most massive clusters. The NSC mass and sizes are summa- rized in Table 2. In order to obtain a reasonable balance between com- putational load and the resolution of our cluster models, we allowed a diﬀerence between the mass of cluster stars, mc, and that of galaxy stars, mg, assuming mg/mc = 8. We ran several tests in order to ensure that the results are not aﬀected by such a choice. The simulations have been carried out using the HiGPUs code (Capuzzo-Dolcetta et al. 2013), a 6th order Hermite integrator with block time-steps that runs on hybrid GPU- CPU platforms. In all the simulations, we set a softening parameter ǫ = 0.02 pc in order to smooth strong gravita- tional interactions. The newly-born NSC contains both “cluster stars,” dragged into the galactic centre from the infalling clusters, and “galaxy stars,” which were already present in the in- ner galaxy and remained trapped within the NSC during its assembly. 2 NUMERICAL MODEL 4 SMBH smaller than 150 pc. The recent ﬁnding of a sig- niﬁcant rotation in the Galactic nuclear cluster (Feldmeier et al. 2014) and its possible connection to the dry-merger scenario (Tsatsi et al. 2017) indicates that the star clusters that contributed to its assembly likely included initial or- bital properties in between models S1 and S2. Gamma-ray and X-ray emission from the Galactic centre 5 0 0.05 0.1 0.15 0.2 0.25 0.1 1 10 N/NNSC v/vsca vsca = 14.7 km s-1 Figure 1. Velocity distribution of stars orbiting within 10 pc. The vertical line represent the mean (straight black line) and the boundaries delimited adding to this value ±2σ (dotted black lines). 0 0.05 0.1 0.15 0.2 0.25 0.1 1 10 N/NNSC v/vsca vsca = 14.7 km s-1 Table 3. NSC surface density parameters model Σ0 R0 ζ M⊙pc−2 pc S1 0.28 ± 0.01 2.1 ± 0.3 0.59 ± 0.10 S2 0.34 ± 0.01 4.1 ± 1.1 0.88 ± 0.26 S3 2.18 ± 0.08 1.2 ± 0.1 0.82 ± 0.06 Col. 1: model name. Col. 2: central surface density. Col. 3: scale radius. Col. 4: surface density slope. Table 3. NSC surface density parameters Col. 1: model name. Col. 2: central surface density. Col. 3: scale radius. Col. 4: surface density slope. The best ﬁt parameters, listed in Table 3, of models S1 and S2 in which an SMBH is present, are in remarkably good agreement with earlier numerical calculations tailored to the Milky Way nucleus (Antonini et al. 2012). However, our cal- culations are characterised by a slightly larger central sur- face density, due to the fact that we are rescaling our models to an NSC 1.5 times heavier than in Antonini et al. (2012). Assuming that the mass and luminosity proﬁles follow the same behaviour, we found that our ζ are compatible with the best-ﬁtting observational estimates, which lie in the range 0.3 −0.8 (Sch¨odel et al. 2014). We ﬁnd further agreement with Antonini et al. (2012) in terms of the 3D density pro- ﬁle. For instance, using a modiﬁed Hubble law (Rood et al. 1972; Antonini et al. 2012), Figure 1. Velocity distribution of stars orbiting within 10 pc. The vertical line represent the mean (straight black line) and the boundaries delimited adding to this value ±2σ (dotted black lines). NSC (which is really just a power-law distribution of stellar density) remains somewhat fuzzy, and the fraction of NSC members that originated in GCs depends on this boundary. ρ(r) = ρ0 " 1 + r r0 2#−1.5 , (5) For instance, in simulation S1 the NSC, as observed in the global surface density proﬁle, consists of a clear over- density extending up to 10 pc characterized by an eﬀective radius Re ≃4.2 pc. 2.1.3 Relaxation timescale Another important parameter to consider in converting the Henize 2-10 galaxy simulations to a Milky Way model is the time scale. The long-term evolution of gravitating systems is generally driven by two-body encounters over a relaxation time-scale (Spitzer 1987) Gamma-ray and X-ray emission from the Galactic centre 5 The mass enclosed within a 10 pc ra- dius around the SMBH is 4.7 × 106 M⊙, as shown in Ta- ble 2, but the GC debris mass deposited inside of 10 pc is ≃2.3 × 106 M⊙. Therefore, in this case the orbitally segre- gated GCs represent 48% of the total NSC mass. The other 52% of the mass is “galaxy stars” in our terminology: bona ﬁde NSC members that were born outside of GCs. (5) the best ﬁt parameters in model S1 are ρ0 = (8.2 ± 0.4) × 104 M⊙pc−3 and r0 = 3.3 ± 0.4 pc. The kinematics of the cluster are also in agreement with observational and numerical estimates, as our NSC models are characterized by a radial velocity dispersion of ∼100 km s−1 at 1 pc from the SMBH. For instance, Figure 3 shows the line-of-sight (LOS) ve- locity proﬁle for our model S1, rescaled to the MW nucleus. The proﬁle that we show is the average of the proﬁles com- puted along the x, y, and z reference axes in our model (the actual observed LOS velocity proﬁle depends on the location of an observer in the Galaxy). More reﬁned meth- ods of inferring the LOS velocity proﬁle exist, for example the kinemetric approach (Krajnovi´c et al. 2006). However, our calculations of the high-energy emission from the inner Galaxy depend only weakly on the NSC kinematics, and our averaged LOS velocity proﬁle agrees well with the observa- tional results of Feldmeier et al. (2014) (c.f. their Figure 13). We can verify whether our “galaxy stars” and former GC stars are bound to the NSC by calculating the velocity distribution within the NSC radius (r ≲10 pc), and labeling as “contaminants” the stars having a velocity larger than a given threshold. We adopt as our contaminants stars with v > 2σ, where σ is the velocity dispersion; at these velocities the stars can travel far from the NSC. Figure 1 shows that only 15% of the “galaxy stars” orbiting in the inner 10 pc have a velocity larger than the threshold. The rest are bona ﬁde NSC members. These simulations conﬁrmed that a dense NSC can form on a very short timescale from clusters that sink in from the inner ≲200 pc of the Galactic bulge. Next, we discuss the strengths and limitations of the rescaling procedure. 2.1.1 Adapting the N-body model to the Milky Way nucleus 2.1.1 Adapting the N-body model to the Milky Way nucleus Our simulations were originally tailored to the Henize 2- 10 galaxy, and followed the evolution of 11 young massive clusters with masses in the range (0.2 −2.6) × 106 M⊙ (Nguyen et al. 2014), orbiting around an SMBH with mass MBH = 2.6 × 106 M⊙(Reines et al. 2011, 2016). Due to its nature, N-body modelling can be easily adapted to diﬀerent systems by simply rescaling the particle mass and positions, and by changing the velocity- and time-scales accordingly. In order to rescale the simulation results to the Milky Way, we rescale the masses and positions to match the observed total mass and eﬀective radius We stress that this deﬁnition of “galaxy stars” diﬀers from the typical observer’s deﬁnition when studying the NSC. Most observers use “galaxy stars” to refer to inter- lopers: stars whose projected positions place them in the NSC, but that in reality are foreground stars very far away from the inner few pc. Our use of the term refers to bona ﬁde members of the NSC that were not initally members of one of the infalling GCs. By tagging each star, we can trace its full dynamical history and attribute it to a source population. Our deﬁnition of the NSC’s size and mass follows Arca- Sedda et al. (2015); we select as NSC members all stars moving inside the “bump” observed in the surface density proﬁle. This is simpler than the typical observational ap- proach. To extract the NSC’s properties, observers usually ﬁt the observed surface brightness with a combination of known proﬁles, like S´ersic and Core-S´ersic (Cˆot´e et al. 2006; Turner et al. 2012), and use these to infer the mass and density proﬁle. In our case the surface density bump pro- vides a rough estimate of NSC size, but the boundary of the mi →MMWNSC MNSC × mi , (2) ri →rMWNSC rNSC × ri . (3) mi →MMWNSC MNSC × mi , (2) ri →rMWNSC rNSC × ri . (3) (2) (3) 3 Clearly, an exactly null angular momentum can be achieved only in special conﬁguration. 5 2.1.2 Surface density and velocity dispersion Figure 2 shows the contribution of star clusters and back- ground to the total surface density proﬁle of the Galactic Centre in our rescaled simulations. It is evident that in all the cases the GCs’ debris dominates over the initial galaxy density in the inner 10 pc. The NSC component can be well described by a simple relation tr = 0.33σ3 G2ρmeﬀln Λ, (6) (6) where σ is the one-dimensional velocity dispersion, ρ is the mean stellar density, meﬀ= ⟨m2⟩/⟨m⟩is the so-called eﬀec- tive mass, deﬁned as the ratio of the mean-squared stellar mass to the mean stellar mass, Σ(R) = Σ0 1 + R R0 −ζ . (4) (4) Arca-Sedda, M. and Kocsis, B. and Brandt, T. D. 6 1000 10000 100000 1e+06 0.001 0.01 0.1 Σ (M⊙ pc-2) R (kpc) S1 GCs+Gal GCs Gal 1000 10000 100000 1e+06 0.001 0.01 0.1 Σ (M⊙ pc-2) R (kpc) S2 GCs+Gal GCs Gal 1000 10000 100000 1e+06 1e+07 0.001 0.01 0.1 Σ (M⊙ pc-2) R (kpc) S3 GCs+Gal GCs Gal 1000 10000 100000 1e+06 1e+07 0.001 0.01 0.1 Σ(R) (M⊙ pc-2) R (kpc) S1 S2 S3 Figure 2. Present day surface density proﬁle for diﬀerent initial conditions shown in Table 3: spherical GC distribution (top left panel, model S1), disk like distribution of GCs (top right panel, model S2), and spherical GC distribution without a SMBH (bottom left panel, model S3). The solid red, blue dashed, and black dotted lines represent the total proﬁle, the star clusters’, and the background galaxy’s contributions, respectively. The bottom right panel represents a comparison between the overall surface densities for the three models investigated. 1000 10000 100000 1e+06 0.001 0.01 0.1 Σ (M⊙ pc-2) R (kpc) S2 GCs+Gal GCs Gal 1000 10000 100000 1e+06 0.001 0.01 0.1 Σ (M⊙ pc-2) R (kpc) S1 GCs+Gal GCs Gal 1000 10000 100000 1e+06 1e+07 0.001 0.01 0.1 Σ (M⊙ pc-2) R (kpc) S3 GCs+Gal GCs Gal 1000 10000 100000 1e+06 1e+07 0.001 0.01 0.1 Σ(R) (M⊙ pc-2) R (kpc) S1 S2 S3 Figure 2. Present day surface density proﬁle for diﬀerent initial conditions shown in Table 3: spherical GC distribution (top left panel, model S1), disk like distribution of GCs (top right panel, model S2), and spherical GC distribution without a SMBH (bottom left panel, model S3). 2.1.2 Surface density and velocity dispersion The solid red, blue dashed, and black dotted lines represent the total proﬁle, the star clusters’, and the background galaxy’s contributions, respectively. The bottom right panel represents a comparison between the overall surface densities for the three models investigated. -100 -50 0 50 100 -4 -2 0 2 4 Vlos (km s-1) Rlos (pc) Feldmeier+(2014) Figure 3. Line-of-sight velocity proﬁle in our model S1 (red ﬁlled squares) compared with observed values provided by Feldmeier et al. (2014) (blue crosses). -100 -50 0 50 100 -4 -2 0 2 4 Vlos (km s-1) Rlos (pc) Feldmeier+(2014) an eﬀective mass of meﬀ∼1 M⊙, the relaxation time at the Sgr A* inﬂuence radius is ∼20 −30 Gyr (Merritt 2010). In direct N-body simulations, the relaxation time is reduced by a factor msim/m∗due to the smaller number of particles as compared to real stellar systems, where msim and m∗are the simulated and actual single stellar masses, respectively. Therefore, in order to mimic the NSC’s long-term evolution, we carried out our simulations up to a fraction msim/m∗of the observed relaxation time-scale. These approximations, widely used in the ﬁeld of numerical simulations, alleviate the large N problem (N ≳108 in reality, see for instance Antonini et al. 2012; Antonini 2013; Perets & Mastrobuono- Battisti 2014; Tsatsi et al. 2017). Upon this approximation, we selected the snapshot corresponding to 12 Gyr to perform our analysis. Figure 3. Line-of-sight velocity proﬁle in our model S1 (red ﬁlled squares) compared with observed values provided by Feldmeier et al. (2014) (blue crosses). 2.1.4 Mass segregation The other important process to take into account is the possible imprint of mass segregation in the observational properties. Indeed, the MSP and CV progenitor stars may have already undergone mass segregation in their parent GC when they reach the Galactic Centre, possibly aﬀecting their distribution within the NSC after formation. The GCs’ in- fall is regulated by dynamical friction, whose timescale de- and ln Λ is the Coulomb logarithm. The SMBH’s gravity dominates the dynamics within its inﬂuence radius rinﬂ= GMBH/σ2. Within this region, ln Λ = ln(rinﬂσ2/2G⟨m⟩) (Merritt 2013a,b). After rescaling the simulations with Equations (2–3), our system is characterized by an inﬂuence radius equal to the Milky Way’s estimated value. Assuming and ln Λ is the Coulomb logarithm. The SMBH’s gravity dominates the dynamics within its inﬂuence radius rinﬂ= GMBH/σ2. Within this region, ln Λ = ln(rinﬂσ2/2G⟨m⟩) (Merritt 2013a,b). After rescaling the simulations with Equations (2–3), our system is characterized by an inﬂuence radius equal to the Milky Way’s estimated value. Assuming Gamma-ray and X-ray emission from the Galactic centre 7 pends on the mass of the GC as ⟨m⟩/mGC. Similarly, mass segregation in dense clusters also operates in a fraction of the relaxation time-scale (Spitzer 1940; Portegies Zwart & McMillan 2002; Arca-Sedda 2016) other hand, the diﬀerences in the star clusters’ initial con- ditions (models S1 and S2) have only a weak impact on the ﬁnal matter distribution. Beyond ∼3 pc the three proﬁles become almost indistinguishable, due to the fact that above this length scale the dominant contribution comes from the background galaxy in this model.4 These ﬁndings are con- sistent with previous models of the Milky Way NSC by An- tonini et al. (2012). In particular, our surface density proﬁle matches their Figure 4 in both the central surface density and the NSC eﬀective radius. tseg = 0.65Gyr ln(0.4M/⟨m⟩) M 105 M⊙ 1/2 1 M⊙ ⟨m⟩ rh 1pc 3/2 , (7) (7) ( ) where ⟨m⟩is the mean stellar mass and rh is the cluster half-mass radius. Since tseg ∝M 0.5 while tdf ∝M −0.67 (Arca-Sedda et al. 2015), the lighter the cluster the higher the probability that it reaches the galactic centre in a mass- segregated state. This is an oversimpliﬁcation of the prob- lem, since the segregation process depends mainly on the in- ternal properties of the cluster, e.g. 2.1.4 Mass segregation core radius, metallicity, mass function, while the cluster infall depends on its orbital properties and the host galaxy structure. In our simulations we cannot account for this eﬀect self-consistently, as we used single mass models for both the clusters and the background galaxy and our mass resolution is much larger than 1 M⊙. As we will discuss in detail in Section 3.2, in order to allevi- ate our blindness of the MSPs and CVs formation history, in our calculations we assume that these sources at formation are either segregated into the parent GC core or completely unsegregated. Since the mixing time in globular clusters is of order 10 times the relaxation time (Meiron & Kocsis 2018) and the outer stars are stripped more easily from GCs, the initial conditions of MSPs and CVs within GCs aﬀects their ﬁnal distribution in the Galactic bulge. The absence of signiﬁcant diﬀerences between the den- sity proﬁles on length scales larger than 10-100 pc could im- ply that the GC initial conditions do not play a signiﬁcant role as far as the gamma ray emission is concerned, which is observed with Fermi with a poorer resolution. The radial proﬁle of hard X-ray emission with NuSTAR has a better resolution of the Galactic Centre, which might oﬀer further constraints on the GC initial conditions. The NuSTAR satel- lite has an angular resolution of 58′′ (HPD, corresponding to 2.2 pc at 8 kpc), a FWHM equal to 18′′ (0.7 pc) and a ﬁeld of view of 6′ (14 pc).5 In the next sections we investi- gate whether the level of mass segregation in the infalling clusters and the presence of the SMBH may alter the ﬁnal distribution of both MSPs and CVs in the very inner region of the Galaxy. 3.1.1 Number of MSPs in the NSC We determine the number of MSPs based on the following phenomenological parameters. • µGCi: the initial fraction of mass in the ith GC in the Galactic bulge compared to the total Galactic bulge mass. • µNSC,GC: the ﬁnal fraction of mass in the NSC com- prised of GC debris. • µNSC,G: the fraction of mass in the NSC already present in the Galactic centre before NSC formation. We assume that µNSC,GC + µNSC,G = 1. • νMSP: the number of MSPs per unit mass in GCs, νMSP = NMSP/MGC. • δi: the ﬁnal fraction of the ith GC mass in the bulge that makes it to the NSC. The rest 1 −δi represents the ﬁnal fraction of GC mass deposited in the bulge due to GC evaporation and tidal disruption of GCs. Thus µNSC,GC = P i δiµGCi. 3.1 The expected number of MSPs and CVs in the infall scenario In this section we provide a crude estimate of the number of MSPs and CVs expected to be found in the Galactic Centre under the hypothesis that the NSC formed by repeated GC infall. 2.2 Selecting MSP and CV candidates in N-body modelling Our N-body simulations are based on single-mass particle representations of both the infalling GCs and the galaxy nu- cleus, while stellar evolution and binary formation are not treated in any way. The number of particles used is suﬃ- ciently high to resolve the stellar distribution in the star clusters’ cores and in the SMBH surroundings, thus provid- ing a statistical basis suﬃciently robust to measure the evo- lution of stellar orbits. We select and label particles as MSP or CV candidates, as discussed in 3.1 and 3.2, and follow their evolution from their initial position within the parent cluster up to their ﬁnal position in the Galactic Centre af- ter the NSC formation. The selection procedure accounts for two important quantities: (1) the fraction of stars that can form an MSP or CV in a massive GC; and (2) the level of mass segregation of the parent GC when it impacts the SMBH. We discuss these aspects in Sections 3.1 and 3.2, respectively. 4 Note that it may come from a large number of primordial GCs that sink to this region from a kpc distance, modelled as a Galac- tic contribution here (Brandt & Kocsis 2015). 5 https://heasarc.gsfc.nasa.gov/docs/nustar/nustar_tech_desc.html. 3 RESULTS AND DISCUSSION i • ηMSP: the ratio between the initial mass in MSPs in the Galactic ﬁeld and that in GCs. Since MSP formation is correlated with the dynamical encounter rate (Bahramian In this section we use our rescaled N-body results to in- vestigate the role of the GCs’ infall in the production of high-energy emission from the Galactic Centre. The bot- tom panel of Figure 2 compares the NSC surface density proﬁles for our three simulations. The most prominent dif- ference between the models is that a central SMBH strongly aﬀects the inner mass distribution: with no SMBH (model S3), the central density is up to ﬁve times larger. On the 4 Note that it may come from a large number of primordial GCs that sink to this region from a kpc distance, modelled as a Galac- tic contribution here (Brandt & Kocsis 2015). 8 Arca Sedda M and Kocsis B and Brandt T D 8 Arca-Sedda, M. and Kocsis, B. and Brandt, T. D. et al. 2013; Hui et al. 2010), the formation eﬃciency in the Galactic ﬁeld is smaller than in GCs, ηMSP < 1. We use the same δi, µGCi and µNSC,G measured from the simulation as for the MSPs. The parameter values in Equation (9) are summarized in Table 5 (see bottom row for IPs). According to Equation (9), the total number of IPs formed in the infalling clusters is NIP,tot = 5994. After the NSC build-up, the expected number of IPs, solely coming from the infalling clusters and deposited inside the inner 10 pc, is NIP = 1823. Given that the Galaxy’s NSC eﬀective radius is rNSC ∼4.2 pc (Sch¨odel et al. 2014) and its ra- dial inner slope is γNSC ≃1 −2 (Sch¨odel et al. 2014), we can calculate the IPs’ mean density assuming that the NSC distribution follows a powerlaw nIP ∝r−γ. This leads to nIP = 0.35−1.6 pc−3. In the next section, we will show that this rough estimate agrees with the IPs’ simulated radial dis- tribution (Figure 6), and it is well below the upper bound on the IP density inferred from observations (nobs ≃1 −3 pc−3, Heard & Warwick 2013; Perez et al. 2015; Hailey et al. 2016). Thus, the simulated number of IPs within 150 pc is consistent with the observationally inferred values within the theoretical uncertainties. 3 RESULTS AND DISCUSSION Thus, we do not expect a signiﬁcant variation in NMSP0. We get νMSP = 775/(8.6 × 106 M⊙) = 9 × 10−5 MSP M−1 ⊙. We measure δi, µGCi and µNSC,G from the simulation. 3.2 Mock catalog of gamma and X-ray sources in N-body simulations 3.2 Mock catalog of gamma and X-ray sources in N-body simulations The main parameter values used in the above calcu- lations are summarized in Table 5 (see top row for CVs). Assuming ηMSP = 0 and substituting the parameters in Abdo et al. (2010) and Table 4 into Equation (8), we obtain NMSP = 1000–1200 within 10 pc from Sgr A, a number in good agreement with semi-analytic calculations and nu- merical modelling presented in the literature (Bednarek & Sobczak 2013; Brandt & Kocsis 2015; Abbate et al. 2017). We run calculations with ηMSP = 0, 0.01, and 0.1. Here ηMSP ∼0.01 is compatible with observational evidence of a much smaller number of MSPs per unit mass in the Galactic ﬁeld, up to ∼100 times that in GCs (Grindlay & Bogdanov 2009). In the following we combine the calculation performed to ob- tain the expected number of MSPs and CVs in our modelled clusters with the data provided by the numerical simulations to study the shape and characteristics of the γ and X-ray emission expected from the Galactic centre. Our N-body models have a suﬃciently large number of particles to ensure a reliable selection of source candidates. In the following, we will focus on model S2, due to the ab- sence of big diﬀerences between S1 and S2 surface density proﬁles which represent spherical and planar initial GC dis- tributions respectively (see Figure 2). We will use models S1 and S3 in the next sections to highlight the role played by the central SMBH. 3 RESULTS AND DISCUSSION Further, we show in the next section, that the surface density and X-ray ﬂux found in our simulations are consistent with observations, suggesting that a dry merger origin of the Galactic NSC is viable to explain the origin of the large population of MSPs and CVs that generate the observed gamma and X-ray emission in the Galactic centre. et al. 2013; Hui et al. 2010), the formation eﬃciency in the Galactic ﬁeld is smaller than in GCs, ηMSP < 1. With these parameters, the total number of MSPs in the NSC can be expressed as NMSP = νMSP ηMSPµNSC,G + X i δiµGCi ! MNSC, (8) where the sum is over all GCs in the Galactic bulge. We determine the parameters as follows. p We follow Abdo et al. (2010) to estimate νMSP using Fermi observations of the gamma-ray ﬂux for ten old and massive GCs, which yields the enclosed number of MSPs, NMSP0. Table 4 summarizes NMSP0 and the mass of the host GCs, derived from literature. Our GC models have similar GC masses, and therefore assume similar numbers of MSPs. The Abdo et al. (2010) sample is comprised of old GCs, with ages ∼10 Gyr. Hence, NMSP0 represents the lower limit of MSP progenitors. Neutron stars form over a time-scale ∼10 −100 Myr, with a substantial fraction of them being ejected due to supernovae kicks. On the contrary, MSPs form after a NS is captured in a binary system and the binary is hardened by dynamical encounters to be spun up into a MSP. This typical time is ∼1 Gyr, larger than the time- scale for NS formation and ejection. Thus, we do not expect a signiﬁcant variation in NMSP0. We get νMSP = 775/(8.6 × 106 M⊙) = 9 × 10−5 MSP M−1 ⊙. We measure δi, µGCi and µNSC,G from the simulation. The Abdo et al. (2010) sample is comprised of old GCs, with ages ∼10 Gyr. Hence, NMSP0 represents the lower limit of MSP progenitors. Neutron stars form over a time-scale ∼10 −100 Myr, with a substantial fraction of them being ejected due to supernovae kicks. On the contrary, MSPs form after a NS is captured in a binary system and the binary is hardened by dynamical encounters to be spun up into a MSP. This typical time is ∼1 Gyr, larger than the time- scale for NS formation and ejection. Table 5. Main parameters for MSPs and IPs number calculation source ν ( M−1 ⊙) f δ µGCS η µNSC,G Nsrc MSPs 9 × 10−5 − 0.5 1.3 0.0 0.5 1911 IPs 1.92 × 10−3 0.08 0.5 1.3 0.0 0.5 3255 Main parameters for MSPs and IPs number calculation Main parameters for MSPs and IPs number calculation Table 6. MSP and CV distributions in our simulations fs l η Nsrc Rc MSPa 1 1 0 2254 MSPb 1 1 0.01 2758 MSPc 0.5 1 0 2254 MSPd 0.5 1 0.1 6953 MSPe 0.5 0.2 0 2254 MSPf 1 103 0 2254 CVa 1 1 0 5994 CVb 1 1 0.01 59577 CVc 1 103 0 5994 CVd 1 103 0.01 59577 CVe 1 103 0.002 16710 Col 1. Model name. Col. 2: fraction of sources within l times the cluster core radius, Rc. Col. 3: radius, in unit of the cluster core radius, within a fraction fs of the sources is enclosed; 103 means that sources are distributed wherever inside the cluster tidal radius. Col. 4: eﬃciency factor as deﬁned in Eqs. 8 and 9. Col. 5: total number of sources. For models with η > 0 (including sources formed in the Galactic ﬁeld), the numbers show sources within the modelled region of the bulge, r < 150 pc. Table 6. MSP and CV distributions in our simulations fs l η Nsrc Rc MSPa 1 1 0 2254 MSPb 1 1 0.01 2758 MSPc 0.5 1 0 2254 MSPd 0.5 1 0.1 6953 MSPe 0.5 0.2 0 2254 MSPf 1 103 0 2254 CVa 1 1 0 5994 CVb 1 1 0.01 59577 CVc 1 103 0 5994 CVd 1 103 0.01 59577 CVe 1 103 0.002 16710 Table 6. MSP and CV distributions in our simulations merical code does not implement any treatment for stellar binaries or tight multiple systems. Table 6 shows our as- sumptions on the MSP and CV populations. Our approach assumes that the MSP and CV populations do not mix with the bulk of the cluster population after they have mass- segregated. Meiron & Kocsis (2018) showed that the mixing time of objects in the cluster is about 10 relaxation times, which is typically larger than the timescale on which the clusters are stripped by galactic tides. We generated ﬁve diﬀerent models, characterised by diﬀerent levels of segregation for MSPs and CVs. Table 5. In these models we also varied the eﬃciency factors ηMSP and ηCV, deﬁned in Eqs. (8) and (9), in order to outline the role of sources born in the Galactic disk. For instance, models MSPa/CVa and MSPb/CVb refer to a population of MSPs and CVs conﬁned initially to the cores of their parent clus- ters. The contribution formed in the Galactic centre is set to ηMSP = ηCV = 0 in MSPa/CVb and 1% in MSPb/CVb. In models MSPc/CVc, MSPd/CVd, MSPe/CVe we set the fraction of sources in the core to be fs = 50%. Model MSPf/CVf represents an unsegregated populations of MSPs and CVs. Col 1. Model name. Col. 2: fraction of sources within l times the cluster core radius, Rc. Col. 3: radius, in unit of the cluster core radius, within a fraction fs of the sources is enclosed; 103 means that sources are distributed wherever inside the cluster tidal radius. Col. 4: eﬃciency factor as deﬁned in Eqs. 8 and 9. Col. 5: total number of sources. For models with η > 0 (including sources formed in the Galactic ﬁeld), the numbers show sources within the modelled region of the bulge, r < 150 pc. Col 1. Model name. Col. 2: fraction of sources within l times the cluster core radius, Rc. Col. 3: radius, in unit of the cluster core radius, within a fraction fs of the sources is enclosed; 103 means that sources are distributed wherever inside the cluster tidal radius. Col. 4: eﬃciency factor as deﬁned in Eqs. 8 and 9. Col. 5: total number of sources. For models with η > 0 (including sources formed in the Galactic ﬁeld), the numbers show sources within the modelled region of the bulge, r < 150 pc. For reference, models CVa refers to a completely segre- gated population of CVs in their parent clusters, while the contribution coming from CVs formed in the Galactic bulge outside of GCs is set to zero. Moreover, Table 6 summa- rizes our choices fot the Galactic ﬁeld contribution, number of sources and fraction of sources contained within a given fraction of the cluster core radius. where mMSP is the MSP mass, F2GeV the GC observed ﬂux, MGC its mass and D its distance from the observer. Following Abdo et al. (2010), we found L2GeV ∼4 × 1035 GeV cm−2 s−1 per MSP. Table 5. Figure 4 shows the observed ﬂux in all the conﬁgura- tions tested as a function of radius and Figure 5 shows the 2D surface map of the gamma-ray intensity emitted by the Galaxy’s NSC in all the models investigated. Diﬀerent lines in Figure 4 show diﬀerent assumptions on the initial inter- nal distribution of MSPs within the globular clusters and the initial number of MSPs outside of globular clusters in the Galaxy as shown by Table 6. We ﬁnd that many of these models are in tension or inconsistent with observations. In particular, if the initial fraction of MSPs in the Galaxy is 3.1.2 Number of CVs in the NSC In each cluster, we randomly selected νjMGCj particles, where the subscript j refers either to MSPs or CVs. The number of sources for each cluster, rescaled to the Milky Way nucleus, is obtained through Equations (8) and (9). All particles in our simulation have the same mass, so any treatment of mass segregation of the MSPs and CVs can only be done in postprocessing. We qualitatively account for this eﬀect by preferentially selecting tracer particles within or beyond a given radius from the cluster center. We ﬁrst ﬁx a radius l in units of the core radius, and then vary the fraction of MSPs and CVs tracing the mass inside (fs) and outside (1 −fs) of l. For example, fs = 0.5 and l = 1 corresponds to half of the MSPs and CVs tracing mass in the core and the other half outside the core, while fs = 1 and l = ∞corresponds to all MSPs and CVs tracing the cluster’s stellar mass. With fs = 1 and l = 1, all of the MSPs and CVs are assumed to have mass-segregated into the cluster’s core. We use a simple phenomenological model to derive the num- ber of IPs in the Milky Way’s NSC, which may be respon- sible for the hard X-ray emission observed in the Galactic Centre: NIP = νCVfIP ηCVµNSC,G + X i δiµGCi ! MNSC. (9) (9) Here, fIP denotes the fraction of IPs among all CVs. Based on observations of the ROSAT Bright Survey, Pretorius et al. (2013) estimated that a fraction fmCV = 0.2 of all CVs in the Solar neighbourhood are magnetic, and about 40% of the local magnetic CVs are IPs. Thus, we assume that fIP = 0.4fmCV = 0.08. Further Ivanova et al. (2006) used Monte-Carlo models of star clusters and found the formation of NCVo = 2490 CVs in 13 massive GCs with lifetimes larger than 12 Gyr. The total mass of GCs was MGCo = 1.3 × 106 M⊙. Thus, we get, νCV = NCVo/MGCo = 1.92 × 10−3 CV M−1 ⊙. We do not account for strong encounters, as our nu- We do not account for strong encounters, as our nu- Gamma-ray and X-ray emission from the Galactic centre 9 9 Table 4. Number of expected MSPs in observed GCs. GC name NMSP0 MGC d F2 Gev ref. (106 M⊙) kpc (10−9 GeV cm−2 s−1) 47 Tuc 33 0.700 4.5 5.6 Marks & Kroupa (2010) ΩCen 19 1.500 5.2 2.8 Marks & Kroupa (2010) M 62 76 1.220 6.8 3.8 Boyles et al. (2011) NGC 6388 180 2.170 9.9 3.4 Boyles et al. (2011) Terzan 5 180 0.374 6.9 12.6 Boyles et al. (2011) NGC 6440 130 0.811 8.5 2.9 Boyles et al. (2011) M 28 43 0.551 5.5 3.8 Boyles et al. (2011) NGC 6541 47 0.572 7.5 0.9 Boyles et al. (2011) NGC 6752 11 0.140 4.0 0.5 Marks & Kroupa (2010) M 15 56 0.560 10.4 - Marks & Kroupa (2010) Col. 1 GC name. Expected number of MSPs (Abdo et al. 2010). Col. 3: GC mass. Col. 4: observed ﬂux at 2 GeV (Cholis et al. 2014). Col. 5: GCs distances (Harris 1996). Col. 6: reference for GC masses. Table 4. Number of expected MSPs in observed GCs. Col. 1 GC name. Expected number of MSPs (Abdo et al. 2010). Col. 3: GC mass. Col. 4: observed ﬂux at 2 GeV (Cholis et al. 2014). Col. 5: GCs distances (Harris 1996). Col. 6: reference for GC masses. umber of MSPs (Abdo et al. 2010). Col. 3: GC mass. Col. 4: observed ﬂux at 2 GeV (Cholis et al. 2014). 1996). Col. 6: reference for GC masses. Col. 1 GC name. Expected number of MSPs (Abdo et al. 2010). Col. 3: GC mass. Col. 4: observed ﬂux Col. 5: GCs distances (Harris 1996). Col. 6: reference for GC masses. 3.3 Comparison of observations with simulations The power emitted in the 2 GeV band from a single MSP Flux for diﬀerent MSP models for the initially disk-like distribution of globular clusters (model S2) and diﬀerent assump- tions on the initial distribution of MSPs within the cluster and the number of MSPs in the galactic ﬁeld. For the deﬁnition of models see Table 6. The large deviation for model MSPd is due to the assumed high Galactic ﬁeld MSP contribution N ∼4000 MSPs (η = 0.1). Note that the modelled region is meaningful within 150 pc (the sampling is exponentially truncated beyond 150 pc). The MSP population from infalling GCs is also underestimated further out due to the neglect of infalling GCs from outside of 200 pc. The black ﬁlled dots represent observed γ-excess reported by Brandt & Kocsis (2015). For instance, the observed gamma-ﬂux is well-ﬁt by model MSPc, which assumes partial mass-segregation within their parent clusters. However, in the extreme limit in which MSPs are completely unsegregated (model MSPf), the re- sulting ﬂux is much lower than observations. Our results sug- gest that MSPs were at least partly segregated when they reached the inner galactic regions. This has interesting implications on the dynamics of the parent clusters. Indeed, according to Equation 7, stars hav- ing a mass in the range ∼10 M⊙will segregate into the par- ent cluster centre in tseg ≃15 Myr, assuming a cluster mass of 106 M⊙. Given the similarity between the tseg and the NSC assembly time-scale, tNSC ≳100 Myr, we expect that the population of MSPs progenitors arrived at the Galactic Centre at least partially segregated, although tseg provides only a crude estimate of the actual segregation time-scale. -6 -4 -2 0 2 4 6 -6 -4 -2 0 2 4 6 y (arcmin) x (arcmin) -11.0 -10.5 -10.0 -9.5 -9.0 -8.5 -8.0 Log F2GeV (10-10 Gev cm-2 s-1 pc-2) Figure 5. Surface map of the 2 GeV intensity produced by the Milky Way’s NSC as calculated from our simulation S2, model MSPa. -6 -4 -2 0 2 4 6 -6 -4 -2 0 2 4 6 y (arcmin) x (arcmin) -11.0 -10.5 -10.0 -9.5 -9.0 -8.5 -8.0 Log F2GeV (10-10 Gev cm-2 s-1 pc-2) Similarly to the study of MSPs, we investigate the role of mass segregation and varying fraction of Galactic CVs using models listed in Table 6. Figure 6 shows the number density distribution of CVs and their averaged density per total volume ⟨nCV⟩. 3.3 Comparison of observations with simulations The power emitted in the 2 GeV band from a single MSP The power emitted in the 2 GeV band from a single MSP can be estimated as L2GeV = F2GeV(4πD2) MGC mMSP, (10) L2GeV = F2GeV(4πD2) MGC mMSP, (10) (10) Arca-Sedda, M. and Kocsis, B. and Brandt, T. D. 10 1e-09 1e-08 1e-07 0.001 0.01 0.1 F (GeV cm-2 s-1) R (kpc) MSPa MSPb MSPc MSPc1 MSPd MSPe MSPf Figure 4. Flux for diﬀerent MSP models for the initially disk-like distribution of globular clusters (model S2) and diﬀerent assump- tions on the initial distribution of MSPs within the cluster and the number of MSPs in the galactic ﬁeld. For the deﬁnition of models see Table 6. The large deviation for model MSPd is due to the assumed high Galactic ﬁeld MSP contribution N ∼4000 MSPs (η = 0.1). Note that the modelled region is meaningful within 150 pc (the sampling is exponentially truncated beyond 150 pc). The MSP population from infalling GCs is also underestimated further out due to the neglect of infalling GCs from outside of 200 pc. The black ﬁlled dots represent observed γ-excess reported by Brandt & Kocsis (2015). The best agreement between observations and the sim- ulations shown in Figure 4 is achieved by model MSPb, which is characterised by having all the MSPs contained within the core radius of their host clusters and having a very small contribution of sources formed in the Galactic nucleus, namely ηMSP = 0.01. Note that in this model 1912 MSPs were brought to the Galactic Centre by GC infall, while the progenitors of 503 MSPs were born in the Galactic Centre before the GC merging process occurred. Thus, our results suggest that before NSC formation the MSPs reside within the core radius of their host clusters and only ∼25% of the total number of MSPs currently in the Galaxy’s NSC have formed in the Galactic nucleus. However, note that sev- eral other models may also be statistically consistent due to the current level of large observational errors shown in Fig- ure 4 and due to the limitations of our models neglecting GCs initially further out. 1e-09 1e-08 1e-07 0.001 0.01 0.1 F (GeV cm-2 s-1) R (kpc) MSPa MSPb MSPc MSPc1 MSPd MSPe MSPf Figure 4. 3.3 Comparison of observations with simulations The power emitted in the 2 GeV band from a single MSP The diﬀerence between the IPs’ distri- bution indicates how the initial IP distribution aﬀects their ﬁnal density proﬁle after NSC formation. As expected, an initially unsegregated IP population is characterised by a less centrally concentrated distribution of IP population in the NSC. Note in Figure 6 that model CVe is characterised by a cored distribution inside 2 pc, while model CVa has a steeper distribution n(r) ∝r−γ, with slope γ ∼0.32 ± 0.03. Comparing models CVa and CVc initially without Galac- tic IPs with models CVd and CVe for which ηCV = 0.01 (see Table 6) shows that in the latter case the CV popu- lation formed in the Galactic centre dominates outside of ∼12 pc. However, within this radius GCs may deliver a dominant population of IPs. Hailey et al. (2016) derived an IP number density of ⟨nIP⟩∼2 −10 pc−3 in the inner 10 pc to match NuSTAR observations, while our simulation models predict an IP density ⟨nIP⟩∼0.2 pc−3, an order of magnitude smaller than the number observations suggest. Nevertheless, this discrepancy does not necessarily rule out the IP interpretation of the NuSTAR data. Indeed, we ar- gue that, the X-ray surface density proﬁle inferred from our simulations is compatible with observations, as well as the simulated X-ray ﬂux morphology is quite similar to the ob- served map. The discrepancy might be due to the strategy followed in Hailey et al. (2016) to infer the number of IPs Figure 5. Surface map of the 2 GeV intensity produced by the Milky Way’s NSC as calculated from our simulation S2, model MSPa. high ηMSP = 0.1 (Model MSPd), the simulation greatly over- predicts the gamma-ray ﬂux at 80 and 200 pc relative to the observed values. Further, an initially unsegregated popula- tion of MSPs in model MSPf produces a low ﬂux in the inner region of the Galaxy < 1 pc at the margin of the ob- servational error. If all the MSPs are contained within their host clusters’ core and the galaxy does not contribute to their population at all (model MSPa), the expected ﬂux is consistent with observations between 10 and ∼80 pc, while it is 7% smaller than the ﬂux observed at 150 pc. How- ever, we must stress here that our numerical model for the galactic bulge extends up to 150 pc at most, being exponen- tially truncated outward. 3.3 Comparison of observations with simulations The power emitted in the 2 GeV band from a single MSP The ﬁlled grey triangle are data adapted from Zhu et al. (2018), based on Chandra observations of X-ray sources in the Galactic Center. Note that 1 deg corresponds to 140 pc. from the observed number of main sequence stars in the Galactic Centre. In particular, they assume that all the bi- naries containing a white dwarf are CV (see their Sect. 7.2), which amounts to an upper limit, since only tight binaries can lead to the formation of a CV. Assuming that the IP interpretation of the hard X-ray foreground is correct, our calculations suggest that at most 10% of these binaries un- dergo a CV phase. We note that Pretorius et al. (2013) sug- gested an IP density nIP = 6.4×10−4 pc−3 calculated within a sphere with radius 150 pc. In our models the number of IPs enclosed within this radius yields to an average density nIP = 1.4 −6.7 × 10−4 pc−3, depending on the choice of ηIP = 0, 0.01, 0.002. in our model, compared to observational results from Hong et al. (2009) (their Figure 6). Since our models take into account only the NSC and the Galactic bulge, we show only these two contributions from Hong et al. (2009). We found an overall agreement between 0.07 and 1 deg, corresponding to ∼10 −140 pc at 8 kpc. The discrepancy outside of 1 deg is due to the adopted exponential truncation in our model, which makes the simulation unreliable outside the trunca- tion radius (150 pc). In particular, the best agreement with observations is achieved with model CVe, in which we as- sumed η = 2 × 10−3. In this model, 2/3 of the total number of IPs within the inner 150 pc formed in the Galactic ﬁeld, while the remaining population originated in star clusters with radially segregated initial proﬁles. We also compared our models with Chandra data (Zhu et al. 2018), ﬁnding a discrepancy in the emission from within 0.1 deg. This dif- ference can be due to our assumption that IPs are the only sources emitting in the X-ray band and that all the IPs in our sample are characterised by a luminosity of 1032 erg s−1. On the other hand, the discrepancy might imply that some- thing in our knowledge of CVs and IPs formation processes We show in Figure 7 the surface ﬂux map in our CVb model. 3.3 Comparison of observations with simulations The power emitted in the 2 GeV band from a single MSP 0.1 1 10 100 1000 10000 100000 1e+06 1e+07 0.001 0.01 0.1 1 10 N (deg-2) R (deg) CVa CVe Hong+09 (NC+bulge) Zhu+18 Figure 8. Surface X-ray proﬁle calculated for model CVa (red line) and CVe (blue line). The ﬁlled black circles represent ob- servational data by Hong et al. (2009) (their Fig. 6), where we included only the contribution from the NSC and the Galactic bulge. The ﬁlled grey triangle are data adapted from Zhu et al. (2018), based on Chandra observations of X-ray sources in the Galactic Center. Note that 1 deg corresponds to 140 pc. in our model compared to observational results from Hong -6 -4 -2 0 2 4 6 -6 -4 -2 0 2 4 6 y (arcmin) x (arcmin) -6.0 -5.5 -5.0 -4.5 -4.0 Log F (keV cm-2 s-1 pc-2) Figure 7. Emitted ﬂux from IPs in our model. -6 -4 -2 0 2 4 6 -6 -4 -2 0 2 4 6 y (arcmin) x (arcmin) -6.0 -5.5 -5.0 -4.5 -4.0 Log F (keV cm-2 s-1 pc-2) Figure 7. Emitted ﬂux from IPs in our model. 1.0e-05 1.0e-04 1.0e-03 1.0e-02 1.0e-01 1.0e+00 1.0e+01 0.001 0.01 0.1 nIP(R) (pc-3) R (kpc) CVa CVc CVd CVe y (arcmin) Figure 7. Emitted ﬂux from IPs in our model. 0.1 1 10 100 1000 10000 100000 1e+06 1e+07 0.001 0.01 0.1 1 10 N (deg-2) R (deg) CVa CVe Hong+09 (NC+bulge) Zhu+18 Figure 8. Surface X-ray proﬁle calculated for model CVa (red line) and CVe (blue line). The ﬁlled black circles represent ob- servational data by Hong et al. (2009) (their Fig. 6), where we included only the contribution from the NSC and the Galactic bulge. The ﬁlled grey triangle are data adapted from Zhu et al. (2018), based on Chandra observations of X-ray sources in the Galactic Center. Note that 1 deg corresponds to 140 pc. 0.1 1 10 100 1000 10000 100000 1e+06 1e+07 0.001 0.01 0.1 1 10 N (deg-2) R (deg) CVa CVe Hong+09 (NC+bulge) Zhu+18 N (deg-2) Figure 6. Top panel: number density proﬁle of CVs in diﬀerent conﬁgurations. Bottom panel: CV cumulative distribution proﬁle. Figure 8. Surface X-ray proﬁle calculated for model CVa (red line) and CVe (blue line). The ﬁlled black circles represent ob- servational data by Hong et al. (2009) (their Fig. 6), where we included only the contribution from the NSC and the Galactic bulge. 3.3 Comparison of observations with simulations The power emitted in the 2 GeV band from a single MSP Thus, the GC material delivered to 150pc from the outer regions is underestimated in the simulation. high ηMSP = 0.1 (Model MSPd), the simulation greatly over- predicts the gamma-ray ﬂux at 80 and 200 pc relative to the observed values. Further, an initially unsegregated popula- tion of MSPs in model MSPf produces a low ﬂux in the inner region of the Galaxy < 1 pc at the margin of the ob- servational error. If all the MSPs are contained within their host clusters’ core and the galaxy does not contribute to their population at all (model MSPa), the expected ﬂux is consistent with observations between 10 and ∼80 pc, while it is 7% smaller than the ﬂux observed at 150 pc. How- ever, we must stress here that our numerical model for the galactic bulge extends up to 150 pc at most, being exponen- tially truncated outward. Thus, the GC material delivered to 150pc from the outer regions is underestimated in the simulation. Gamma-ray and X-ray emission from the Galactic centre 11 11 1.0e-05 1.0e-04 1.0e-03 1.0e-02 1.0e-01 1.0e+00 1.0e+01 0.001 0.01 0.1 nIP(R) (pc-3) R (kpc) CVa CVc CVd CVe 1.0e+02 1.0e+03 1.0e+04 1.0e+05 0.001 0.01 0.1 NIP(R) R (kpc) CVa CVc CVd CVe Figure 6. Top panel: number density proﬁle of CVs in diﬀerent conﬁgurations. Bottom panel: CV cumulative distribution proﬁle. 1.0e-05 1.0e-04 1.0e-03 1.0e-02 1.0e-01 1.0e+00 1.0e+01 0.001 0.01 0.1 nIP(R) (pc-3) R (kpc) CVa CVc CVd CVe 1.0e+02 1.0e+03 1.0e+04 1.0e+05 0.001 0.01 0.1 NIP(R) R (kpc) CVa CVc CVd CVe Figure 6. Top panel: number density proﬁle of CVs in diﬀerent conﬁgurations. Bottom panel: CV cumulative distribution proﬁle. from the observed number of main sequence stars in the Galactic Centre. In particular, they assume that all the bi- naries containing a white dwarf are CV (see their Sect. 7.2), which amounts to an upper limit, since only tight binaries can lead to the formation of a CV. Assuming that the IP interpretation of the hard X-ray foreground is correct, our calculations suggest that at most 10% of these binaries un- dergo a CV phase We note that Pretorius et al (2013) sug -6 -4 -2 0 2 4 6 -6 -4 -2 0 2 4 6 y (arcmin) x (arcmin) -6.0 -5.5 -5.0 -4.5 -4.0 Log F (keV cm-2 s-1 pc-2) Figure 7. Emitted ﬂux from IPs in our model. Arca-Sedda, M. and Kocsis, B. and Brandt, T. D. Arca-Sedda, M. and Kocsis, B. and Brandt, T. D. 12 Here, Γret is the BH retention fraction. BHs formed in clus- ters may be ejected immediately due to a large natal kick, or they may be ejected later due to dynamical interactions as they mass segregate to the core and undergo strong scat- terings. The retention fraction here is the fraction of BHs that remain bound to the cluster until it reaches the NSC. Although uncertain, this parameter is thought to be ≳0.5 on both numerical (Repetto & Nelemans 2015; Morscher et al. 2015; Mandel 2016; Peuten et al. 2016) and observational (Strader et al. 2012; Chomiuk et al. 2013; Miller-Jones et al. 2015; Bahramian et al. 2017; Giesers et al. 2018) grounds. We assume a Kroupa (2001) initial mass function and cal- culate νBH as the number of stars with initial masses above 18 M⊙. In this way we obtain a ratio of BHs to stellar mass of νBH ≃3.5 × 10−3 M−1 ⊙. Table 7. Number of CVs over total number of stars Γ10 Γ100 r < 10 pc r < 100 pc CVa 1.69 × 10−4 2.69 × 10−5 CVb 1.79 × 10−4 8.10 × 10−5 CVc 1.10 × 10−4 2.57 × 10−5 CVd 1.14 × 10−4 8.03 × 10−5 CVe 1.09 × 10−4 3.65 × 10−5 Col 1. Model name. Col. 2: fraction of CVs over the fraction of stars in a 10 pc volume. Col. 3: same as column 2, but in a 100 pc volume. Table 7. Number of CVs over total number of stars Col 1. Model name. Col. 2: fraction of CVs over the fraction of stars in a 10 pc volume. Col. 3: same as column 2, but in a 100 pc volume. Col 1. Model name. Col. 2: fraction of CVs over the fraction of stars in a 10 pc volume. Col. 3: same as column 2, but in a 100 pc volume. is missing. Indeed, it is possible either that i) the number of CVs in GCs is smaller than inferred from observations, or ii) that the fraction of CVs that turn into IPs is smaller than expected. For instance, a recent paper based on Chandra observations of Galactic GCs suggests that the population of CVs forming in GCs could be small compared to the ﬁeld (Cheng et al. 2018). Arca-Sedda, M. and Kocsis, B. and Brandt, T. D. ⊙ We measure the fraction of GC mass transported to the galactic centre δi directly from the simulations, while we assume that the eﬃciency of BH formation is the same both in the galaxy ﬁeld and the cluster, thus implying ηBH = 1. Assuming a Γret = 0.5 retention probability, our results suggest that GCs deposit ( g ) Hong et al. (2009) calculated the number of CVs within ∼1 kpc, normalized to the total number of stars, needed to ascribe the Milky Way’s hard X-ray emission to IPs. They found that this quantity should be in the range ΓIP ∼ (1.6 −9.5) × 10−5. In order to compare with observations, we calculate the same fraction, assuming that IPs are only 0.8% of the whole CV population (Pretorius et al. 2013). We ﬁnd that in the inner 100 pc, Γ100 = (2.5 −8) × 10−5, in agreement with the predictions based on observations. More recently, Zhu et al. (2018) reported an enhanced abundance of CVs in the central 10 pc with respect to the outside by a factor 2, compatible with our ﬁndings summarized in Table 7. Thus, we conclude that the population of IPs dominating the X-ray emission in the Galactic centre could have mostly originated in GCs. NBH = 2.4 × 104 BHs into the NSC, while a similar number of BHs should form directly in the galactic nucleus while the NSC grew up. Once deposited into the Galactic Centre, these BHs will segregate into the deepest NSC regions due to dynamical friction, which operate very eﬃciently for heavy objects like stellar mass BHs, becoming the most likely progenitors for the observed population of LMXBs. 4.2 Implication of a NSC wet origin for the γ ray excess If the NSC formed according to the wet scenario, its for- mation would have occurred by gas fragmentation around the SMBH. In this case, the NSC would behave similarly to a very massive and dense star cluster. Assuming a total mass MNSC = 2.5 × 107 M⊙and half-mass radius rh ≃2 pc, the NSC is expected to undergo mass segregation in a fraction of its relaxation time, ∼200 Myr according to Equation (7). This is a clear oversimpliﬁcation since the NSC will grow in time, thus implying that MNSC and rh are time-varying quantities. Therefore, we caution that these are rough estimates, and represent a useful point of comparison for the next generation of numerical models. These models will hopefully have suﬃcient resolution to discern the mo- tion of actual MSPs or CVs candidates in galactic nuclei. 3.3 Comparison of observations with simulations The power emitted in the 2 GeV band from a single MSP We limited the ﬁeld of view in this case to 6.4 ar- cmin, in order to compare with observations provided by NuSTAR (Mori et al. 2015). A qualitative comparison with the inner galactic regions in the 20-40 keV energy range (Fig. 5 Mori et al. (2015), shows similarity between the X- ray image from our simulations, rescaled to the MW centre, and the observed one. Note that the simulated IPs’ mor- phology is qualitatively consistent with the observed X-ray image, although a more rigorous comparison is diﬃcult due to the fact that the observed features are sensitive to the initial conditions that aﬀect NSC formation. Interestingly spiral streamlike structures are visible in the simulation im- age (see e.g. between −5′ < x < −3′ and −2′ < y < 0′). Figure 8 shows the number of sources per square degree 4 DISCUSSION In the case of model S2, instead, the ﬂattening ratio is smaller, qf ∼0.52, if we look in the plane perpendicular to the GCs initial or- bital plane, while it rises up to qf = 0.7 −0.9 if we look in the parallel plane. Since the mass of MSP progenitors is signiﬁcantly higher than the average stellar mass in stellar system, they are expected to segregate into the NSC’s central region over a dynamical friction time-scale (df), provided that this is shorter than the stellar lifetime. Indeed, the zero age main sequence mass range of a star which evolves into a neutron star is 7 −20 M⊙(Belczynski & Taam 2008), and the av- erage stellar mass is meﬀ∼0.6 M⊙for a Kroupa (2001) mass function. The stellar lifetimes may be calculated using the SSE code for modelling stellar evolution (Hurley et al. 2000), which gives 56 Myr for 7 M⊙and 11 Myr for 20 M⊙. Following Arca-Sedda (2016) (but see also Arca-Sedda & Capuzzo-Dolcetta (2014b) for details), and assuming that the MSP progenitor stars were orbiting within the NSC core radius r∗≃rNSC, on a nearly circular orbit, the df time can be calculated as: In particular, the original disc-like distributions of GCs in model S2 is reﬂected also in its NSC morphology. Figure 11 shows the time evolution of its three principal moments of inertia Ii, where I1 is the component perpendicular to or- bital plane of clusters in conﬁguration S2. It is worth noting that all three models are axisymmetric within this prefer- ential plane (I2 = I3), but Model S2 shows an axisymmetic anisotropy with I1/I3 ∼0.3. tdf(m∗) ≃0.3Myr g(e, γNSC) m∗ MNSC −0.67 , (12) Therefore, gamma-ray imaging of the Galactic centre with a resolution of 6.4 arcmin carries information on the initial GC population that formed the NSC. The stellar dis- tribution around the SMBH is expected to be a combination of infalling GC debris and the Galactic background. Simi- larly, the gamma-ray ﬂux is expected to have a contribution from these two channels hinting at the relative fraction of “dry” and “wet” origins of the NSC. To examine these possi- bilities, we calculate the cumulative ﬂux, F, at 2 Gev similar to that in Figure 5 above but for all three initial conditions S1, S2, S3 for diﬀerent values of ηMSP. 4 DISCUSSION 0.01 0.1 1 10 100 1000 0.1 1 10 100 Fwet/Fdry r/RcNC Rmax/RcNC = 10 ; nwet = 1.0 Rmax/RcNC = 1 ; nwet = 1.0 This assumption for the wet formation scenario directly implies that the MSPs’ radial distribution follows the NSC radial distribution. Again, using these simulations, we can- not directly account for mass segregation of stars in the NSC, so we assume that a fraction nMSP,wet of stars is en- closed within Rmax times the NSC core radius rcNSC, which for the Milky Way’s NSC is rcNSC ≃1 pc. We investigated two diﬀerent cases: i) the whole popula- tion of MSPs is fully segregated inside the NSC core radius, i.e. Rmax = rcNSC and nwet = 1; ii) the MSPs are distributed within Rmax = 10rcNSC and nwet = 1. Figure 9 shows the gamma ray ﬂux calculated in the in-situ scenario, Fwet, normalized to the values obtained for model MSPa under the dry-merger scenario assumptions, in the three cases investigated. Figure 9. Ratio between the MSPs γ ﬂuxes in the wet and dry NSC formation scenarios, for two diﬀerent levels of MSPs segre- gation in the NSC formed in-situ. Surprisingly, we found signiﬁcant diﬀerences between dry- and wet- γ ﬂuxes emitted from the inner 10 pc. If the MSPs population is completely segregated in the “in- situ NSC”, we found that the ﬂux is up to 100 times those emitted from MSPs delivered from orbitally segregated star clusters. However, if we assume that the whole population of MSPs is mixed within the inner 10 pc and follow the same radial distribution of background stars, the dry- and wet- scenarios produce similar results. MSP ﬂux morphology, shows diﬀerences among diﬀerent star cluster initial distributions. The NSC ﬂattening ratio qf, calculated as the ratio be- tween the minor and major axis of the ellipsoid enclosing the NSC, varies depending on the model considered. In our model S1, where clusters initial orbits have diﬀerent orien- tations, we found qf = 0.6 −0.8, independently of the plane considered. It is worth noting that this value is really close to the Galactic NSC observed ﬂattening (Sch¨odel et al. 2014; Chatzopoulos et al. 2015; Fritz et al. 2016). 4 DISCUSSION In Figure 12, we com- pare the results with observational estimates by Abazajian et al. (2014) (see also Figure 1 in Brandt & Kocsis 2015 for a comparison with the modelled cumulative ﬂux). (12) where g(e, γNSC) is a smooth function of the NSC inner den- sity slope and the star orbital eccentricity as given by Arca- Sedda et al. (2015). Substituting the properties of our NSC, we ﬁnd tdf(7 M⊙) = 31 Myr and tdf(20 M⊙) = 15 Myr, thus comparable to the stellar evolution time-scale above. This suggests that it MSPs progenitors can be partially seg- regated into the NSC, if formed in-situ therein. 4 DISCUSSION 4.1 The link between the NSC formation history and the Galactic centre BH population Our approach shows that many compact sources can be de- posited into the Galactic Centre in the course of the NSC’s formation. The recent discovery of 12 low-mass X-ray bi- naries (LMXBs) orbiting around 1 pc from Sgr A (Hailey et al. 2018), raised further questions about the evolution of our Milky Way centre. As discussed by Generozov et al. (2018), these sources might have formed in tidal capture by single BHs in the dense environment characterizing the NSC. The expected population of MSPs in a NSC formed en- tirely in-situ is uncertain. Escape speeds from the NSC are much higher than for GCs, so the NSC should retain a larger fraction of its neutron stars. Higher velocity dispersions also reduce the rate of strong encounters and may therefore in- hibit MSP formation. It is not clear whether these eﬀects combine to increase or decrease the MSP population per unit stellar mass relative to that seen in GCs (Faucher-Gigu`ere & Loeb 2011; Dexter & O’Leary 2014). As a simple baseline model, we assume that they cancel out and produce a sim- ilar MSP abundance per unit mass as that seen in GCs, or NMW ∼2450 MSPs. We then produce gamma ray predic- tions from the wet formation scenario by randomly selecting A “wet” NSC origin, in which the stars formed in situ, will leave a large population of BH remnants near the Galac- tic centre. In this section we explore whether a dry-merger scenario can produce a population of remnants compatible with the inferred BH population, which could number as high as 20,000 (Miralda-Escud´e & Gould 2000). We use the same approach as for MSPs and CVs to infer the number of BHs delivered to the NSC by infalling clusters: NBH = ΓretνBH ηBHµNSC,G + X i δiµGC,i ! MNSC (11) (11) Gamma-ray and X-ray emission from the Galactic centre 13 NMSP,wet ≡NMW particles within the spatial region enclos- ing the NSC. 0.01 0.1 1 10 100 1000 0.1 1 10 100 Fwet/Fdry r/RcNC Rmax/RcNC = 10 ; nwet = 1.0 Rmax/RcNC = 1 ; nwet = 1.0 Figure 9. Ratio between the MSPs γ ﬂuxes in the wet and dry NSC formation scenarios, for two diﬀerent levels of MSPs segre- gation in the NSC formed in-situ. 4.3 The role of a central SMBH -20 -10 0 10 20 -20 -10 0 10 20 y (pc) x (pc) -12.0 -11.5 -11.0 -10.5 -10.0 -9.5 -9.0 -8.5 -8.0 Log F2GeV (Gev cm-2 s-1) -20 -10 0 10 20 -20 -10 0 10 20 y (pc) x (pc) -12.0 -11.5 -11.0 -10.5 -10.0 -9.5 -9.0 -8.5 -8.0 Log F2GeV (Gev cm-2 s-1) -20 -10 0 10 20 -20 -10 0 10 20 z (pc) x (pc) -12.0 -11.5 -11.0 -10.5 -10.0 -9.5 -9.0 -8.5 -8.0 Log F2GeV (Gev cm-2 s-1) -20 -10 0 10 20 y (pc) -11.5 -11.0 -10.5 -10.0 -9.5 -9.0 -8.5 -8.0 Log F2GeV (Gev cm-2 s-1) 0.01 0.1 1 10 100 10 100 I1,2/I3 t (Myr) S1 I1:I3 I2:I3 0.1 1 10 100 10 100 I1,2/I3 t (Myr) S2 I1:I3 I2:I3 0.001 0.01 0.1 1 10 100 10 100 I1,2/I3 t (Myr) S3 I1:I3 I2:I3 Figure 11. Principal axes of inertia in our numerical models of the inner 10pc, where the NSC dominates the matter distribution. decrease of the ﬂux by a factor ∼2 for model S3 relative to that in model S1. In Figure 12, we can identify two important regions: in- side 10 pc the gamma ray ﬂux carries information on both the dry and wet NSC formation pathways, while in the outer region the results are consistent with only a small contri- bution from the Galactic background. The results in the 0.01 0.1 1 10 100 10 100 I1,2/I3 t (Myr) S1 I1:I3 I2:I3 0.1 1 10 100 10 100 I1,2/I3 t (Myr) S2 I1:I3 I2:I3 0.001 0.01 0.1 1 10 100 10 100 I1,2/I3 t (Myr) S3 I1:I3 I2:I3 Figure 11. Principal axes of inertia in our numerical models o the inner 10pc, where the NSC dominates the matter distribution 0.01 0.1 1 10 100 10 100 I1,2/I3 t (Myr) S1 I1:I3 I2:I3 0.1 1 10 100 10 100 I1,2/I3 t (Myr) S2 I1:I3 I2:I3 0.001 0.01 0.1 1 10 100 10 100 I1,2/I3 t (Myr) S3 I1:I3 I2:I3 Figure 11. 4.3 The role of a central SMBH In this section we focus on gamma-ray emission, to deter- mine the role played by the GCs’ initial conditions with and without an SMBH (S1, S2, S3, see Section 2). The results in our three models are quite similar outside of the NSC (r > 10 pc), while they exhibit interesting diﬀer- ences in the inner few pc, as shown in Figure 12. The high end of the distribution is compatible with a small contribu- tion coming from the GC, with an upper limit of η ≃0.01, while in the inner pc the presence of the SMBH causes a Figure 10 shows the Galactic centre emission pro- ﬁle. The map shows the inner 25 × 25 pc region or 11 arcmin×11 arcmin around Sgr A*. The Galactic centre Arca-Sedda, M. and Kocsis, B. and Brandt, T. D. 14 14 Arca Sedda, M. and Kocsis, B. and Bran -20 -10 0 10 20 -20 -10 0 10 20 y (pc) x (pc) -12.0 -11.5 -11.0 -10.5 -10.0 -9.5 -9.0 -8.5 -8.0 Log F2GeV (Gev cm-2 s-1) -20 -10 0 10 20 -20 -10 0 10 20 y (pc) x (pc) -12.0 -11.5 -11.0 -10.5 -10.0 -9.5 -9.0 -8.5 -8.0 Log F2GeV (Gev cm-2 s-1) -20 -10 0 10 20 -20 -10 0 10 20 z (pc) x (pc) -12.0 -11.5 -11.0 -10.5 -10.0 -9.5 -9.0 -8.5 -8.0 Log F2GeV (Gev cm-2 s-1) -20 -10 0 10 20 -20 -10 0 10 20 y (pc) x (pc) -12.0 -11.5 -11.0 -10.5 -10.0 -9.5 -9.0 -8.5 -8.0 Log F2GeV (Gev cm-2 s-1) Figure 10. Surface ﬂux map in the three models investigated The panels show respectively model S1, model S2 in the xy plane model S2 in the xz plane, and model S3. 4.3 The role of a central SMBH Cumulative ﬂux distribution calculated from mode S1 (top) S2 (centre), S3 (bottom panel), compared with observe γ excess (black ﬁlled dots Brandt & Kocsis 2015), as a function the projected distance to the SMBH. The red shaded region re resents the cumulative ﬂux assuming that it comes only from th orbitally segregated clusters. The region width encloses a fact of 50% error in the calculation. The dotted curves are obtaine assuming that a fraction of the enclosed galaxy mass contribut to the ﬂux (assuming η = 0.01) 1e-09 1e-08 1e-07 0.001 0.01 F (GeV cm-2 s-1) R (kpc) S1 S2 S3 Figure 13. Cumulative ﬂux in the three models investigated. 1e-09 1e-08 1e-07 0.001 0.01 F (GeV cm-2 s-1) R (kpc) S1 S2 S3 Figure 13. Cumulative ﬂux in the three models investigated. 1e-09 1e-08 1e-07 0.001 0.01 0.1 F (GeV cm-2 s-1) R (kpc) S1 F (GeV cm-2 s-1) 1e-09 1e-08 1e-07 0.001 0.01 0.1 F (GeV cm-2 s-1) R (kpc) S2 Figure 13. Cumulative ﬂux in the three models investigated. deeper potential well. Regarding the second point, in deriv- ing the cumulative ﬂux we assumed that all the GCs have the same level of segregation. Accounting for diﬀerent segre- gation status may decrease the ﬂux, as expected comparing unsegregated (MSPf) and fully segregated models (MSPa) (see Figure 4). Finally, the third point is related to unknown number of MSP formed in the Galactic background, that af- fects the cumulative ﬂux outside 10 pc. F (GeV cm-2 s-1) p We ﬁnd that the presence of an SMBH in the Galac- tic centre and the GCs initial conditions cause a noticeable variation of the emission only within 10 pc from the Galactic centre. The γ ray ﬂux increases by a factor ∼10 within 1-2 pc in model S3, which do not contain any SMBH. The reason for such a diﬀerence is related again to the NSC formation process. Indeed, when the SMBH is absent, tidal forces aris- ing from the Galactic centre are signiﬁcantly smaller. As a consequence, the GCs tidal stripping is less eﬀective in the inner region, allowing for the formation of an NSC charac- terized by an eﬀective radius smaller than in the other two models (see Table 2). Therefore, detailed observations of the inner regions of external galaxies that underwent GC-SMBH interactions can potentially help in arguing for the presence of a central SMBH therein. 4.3 The role of a central SMBH Principal axes of inertia in our numerical models o h i h h NSC d i h di ib i -20 -10 0 10 20 -20 -10 0 10 20 y (pc) x (pc) -12.0 -11.5 -11.0 -10.5 -10.0 -9.5 -9.0 -8.5 -8.0 Log F2GeV (Gev cm-2 s-1) 0.01 0.1 1 10 100 10 100 I1,2/I3 t (Myr) S1 I1:I3 I2:I3 (p ) -20 -10 0 10 20 -20 -10 0 10 20 y (pc) x (pc) 0.1 1 10 100 10 100 I1,2/I3 t (Myr) S2 I1:I3 I2:I3 t (Myr) 0.001 0.01 0.1 1 10 100 10 100 I1,2/I3 t (Myr) S3 I1:I3 I2:I3 -20 -10 0 10 20 -20 -10 0 10 20 z (pc) x (pc) I1,2/I3 -12.0 -11.5 -11.0 -10.5 -10.0 -9.5 -9.0 -8.5 -8.0 Log F2GeV (Gev cm-2 s-1) (p ) -20 -10 0 10 20 -20 -10 0 10 20 y (pc) x (pc) Figure 11. Principal axes of inertia in our numerical models of the inner 10pc, where the NSC dominates the matter distribution. decrease of the ﬂux by a factor ∼2 for model S3 relative to that in model S1. y (pc) In Figure 12, we can identify two important regions: in- side 10 pc the gamma ray ﬂux carries information on both the dry and wet NSC formation pathways, while in the outer region the results are consistent with only a small contri- bution from the Galactic background. The results in the three models investigated look quite similar outside the NSC (r > 10 pc), while they exhibit interesting diﬀerences in the inner few pc. This is highlighted by Figure 13, which shows the cumulative ﬂux in three models with η ≃0.01. Figure 10. Surface ﬂux map in the three models investigated. The panels show respectively model S1, model S2 in the xy plane, model S2 in the xz plane, and model S3. The shaded region in Figure 12 covers the allowed re- gion assuming a 50% error in our calculations which may be Gamma-ray and X-ray emission from the Galactic centre 15 Gamma ra 1e-09 1e-08 1e-07 0.001 0.01 0.1 F (GeV cm-2 s-1) R (kpc) S1 1e-09 1e-08 1e-07 0.001 0.01 0.1 F (GeV cm-2 s-1) R (kpc) S2 1e-09 1e-08 1e-07 0.001 0.01 0.1 F (GeV cm-2 s-1) R (kpc) S3 Figure 12. 4.4 Caveats In our analysis we have shown that a population of MSPs and CVs dragged from infalling GCs in the NSC “dry- merger” scenario can account for most of the observed hard X-ray and gamma-ray excess ﬂux coming from the innermost region of the Milky Way centre, r ≲10 −100 pc, while the emission observed outside ∼100 pc is likely due to sources born in-situ. On the other hand, our numerical simulations suﬀer several limitations that are dictated by the current status of numerical modelling of galactic dynamics. • Regarding the gamma-ray emission, our results sug- gest that nearly 80% of the ﬂux emitted from the inner ∼100 −150 pc can be ascribed to MSPs well segregated in their parent stellar clusters, while the remaining 20% can be associated with sources formed in-situ, which dominate the gamma-ray proﬁle outside 20 pc. These results are mostly independent of the clusters’ initial orbital properties. • The best agreement with the observed gamma-ray emis- sion is achieved assuming that the MSPs’ progenitors pop- ulate their host clusters’ core during the NSC assembly. An originally unsegregated MSP population leads to a ﬁnal dis- tribution that produces a weaker emission than observed in the range 1–10 pc. Due to the fact that each of the presented simulations took several months to be completed, the limited number of models provided does not allow us to investigate the role of the GCs’ mass function or to determine the impact of a GCs’ initial radial distribution signiﬁcantly diﬀerent from that of the background Galaxy. As shown in Section 2.1.1, the resulting NSCs in two models S1 and S2 are both con- sistent with previous results and observations of the Milky Way NSC, thus suggesting that these assumptions have a minor impact on the NSC ﬁnal properties. • The CV number density inferred from our simulations is consistent with observational estimates, while their spa- tial density proﬁle depends strongly on the level of initial mass segregation in their host clusters. If CVs were initially unsegregated in their parent clusters, their density proﬁle after NSC formation would be ﬂat, while if the whole CVs population is concentrated within the host cluster radius af- ter NSC formation, we get a ﬁnal power-law density proﬁle, with slope ∼0.32. 5 CONCLUSION In this paper we investigated possible links between the NSC’s origin of the Galaxy and the intense ﬂux observed in the gamma-ray and hard X-ray bands by the Fermi and NuS- TAR satellite. Using state-of-the-art numerical direct N- body simulations, we modelled the NSC dynamical forma- tion process by orbital decay and merger of massive star clus- ters. We investigated the possible conﬁgurations of MSPs and CVs in the newly born NSC, delivered in the Galactic centre by the infalling clusters. Our main results are summarized as follows. • We showed that the dry-merger scenario of GCs pro- vides a suitable mechanism for the deposition of a large num- ber of MSPs and CVs in the Galactic centre. The predicted numbers of MSPs and CVs (particularly IPs) are consistent with the gamma-ray and X-ray observations, assuming that they formed in dense star clusters that underwent orbital decay. • We showed that the dry-merger scenario of GCs pro- vides a suitable mechanism for the deposition of a large num- ber of MSPs and CVs in the Galactic centre. The predicted numbers of MSPs and CVs (particularly IPs) are consistent with the gamma-ray and X-ray observations, assuming that they formed in dense star clusters that underwent orbital decay. Figure 14. Flux emitted from diﬀerent galactic centres with dif- ferent SMBHs masses, normalized to the value calculated in ab- sence of an SMBH, as a function of the SMBH mass. • We found that GCs can deliver up to ∼24, 000 BHs to the NSC at the Galactic center. This population is added to the BHs that formed in the NSC. 4.3 The role of a central SMBH 16 0 0.2 0.4 0.6 0.8 1 1000 10000 100000 1e+06 1e+07 1e+08 FSMBH MSMBH (M⊙) α = 1.0 α = 2.0 α = 3.0 Figure 14. Flux emitted from diﬀerent galactic centres with dif- ferent SMBHs masses, normalized to the value calculated in ab- sence of an SMBH, as a function of the SMBH mass. 0 0.2 0.4 0.6 0.8 1 1000 10000 100000 1e+06 1e+07 1e+08 FSMBH MSMBH (M⊙) α = 1.0 α = 2.0 α = 3.0 4.3 The role of a central SMBH This can be particularly interest- ing in dwarf spheroidal galaxies (dSph), where the relatively low density can prevent the formation of an SMBH seed, depending on the matter distribution in the galaxy (Arca- Sedda & Capuzzo-Dolcetta 2017a). For instance, in the sim- plest approximation in which the emission from the inner 1 pc is connected to the SMBH through a simple power-law, FSMBH ∝( MSMBH M0 + 1)−α, where the scaling factor is de- ﬁned as M0 = 2.6 × 106 M⊙, it would be possible to infer the mass of a central SMBH if it exceeds 5×104 M⊙, almost independently on value of α, as shown in Figure 14. 1e-09 1e-08 1e-07 0.001 0.01 0.1 F (GeV cm-2 s-1) R (kpc) S3 F (GeV cm-2 s-1) Figure 12. Cumulative ﬂux distribution calculated from models S1 (top) S2 (centre), S3 (bottom panel), compared with observed γ excess (black ﬁlled dots Brandt & Kocsis 2015), as a function of the projected distance to the SMBH. The red shaded region rep- resents the cumulative ﬂux assuming that it comes only from the orbitally segregated clusters. The region width encloses a factor of 50% error in the calculation. The dotted curves are obtained assuming that a fraction of the enclosed galaxy mass contributes to the ﬂux (assuming η = 0.01) Moreover, the emission caused by GCs initially orbit- ing in the same plane should be a few times larger than that caused by GCs moving in the Galactic bulge. Such a diﬀerence is quite below the current Fermi resolution, never- theless these ﬁndings can be interesting for next generation of γ ray detectors, either space-based, such as the forthcom- ing ASTROGAM (Tatischeﬀet al. 2016), CALET (Kisaka & Kawanaka 2013) and PANGU (Wu et al. 2014) space missions, or the CTA telescope (Bednarek et al. 2016) (see Kn¨odlseder 2016 for a detailed review on the perspectives of future gamma-ray astronomy). due to i) the uncertainties in the number of MSPs, ii) the level of initial segregation, and iii) the contribution of galac- tic MSPs. Regarding the ﬁrst point, if the GCs reach the Galactic centre before NS form with a velocity dispersion σ = 190 km s−1 (Phinney & Kulkarni 1994), the number of retained neutron stars can increase by a factor up to ∼1.5 with respect to our previous calculation, due to the NSC Arca-Sedda, M. and Kocsis, B. and Brandt, T. D. 4.4 Caveats O., SivakoﬀG. R., Gladstone J. C., 2013, ApJ, 766, 136 the morphology of the X-ray and gamma-ray ﬂuxes, but poorly aﬀect the observed cumulative ﬂux distribution in these bands. Star clusters initially distributed accordingly to the Galactic background lead to a triaxial NSC, while a more disky structure forms when the clusters move on co- planar orbits. Bahramian A., Heinke C. O., Tudor V., Miller-Jones J. C. A., Bogdanov S., Maccarone T. J., Knigge C., SivakoﬀG. R., Chomiuk L., Strader J., Garcia J. A., Kall- man T., 2017, MNRAS, 467, 2199 J. C. A., Bogdanov S., Maccarone T. J., Knigge C., SivakoﬀG. R., Chomiuk L., Strader J., Garcia J. A., Kall- man T., 2017, MNRAS, 467, 2199 • The central SMBH aﬀects the gamma-ray emission in the inner 10 pc, a limit well below the current FERMI resolu- tion. The absence of a central SMBH leads to the formation of a denser NSC characterised by a ﬂux 5–10 times larger in the inner 1–2 pc than in galaxies with SMBHs. This has interesting implications for the mass range of dwarf galaxies, where the formation of an SMBH may be more diﬃcult due to the lower densities. • The central SMBH aﬀects the gamma-ray emission in the inner 10 pc, a limit well below the current FERMI resolu- tion. The absence of a central SMBH leads to the formation of a denser NSC characterised by a ﬂux 5–10 times larger in the inner 1–2 pc than in galaxies with SMBHs. This has interesting implications for the mass range of dwarf galaxies, where the formation of an SMBH may be more diﬃcult due to the lower densities. 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The work was done in the footsteps of the “MEGaN project: modelling the evolution of galactic nuclei”, funded by the University of Rome Sapienza through the grant 52/2015. This project has received funding from the Euro- pean Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme ERC- 2014-STG under grant agreement No 638435 (GalNUC) and from the Hungarian National Research, Development, and Innovation Oﬃce grant NKFIH KH-125675 (to B.K.). This work was performed in part at the Aspen Center for Physics, which is supported by National Science Foundation grant PHY-1607761. Boyles J., Lorimer D. R., Turk P. J., Mnatsakanov R., Lynch R. S., Ransom S. M., Freire P. C., Belczynski K., 2011, ApJ, 742, 51 Brandt T. D., Kocsis B., 2015, ApJ, 812, 15 Calore F., Di Mauro M., Donato F., Hessels J. W. 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The ongoing rapid advance in GPU architectures and dedicated programming can eventually boost the level of resolution achievable, and can lead in the near future to lower particle mass values, closer to reality. Numerical codes implementing stellar evolution and strong encounters already exist (Aarseth 2003; Spurzem 2001; Petts et al. 2015; Arca-Sedda 2016), but they are still limited to a relatively low number of particles, allowing to model star clusters rather than galactic nuclei (Wang et al. 2016). • The X-ray surface brightness proﬁle inferred from our simulations of the CV population agrees with observations. The best agreement is achieved assuming that ∼25% of the CVs in the Galactic centre come from orbitally decayed star clusters, while the remaining X-ray ﬂux is emitted by CVs formed in-situ. The CVs that originated in star clusters (dry merger channel) dominate the emission in the inner 20 pc, while the locally formed sources dominate outside of the NSC. 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https://openalex.org/W2760823425	https://durham-repository.worktribe.com/preview/1295948/31390.pdf	English	null	Investment horizon and corporate social performance: the virtuous circle of long-term institutional ownership and responsible firm conduct	European journal of finance	2,019	cc-by	21,750	Investment Horizon and Corporate Social Performance: The Virtuous Circle of Long-Term Institutional Ownership and Responsible Firm Conduct Ioannis Oikonomou ICMA Centre, Henley Business School, University of Reading, UK Chao Yin ICMA Centre, Henley Business School, University of Reading, UK Lei Zhao ESCP Europe Business School, Paris, France Labex Refi, Paris, France 2 US SIF: The Forum for Sustainable and Responsible Investment is a United States-based membership association that promotes environmentally and socially sustainable investment practices: http://www.ussif.org/about 1 Abstract: We investigate the relationship between corporate social performance and institutional ownership. We distinguish between long-term and short-term institutional investors using holdings-based measures which directly capture the investment horizon of each institution. Our analysis shows that long term institutional investment is positively related to corporate social performance (mainly by an avoidance of investing in firms with significant controversies) whereas short-term institutional investment is negatively related to corporate social performance. Further investigation reveals that increased holdings of a firm by long-term investors are positively associated with its future corporate social performance. Hence, we provide evidence of a “virtuous circle” between long term investment and social responsibility. Keywords: corporate social responsibility; CSR; CSP; sustainability; institutional investors; investment horizon JEL Classification: G23, G31, M14 The authors would like to thank attendees at the 5th Paris Financial Management conference and the PRI Academic Network Conference 2017, Chris Brooks and two anonymous referees for comments that helped improve the paper. 2 1 The acronym SRI is nowadays also used for Sustainable and Responsible Investing. Though subtle differences can be argued to exist between the two terms, the concept is largely the same. p y y p www.ussif.org/about 1. Introduction Corporate social responsibility (CSR) is still on the rise. The notion has evolved from being an interesting yet peripheral issue that mostly business ethics academics and activists would push for to being a key business practice. Companies increasingly recognize the importance of effective CSR practices which help in building trusting, cooperative long-term relationship with crucial stakeholders (Jones, 1995). Hence , CSR has moved from the sphere of moral philosophy to a strategic management consideration (Clarkson, 1995). Credible business sources provide factual support to this evolution. According to a recent survey conducted by PwC and based on 1,409 (anonymised) interviews of CEOs across the world, 85% recognise that their companies are expected to address wider stakeholder issues, 67% state that “our purpose is centred on creating value for wider stakeholders” and 64% claim that “Corporate responsibility is core to everything we do” (PwC, 2016). The numbers become even higher when CEOs are asked to answer to what extent these statements will be true five years after the interviews, which signifies the acknowledgement of the rising strategic significance of CSR for business. Inevitably, the accounting and financial aspects of CSR have followed similarly increasing trends. More than 7,800 companies published CSR or sustainability reports in 2015, an increase of 30% compared to 2010 (Institutional Investor, 2015). As for Socially Responsible Investing (SRI1 - i.e. the practice of incorporating environmental, social, governance and ethical considerations into the mainstream investment process), the growth has been nothing short of remarkable. According to a report of the US SIF foundation2, more than $8.7 trillion assets under management in the US alone fell under the umbrella of sustainable, responsible and impact investing in 2016. This 3 compares to a little more than $2 trillion in 2005 (a percentage increase of more than 400% in 11 years) and about $0.5 trillion in 1995 (an increase in excess of 1,700%). In an attempt to investigate whether increased levels of measurable Corporate Social Performance (CSP3) are aligned with improved firm profitability, market valuations and superior risk management (which would explain the above-mentioned trends in favour of CSR), a plethora of academic studies has focused on the links between CSP and various attributes of financial performance – paying particular attention on whether CSR is priced in the marketplace. 4 ESG stands for Environmental, Social and Governance performance and is often used instead of CSR or CSP in recent literature. CFP stands for Corporate Financial Performance and it is a generic term used in the literature to encapsulate all the financial metrics that researchers have employed and tested whether they are correlated with CSP. 3 The term CSP is usually used to capture the outcome-based measurement of a firm’s stance towards CSR- related issues. In this paper, we will use CSR as the acronym for the main concept and CSP for variables related to its measurement. 1. Introduction The literature is now rich and diverse in the facets of CSR which are studied, the datasets and methodologies employed, the operationalizations of both CSR and financial performance, the periods of operation, sectors and domicile countries of the sample firms. This variability makes comparisons of results of different studies a challenging task and unanimous conclusions almost impossible to draw (Griffin and Mahon, 1997). Nevertheless, both vote-counting literature reviews (Margolis and Walsh 2003) and statistical meta-analyses (Orlitzky, Schmidt, and Rynes, 2003; Margolis, Elfenbein, and Walsh, 2009; Schröder, 2014; Friede, Busch, and Bassen, 2015) clearly point to modest, albeit positive, associations between CSR and increased financial performance (or reduced risk). Most recently, Friede, Busch and Bassen (2015) statistically combined the results of about 2,200 individual papers in the area and found that “Roughly 90% of studies find a nonnegative ESG–CFP4 relation. More importantly, the large majority of studies reports positive findings”. Yet, even though we now know a reasonable amount about the nature of the links between CSP and financial performance, we have uncovered very little about the characteristics of the 3 The term CSP is usually used to capture the outcome-based measurement of a firm’s stance towards CSR- related issues. In this paper, we will use CSR as the acronym for the main concept and CSP for variables related to its measurement. 3 The term CSP is usually used to capture the outcome-based measurement of a firm’s stance towards CSR- related issues. In this paper, we will use CSR as the acronym for the main concept and CSP for variables related to its measurement. 4 ESG stands for Environmental, Social and Governance performance and is often used instead of CSR or CSP in recent literature. CFP stands for Corporate Financial Performance and it is a generic term used in the literature to encapsulate all the financial metrics that researchers have employed and tested whether they are correlated with CSP. 4 people and organizations which have made the choice of investing in CSR (and divesting or avoiding investments in firms with socially and environmentally controversial track-records). The number of studies which, directly or indirectly, investigate what investors’ traits act as drivers, moderatos or mediators of the demand for CSR is very small (Bollen, 2007; Haigh, 2007; Bauer and Smeets, 2015 are notable exceptions) despite the academic and practical importance of this theme. 5 For a full list of the PRI Principles the interested reader is directed to https://www.unpri.org/about/the-six-principles https://www.unpri.org/about/the-six-principles 1. Introduction In other words, we know very little about who, how and why one invests in Corporate Social Responsibility. In this paper, we aim to fill part of this knowledge gap within this admittedly wide-spreading field by focusing on the role that investment horizon plays on the demand for CSR by institutional investors. Institutional ownership of stocks has long been shown to influence both the pricing and volatility of these assets (Bushee and Noe, 2000; Gompers and Metrick, 2001). But more specifically, within the framework of SRI, institutional ownership has become increasingly important. This is clearly reflected both in the overall magnitude of the assets under professional management invested in SRI funds (the US SIF data previously mentioned is indicative of this) – the demand for which comes primarily from institutional investors– and in the increasing number of signatories of the United Nations-backed Principles for Responsible Investment. This initiative has managed to secure the commitment of more than 1,600 asset owners, investment managers and financial service providers who pledge to “incorporate environmental, social and governance issues into their investment analysis and decision making processes”5. In focusing on institutional preferences for CSR, our aim is twofold. We first explore whether the widely used claim that the benefits of CSR tend to accrue in the long run is convincing for market participants, which would mean that stocks of firms with high (low) corporate social performance tend to be preferred by institutions which have longer (shorter) investment horizons 5 For a full list of the PRI Principles the interested reader is directed to // / / 5 and keep their holdings unchanged for longer (shorter) periods. Secondly, previous studies have shown that a higher proportion of long-term institutional ownership decreases managerial myopia and reduces pressures to corporate executives to meet short-term goals (Bushee, 1998). Hence, it would be reasonable to assume that firms with higher levels of long-term institutional owners have a greater capacity to utilise corporate resources in an effort to increases the firm’s CSP in the future – and manage to do so. We investigate if this is indeed the case. Our work contributes to the existing literature in three significant ways. Firstly, although studies on institutional investors often treat them as a homogenous group with similar objectives, investors’ differing investment horizons can affect their decisions. 1. Introduction Thus, differentiating long-term from short-term ownership is essential for a better understanding of the important role that investment horizon plays on investment agendas of investors. Mixing ownership with different investment horizons together, Dyck et al. (2018) fail to detect that higher CSR is attractive to institutional investors. In contrast, we provide evidence in line with the frequently theorized but very rarely empirically tested hypothesis that the beneficial, value-creating or risk-reducing, financial effects of high CSP accrue in the long-term – and hence, firms with high CSP should be more attractive to more long-term investors. Secondly, investment horizon could also be an important factor in influencing investors’ corporate policy-making decisions involving CSR activities. Different institutional investors may have different attitudes toward CSR and mixing short-term and long-term investors may lead to inconsistent conclusions (see Harjoto and Hoje, 2011; and Borghesi et al., 2014). In this study, we separate investors with different expected holding periods and investigate the extent to which long- term institutional ownership is associated with future improvements in the social, environmental and governance performance of their holding firms. Therefore, we also explore a parallel mechanism running in the opposite direction of the link between CSR and institutional ownership. 6 Thirdly, a crucial step in understanding the relationship between investment horizon and CSP is to accurately measure the former. In the finance literature, a direct holdings-based measure — churn rate — has been shown to accurately capture investment horizon and has been used widely in different strands of research (see, for example, Gaspar, Massa and Matos, 2015; Yan and Zhang, 2009; Switzer and Wang, 2017). To the best of our knowledge, this is the first study that makes use of the, arguably more accurate, churn rate as a direct measure of distinguishing between long- term and short-term investors in CSR studies with US data. This improved investment horizon measure enables us to provide new empirical evidence about the IO-CSR relationship. In the extant literature, institutional investors have been categorized into short-term versus long-term either based on their operational/legal identity (e.g. Cox, Brammer, and Millington, 2004) , or based on the classification defined in Bushee (2001) (see for example Boubaker et al., 2017). 1. Introduction As we demonstrate in Section 3.3 of this study, both of these methods can lead to vastly different categorizations compared to the direct use of the churn rate and, we argue, to the introduction of more noise in the analysis. Therefore, we consider this methodological contribution to be modest but impactful. We show that long term institutional investment is positively related to corporate social performance whereas short-term institutional investment is negatively related to corporate social performance. Further investigation reveals that increased holdings of a firm by long-term investors are positively associated with future levels of corporate social performance. Hence, we provide evidence of a “virtuous circle” between long term investment and CSP, in line with the more generic findings of Waddock and Graves (1997) regarding the CSP-financial performance link. Our results are useful for understanding what type of investor is attracted to CSR as well as pinpointing investment horizon as one of the factors that leads to an institutional shift towards CSR at the firm level. Consequently, they are useful for firm managers, investment funds, regulators and the wider activist community advocating for increases in CSR. 7 7 The remainder of the paper is structured as follows. Section 2 provides the details of the literature exploring the institutional demand for SRI and develops the framework of the hypotheses tested in the study. Section 3 describes the datasets we use and the methodology we employ. Section 4 reports and explains the empirical results of the study and Section 5 provides a concluding discussion. 2. Related literature and development of hypotheses The role of institutional ownership has become much more prominent in the last decades. Aggregated data demonstrative of the shift in the overall ownership of stocks from retail investors to institutions are not available, but various estimates suggest that US retail investors owned approximately 90% of the stock market up until 1950 whereas the relevant percentage in recent years is in the vicinity of just 30-35% (Evans, 2009). The percentage of institutional ownership must have therefore correspondingly increased by a huge amount (55% to 60%) over the same period. The importance of this evolution becomes evident when one considers academic findings which suggest that institutional investors are less influenced by “attention grabbing” stocks (Barber and Odean, 2008), tend to be less myopic than individuals in terms of the strategies their holding firms are employing (Bushee, 1998), play an important role in determining executive compensation (Hartzell and Starks, 2003) and, ultimately, significantly influence equity prices (Gompers and Metrick, 2001; Boehmer and Kelley, 2009). In spite of all the aforementioned evidence, very few aspects of the relationship between institutional equity ownership and CSR have been studied. Graves and Waddock (1994) are among the first to look into this in the early era of responsible investment and cannot find evidence that CSR has a discernible impact on the percentage of firm shares held by institutions. But this conclusion may very well be a result of the heterogeneity in the characteristics of institutional investors. Different types of investing entities have different priorities, preferences, risk tolerances and investment horizons and hence they may have different attitudes towards CSR. Thus, when 8 including all institutional investors in one category, irrespective of their very different characteristics, no generalizable conclusions, can be drawn. Recognising this, subsequent studies on the same field looked at different types of institutional investors separately. Johnson and Greening (1999) find that pension fund holdings are positively associated with increased levels of diversity in the workplace and have better relationships with local communities and employees whereas none of these occur for the holdings of mutual funds and investment banks. Similarly, Cox, Brammer, and Millington (2004) focus in the UK market and split institutional investors into a group comprising of pension funds, assurance funds and charitable funds and a group made up of unit trusts and investment trusts. 2. Related literature and development of hypotheses They find that the majority of the investors in the former group (which they label as being long-term oriented investors) have holdings which are positively associated with CSP. These results are broadly verified by Cox and Wicks (2011) who use a categorisation of institutional investors as “dedicated” versus “transient”. Most recently, Harjoto, Jo, and Kim (2015) go a step further by investigating the functional form of the link between institutional ownership and CSP and the potentially mediating role of institutional investment in influencing the association of CSP and financial risk. They find a curvilinear (reverse U shape) relationship between the two, meaning that there is a perceived optimal level of CSP above which institutional investors may not wish to increase their ownership in a firm. But the main takeaway from their study is that “CSR decreases stock return volatility at a decreasing rate through its effect on institutional ownership” – a very interesting and novel observation. 9 Although all of the above-mentioned papers recognize the importance of institutional ownership in the constantly evolving field of SRI, they do not attempt to explicitly test the impact that the investment horizon of institutional owners has on their preferences for CSP. Earlier empirical studies simply make no distinction between different types of institutional owners. In this study, we consider two types of institutional investors: long-term and short-term. By definition, long-term investors intend to hold their shares for a long-time period, whereas short- 9 term investors trade at a higher frequency. Consequently, long-term investors care about the fundamental value of the stock while short-term investors only pay attention to short-term market price fluctuation, which may or may not correctly reflect the change of the firm’s fundamental value. The stock price may temporarily deviate from its fundamental value simply because it takes time for the market to incorporate new information (e.g. the change of the firm’s CSR score) into the stock price. It follows that an inefficient stock market drives a wedge between short-term and long-term investors. p , y 7 The Bushee classification uses a factor and cluster analysis approach to classify institutional investors, based on a large number of trading behaviour variables and portfolio characteristics (e.g. portfolio turnover, diversification, and momentum trading). 6 However, if the stock market is efficient, the stock price at any time point equals its fundamental value. As a result, the interests of short-term investors and long-term investors are aligned perfectly, which are to maximize the present value of all future cash flows of the firm. Therefore, under the efficient market assumption, investment horizon does not really matter. 6 However, if the stock market is efficient, the stock price at any time point equals its fundamental value. As a result, the interests of short-term investors and long-term investors are aligned perfectly, which are to i i th t l f ll f t h fl f th fi Th f d th ffi i t k t 6 However, if the stock market is efficient, the stock price at any time point equals its fundament However, if the stock market is efficient, the stock price at any time point equals its fundament As a result, the interests of short-term investors and long-term investors are aligned perfectly, whic maximize the present value of all future cash flows of the firm. Therefore, under the efficient assumption, investment horizon does not really matter. 2. Related literature and development of hypotheses Unlike long-term investors, short-term investors do not appreciate CSR activities because it is (or believed to be) likely that the value of such activities will only be incorporated in the stock price in the long term.6 Cox, Brammer, and Millington (2004) note the significance of making a distinction between short-term and long-term institutional investors but their categorization depends on the legal or operating nature of each institution instead of their actual investing and trading behaviours (i.e. how often and how much they tend to rebalance the assets in their portfolios). More recently, Cox and Wicks (2011) and Boubaker et al. (2017) rely on the classification (dedicated versus transient) defined in Bushee (2001) to distinguish between institutional investors with long versus short horizon (based on investors’ trading behaviour variables and portfolio characteristics), when examining the impact of investment horizon on CSR. Although the Bushee classification intends to capture the substantive differences in trading and governance behaviour within types of investors (e.g. pension funds, bank trusts and others) and represents a step forward in more accurately measuring investment horizon, it, unlike our measure, is more intricate and not entirely based on trading turnover. In that sense, it is not a “pure” measure of investment horizon.7 As 10 we will show in Section 3.3, a considerable amount of long-term ownership (measured by actual holdings and trading data) is classified as short-term (transient) under the Bushee classification and vice versa. To the best of our knowledge, the study of Li and Lu (2015) is the only other paper in this area which employs a direct measurement of institutional investor horizon based on actual holdings. However, the setting of this study is based on evidence from Chinese firms where a very large proportion of institutional ownership comes from the state and in fact the authors verify that environmental performance seems to only be positively related to institutional ownership for state owned enterprises. Our analysis also explicitly uses a direct measurement of institutional equity holdings and trading turnover to distinguish between short-term and long-term investors. However, it is based on US data where government/state ownership of publicly traded firms is much less important and hence institutional investment patterns are arguably more reflective of the true preferences for CSP in the marketplace. Our ex ante hypotheses are that higher CSP will be positively associated to long-term institutional holdings and negatively associated to short-term institutional holdings. 2. Related literature and development of hypotheses A significant body of conceptual academic work in strategic management has provided a framework that supports our assertions. Looking at corporate social responsibility from the perspective of the resource-based view of the firm, the work of Barney (1991) and Barney and Hansen (1994) suggests that corporate efforts to improve social welfare can create valuable reputational capital for the firm and add to its relational wealth with suppliers, employees, clients and other stakeholders. These efforts to increase CSP constitute complex social resources that are rare and hard to replicate, hence can lead to long–term, sustainable advantages. Barney and Hansen (1994) note that the networks of relations created via this avenue “are developed over long periods of time” p.184) so it would be sensible to assume that the relative impacts in the value of the firm also accrue in the long-run. Consequently, we would expect institutional investors with long term horizons to have a higher preference for higher CSP firms. 11 11 Jones (1995) looks at firms as a nexus of contracts and provides an extensive conceptual framework which suggests that opportunism and self-interest can prevent firms from developing and maintaining long-term, mutually beneficial relationships with their stakeholders, thus leading to higher monitoring costs, inefficient contracting and, ultimately, a competitive disadvantage. Combining this work with Godfrey's (2005) arguments that CSR provides evidence of “good corporate character” in favour of the firm and helps in building the aforementioned long-term relationship further reinforces the point that the value of CSP is more relevant to long-run measurements of firm performance. Along very similar lines, Waddock and Graves (1997) note that “such resource allocations may be strategically linked to improvements in long-term image and relationships with the communities with which it (the firm) must interact”. All of these arguments and positions are strongly reiterated in the work of Hillman and Keim (2001). 2. Related literature and development of hypotheses The authors argue that at least some strategic aspects of high CSP can be value creating in the long-run as the firm builds strong links with its primary stakeholders: “Relations with primary stakeholders…customers, employees, suppliers, community residents and the environment—can constitute intangible, socially complex resources that may enhance firms’ ability to outperform competitors in terms of long-term value creation.” Given all the above, we expect that higher CSP will be a desirable characteristic for institutional investors who anticipate their investments to reap benefits in the long-run and as such tend to hold on to their equity for longer periods (i.e. have a lower trading turnover). We also expect the opposite to be true for institutional stock owners with short-run investment horizons: Hypothesis 1: High (low) CSP is associated with longer (shorter) investment horizons and lower (higher) stock turnover. The academic literature on the financial effects of CSP has often made the case that there is some variability in their magnitude according to the nature of social, governance and environmental actions (or inactions) on the part of the firm. More specifically, there are multiple studies which argue that a firm going “the extra mile” in terms of CSP and being proactively engaged in various 12 socially beneficial initiatives is not necessarily significantly rewarded through the marketplace. On the other hand, firms associated with social/environmental controversies are highly likely to, literally and figuratively, pay the price of their irresponsibility. For example, Meijer and Schuyt (2005) show that consumers are willing to boycott a firm if its CSP is particularly low but, on the other hand, high levels of CSP do not bring about measurable increases in product sales. More broadly, Lankoski (2009) reiterates the existence of a negativity bias (the phenomenon according to which negative actions are perceived as more impactful and are weighed more heavily than positive actions) in the CSP-firm performance link. She argues and shows that “the economic impacts of corporate responsibility are more positive for issues reducing negative externalities than for issues generating positive externalities” (p. 218). More recently, Kappou and Oikonomou (2016) investigate the “social index effect” and find that although deletions of stocks from a socially responsible index (caused by various social, environmental or ethical controversies) are associated with statistically significant negative abnormal returns, additions to the index do not manifest in any measurable financial result. 2. Related literature and development of hypotheses Motivated by the above findings we further posit: Hypothesis 2: The positive association of high CSP and longer investment horizons is predominantly driven by an avoidance or underweighting of firms with significant social/environmental controversies rather than an overweighting of firms with significant respective strengths. The implications of our study are not restricted to the arena of capital markets but instead can spread into the field on corporate decision making on the part of managers and influence the way business is conducted. Due to this, we find it useful to investigate whether the relationship between institutional ownership and CSP also runs from the former to the latter. The often cited “myopic institutions theory” (Hansen and Hill, 1991; Graves and Waddock, 1994) argues that higher institutional investment invariably creates pressures to meet short-term earnings and stock price goals. This, in turn, leads to reductions in innovative practices which require immediate investments but have long-term cash flow effects – such as R&D or practices increasing CSP. 13 Bushee (1998) provides some support to this theory by empirically demonstrating that it is true only for institutions that have a higher portfolio turnover and engage in momentum trading, i.e. they could be de facto characterised as short-term investors. Otherwise, institutional ownership is actually positively associated to corporate projects yielding long-term benefits. Based on the above we posit: Hypothesis 3: Long-term institutional ownership is positively related to subsequent increases in the CSP of the owned firms. To our knowledge, the only previous study to have looked at a relationship running from institutional ownership to CSP is that of Dam and Scholtens (2012). The authors use data from one year (2005) and 16 different countries and provide mixed evidence regarding the sign of this relationship. Perhaps one reason for this is that there is no distinction made between long-term and short-term investors. Our study addresses this issue. In the following section we present the datasets, variables, and methodologies we use in order to test our hypotheses. 8 Starting with the S&P 500 Index firms and the Domini 400 Social Index firms in 1991, KLD has expanded its coverage to incorporate the largest 1,000 US companies by market value since 2001, an expansion which advanced further in 2003 with the inclusion of the 3,000 largest US firms. 3.1. Data and sample construction Our sample is constructed with a variety of data sources. We start with a sample of firms covered by the Kinder, Lydenberg, and Domin (KLD) STATS database (now owned by MSCI) from 1991 to 2012. KLD contains detailed information on US firms’ CSR activities and is arguably the most comprehensive and certainly the most widely-used source of data for research in CSR. The database uses sources both internal to the firm (e.g. annual reports) and external (e.g. articles in the business press) to conduct yearly assessments of the social performance of the 3,000 largest 14 14 US publicly traded companies by market capitalization.8 We then merge the KLD data with the institutional ownership data obtained from Thomson Reuter’s 13F database, which contains quarterly institutional holdings for all common stocks traded on NYSE, AMEX, and NASDAQ.9 We delete observations with overall institutional ownership over 100%, which reduces the number of observations by less than 1%.10 We obtain data on firms’ characteristics from the Compustat database, and data on stock price, stock return, trading volume, and firm age from the Centre for Research in Security Prices (CRSP) database. The final sample consists of 22,801 firm-year observations, representing 3,714 US firms over the 1991-2012 period. US publicly traded companies by market capitalization.8 We then merge the KLD data with the institutional ownership data obtained from Thomson Reuter’s 13F database, which contains quarterly institutional holdings for all common stocks traded on NYSE, AMEX, and NASDAQ.9 We delete observations with overall institutional ownership over 100%, which reduces the number of observations by less than 1%.10 We obtain data on firms’ characteristics from the Compustat database, and data on stock price, stock return, trading volume, and firm age from the Centre for Research in Security Prices (CRSP) database. The final sample consists of 22,801 firm-year observations, representing 3,714 US firms over the 1991-2012 period. 9 The Security and Exchange Commission (SEC) requires that all institutions operating in the US with discretion over 13F securities worth $100 million or more report all equity holdings greater than 10,000 shares (or $200,000) to the SEC at a quarterly frequency. 11 Following Servaes and Tamayo (2013), we exclude corporate governance from our CSP construction because it is a mechanism that aligns the interest between shareholders and managers rather than a concern that deals with social objectives and stakeholders other than shareholders. Human rights has also historically considered to be too broad of a category within KLD and not related to a particular group of stakeholders so is also excluded from the analysis. 3.2. Measuring CSP We employ the KLD database to construct our CSP measures. KLD assesses firms with regard to their strengths and concerns on a variety of dimensions of CSR. More specifically, companies are rated on multiple categories, including seven “qualitative issue areas” (these being community, diversity, employee relationship, environment, product, human rights and corporate governance) as well as six “controversial business issues” (which examine the extent to which a firm is involved with military contracting, nuclear power, firearms, alcohol, tobacco, or gambling). The qualitative 8 Starting with the S&P 500 Index firms and the Domini 400 Social Index firms in 1991, KLD has expanded its coverage to incorporate the largest 1,000 US companies by market value since 2001, an expansion which advanced further in 2003 with the inclusion of the 3,000 largest US firms. 9 The Security and Exchange Commission (SEC) requires that all institutions operating in the US with discretion over 13F securities worth $100 million or more report all equity holdings greater than 10,000 shares (or $200,000) to the SEC at a quarterly frequency. 10 There are several reasons which could lead to a nominal institutional ownership rate being higher than 100% for a given firm. First of all, when investors share investment discretion, the security may be double counted (once for each institution). Secondly, when investors short sell a security, it will be recorded as a holding for both the lender and the borrower (short-seller) which will also lead to an overstatement of ownership. Thirdly, sometimes a firm’s financial reporting date and institutional investors reporting date will not match perfectly. In this case, if a firm’s total shares outstanding changed dramatically during this time gap, the base of ownership calculation could cause some data errors (Striewe, Rottke, and Zietz 2013). To minimize the effects of these factors, we follow the same treatment as in Yan and Zhang (2009). Our results are robust when keeping those observations with more than 100% total institutional ownership in our sample. 15 dimension indicators include both strengths and concerns of the same category, whereas the controversial business issues by definition are only rated on concerns. All ratings are binary, with 1 representing the presence of a particular strength/concern and 0 representing its absence. y 12 Following Hillman and Keim (2001) and Oikonomou, Brooks, and Pavelin (2012), we assume that each type of social action is given equal weighting so that employee programs, for example, are considered just 3.2. Measuring CSP Following much of the literature, including Hillman and Keim (2001) and Oikonomou, Brooks and Pavelin (2012), we do not consider the controversial business issues and concentrate on the five main CSP qualitative issue areas: community, diversity, employee relationship, environment, and product.11 The fact that the number of strengths and concerns within each CSP category has evolved over time as KLD refined the database makes it difficult to directly compare strengths (concerns) across years. Therefore, we scale the strengths and concerns of each category to obtain two indices that range from 0 to 1. To be more specific, within a particular qualitative dimension for each firm-year we calculate adjusted dimension-level strength (concern) scores by adding all the ratings of the indicators for the strengths (concerns) and then dividing the sum by the maximum possible number of strengths (concerns). Then we compute dimension-level CSP scores as the net difference between adjusted dimension-level strength and concern scores for all five qualitative dimensions studied in the paper. The five dimension-level CSP scores are denoted as Community score (COM_CSP), Diversity score (DIV_CSP), Employee score (EMP_CSP), Environment score (ENV_CSP), and Product score (PRO_CSP). Finally, we construct three aggregate CSP measures: overall strengths (AGG_S), overall concerns (AGG_C) and overall CSP (AGG_CSP). To calculate AGG_S (AGG_C), we simply sum the adjusted dimension-level strengths (concerns) across the five categories and then divide the sum by five.12 To calculate AGG_CSP, we subtract the AGG_C from the AGG_S. 16 as important as product safety and quality. Though not a perfect solution, in the absence of up to date data on the relative importance of each dimension this is what the literature has been employing. 3.3. Measuring Institutional Ownership Finally, short-term (long-term) institutional ownership (hereafter 𝑆𝐼𝑂 and 𝐿𝐼𝑂) is constructed as the ratio between the number of shares held by short-term (long-term) investors and the total number of shares outstanding. Appendix 1 summarizes definitions and data sources of various CSP and institutional ownership measures. 3.3. Measuring Institutional Ownership We construct three institutional ownership measures. For a particular firm, we first measure its total institutional ownership (hereafter 𝑇𝐼𝑂) as the ratio between the number of shares held by institutional investors and the total number of shares outstanding. We then further classify institutional investors into short-term and long-term investors based on their portfolio turnover during the past four quarters. Short-term investors, by definition, buy and sell their investments frequently, which is reflected in high portfolio turnover. In contrast, long-term investors tend to hold their positions unchanged for a relatively long time period and thus are associated with low portfolio turnover. Therefore, portfolio turnover de facto serves as an intuitive criterion to distinguish long term investors from their short-term peers. Following Gaspar, Massa and Matos (2005), for each institutional investor 𝑖 at time 𝑡 we calculate churn rate (𝐶𝑅𝑖,𝑡), a measure of how frequently the investor rotates her positions on all the stocks of her portfolio. More precisely, in quarter 𝑡, investor 𝑖’s churn rate is: 𝐶𝑅𝑖,𝑡= ∑ \|𝑁𝑗,𝑖,𝑡𝑃𝑗,𝑡−𝑁𝑗,𝑖,𝑡−1𝑃𝑗,𝑡−1 −𝑁𝑗,𝑖,𝑡−1∆𝑃𝑗,𝑡\| 𝑗∈𝑄 ∑ 𝑁𝑗,𝑖,𝑡𝑃𝑗,𝑡+ 𝑁𝑗,𝑖,𝑡−1𝑃𝑗,𝑡−1 2 𝑗∈𝑄 (1) (1) where 𝑄 represents the set of companies held by investor 𝑖. 𝑃𝑗,𝑡 and 𝑁𝑗,𝑖,𝑡 are the price and the number of shares, respectively, of company 𝑗 held by institutional investor 𝑖 at time 𝑡. ∆𝑃𝑗,𝑡 represents the price change of share 𝑗 between time 𝑡−1 and 𝑡. At time 𝑡, if 𝑁𝑗,𝑖,𝑡= 𝑁𝑗,𝑖,𝑡−1 for all 𝑗, it means that investor 𝑖 does not change her portfolio at all during the period and thus her 17 churn ratio is equal to zero as the numerator of Equation (1) becomes zero, suggesting that she is a long-term investor. churn ratio is equal to zero as the numerator of Equation (1) becomes zero, suggesting that she is a long-term investor. Next, we calculate each investor’s average churn rate over the past four quarters: Next, we calculate each investor’s average churn rate over the past four quarters: 𝐴𝑉𝐺_𝐶𝑅𝑖,𝑡= ∑𝐶𝑅𝑖,𝑡−𝑗 3 0 4 (2) 𝐴𝑉𝐺_𝐶𝑅𝑖,𝑡= ∑𝐶𝑅𝑖,𝑡−𝑗 3 0 4 (2) Based on the average churn rate, at each year end we sort all investors into three tertiles. Those ranked in the top tertile with the highest 𝐴𝑉𝐺_𝐶𝑅𝑖,𝑡 (top 33.3%) are classified as short-term institutional investors and those ranked in the bottom tertile are categorised as long-term investors. INSERT TABLE 1 ABOUT HERE We have a total of 4,588 unique institutional investors with holdings in at least one firm in one year of our sample. It is worth noting that the average churn rate for short-term investors across all years is 15% whereas for long-term investors it is just 2.2%. This essentially translates to short- term investors rebalancing their holding at a pace of nearly 7 times faster than their long-term peers – a truly sizable differential. In addition, an important observation that should be highlighted is that our classification of long-term (short-term) investors leads to substantially different proportions of long-term (short-term) ownership, compared with the methods used by the traditional classifications (fiduciary duty-based classification and Bushee classification). For example, Pension Funds are thought of as being typical examples of long-term institutional 18 owners. Yet Table 1 shows that less than half (48.53%) of corporate pension funds are actually classified as long-term investors based on their churn rate, whereas a very significant proportion of them (25.23%) are actually short-term investors. The misclassification is even more dramatic when looking at insurance companies. Only about one third of them (33.89%) appear to be true long-term investors based on their trading turnover while nearly half (48.52%) are actually short- term investors. The Bushee classification can also lead to mischaracterisations as demonstrated in Panel B of Table 1. Only 30.93% of the Dedicated Investors and 43.34% of Quasi Indexers under the Bushee classification are long-term investors in the purest sense (i.e. according to turnover of holdings). As such, we argue that any previous work in the area that makes use of this classification method, possibly introduces a significant amount of noise in its subsequent empirical analysis – which may also mean drawing misleading conclusions. For example, Boubaker et al. (2017) use the Bushee classification and label both Dedicated Investors and Quasi Indexers as long-term investors. As we have shown, in our sample the application of the same logic would have led to a vastly different (and, we would argue, noisier) classification of investors compared to the use of the churn rate. p y 14 Including the lagged dependent variable could result in biased coefficient estimates if the true data generating process is static. To alleviate this concern, we remove the lagged dependent variable and re-estimate all regression specifications. Our (unreported) results are robust to the changes. We thank an anonymous reviewer for pointing this out to us. 13 Analytical descriptions of all the key dependent and independent variables have been placed in Appendix 1 for the sake of parsimony. y p 1 for the sake of parsimony. 13 Analytical descriptions of all the key dependent and independent variables have been placed i 1 f h k f i y p y p p 1 for the sake of parsimony. 3.4. Methodology With the comprehensive firm-level data retrieved from multiple sources, we are interested in three main questions regarding the relationship between investment horizon and CSP. First, does heterogeneity in terms of investment horizon among institutional investors play a significant role in determining their preferences for CSP? Second, if long-term investors do prefer firms with a higher CSP score as the theory would suggest, do they have equal appetite for seeking strengths and for avoiding concerns? Lastly, if the benefit of activities improving CSP indeed accrues in the long run as literature claims, is there empirical evidence that long-term investors promote higher CSP once they become shareholders? 19 19 To examine the first question, we conduct regression analyses by employing three different institutional ownership (𝐼𝑂) measures as dependent variables. More specifically, our empirical framework is based on the estimation of the following prediction model: 𝐼𝑂𝑖,𝑡= 𝛼+ 𝛽1AGG_CSP𝑖,𝑡−1 + 𝛄𝐗𝒊,𝒕−𝟏+ 𝜀𝑖,𝑡 (3) (3) In Equation (3), the subscripts 𝑖 and 𝑡 denotes the firm and the time (year), respectively. 𝐼𝑂 is either 𝑇𝐼𝑂, 𝑆𝐼𝑂, or 𝐿𝐼𝑂 corresponding to total institutional ownership, short-term and long- term institutional ownership respectively13. Our variable of interest is AGG_CSP, our measure of overall CSP constructed using KLD data. The sign and significance of the coefficient 𝛽1 reveals the relationship between CSP and a particular 𝐼𝑂 measure. 𝐗 is a vector of control variables and 𝛄 is a coefficient vector. The first control variable we include in 𝐗 is the lagged dependent variable (𝐼𝑂𝑖,𝑡−1). Allowing for dynamics in 𝐼𝑂 is crucial for recovering consistent estimates of 𝛽1 if 𝐼𝑂 is serially correlated.14 Prior research shows that certain firm characteristics are associated with institutional investors’ investment preference and thus should be controlled to mitigate the problem of possible spurious relationship between 𝐼𝑂 and CSP (see Gompers and Metrick, 2001; Yan and Zhang, 2009; Harjoto, Jo, and Kim, 2015). Specifically, institutional investors are documented to take into account prudence, stock liquidity, transactions costs, and expected future returns when they make their investment decisions. 3.4. Methodology We also include 𝑁𝑜𝑛−𝑙𝑜𝑛𝑔−𝑡𝑒𝑟𝑚 𝐼𝑂 (NLIO) to capture the impact of other institutional ownership (including short-term and medium-term ownership) on future CSP. In addition, we control for industry fixed effects and year fixed effects as in equations (3) and (4). Appendix 2 summarizes definitions and data sources for these control variables. 3.4. Methodology Therefore, following Yan and Zhang (2009) we include three groups of control variables in 𝐗: 1) Size (MV), firm age (AGE), dividend yield (DY), S&P 500 index membership (S&PIDX), leverage (DTA), stock risk (both systematic risk (BETA) and idiosyncratic risk (IRISK)) to control for prudence; 2) Share price (PRC) and stock 20 turnover (TOV) to control for liquidity and transactions costs; and 3) Past returns (RET), earnings per share (EPS), and book-to-market ratio (BM) to control for expected future returns (see Fama and French, 1992). To account for industry specific factors that may affect the relationship between 𝐼𝑂 and CSP, we include industry dummy variables, which are constructed based on the two-digit Standard Industrial Classification (SIC) code. We also add year dummies in 𝐗 to account for changing economic conditions and more importantly the observed evolution of CSP-related recognitions and practices. Appendix 2 summarizes definitions and data sources for these control variables. Equation (3) looks at the overall CSP indicator, which summarizes strengths and concerns into one single figure and consequently prevents us from exploring investors’ potentially different attitudes towards firms’ socially beneficial and controversial activities. Thus, we replace the AGG_CSP variable with two variables AGG_S and AGG_C, representing social strengths and concerns respectively: 𝐼𝑂𝑖,𝑡= 𝛼+ 𝛽1AGG_S𝑖,𝑡−1 + 𝛽2AGG_C𝑖,𝑡−1 + 𝛄𝐗𝒊,𝒕−𝟏+ 𝜀𝑖,𝑡 (4) (4) In Equation (4), we are interested in variables AGG_S and AGG_C, which enable us to breakdown overall CSP and allow for asymmetric effects of strengths and concerns on future institutional ownership. In Equation (4), we are interested in variables AGG_S and AGG_C, which enable us to breakdown overall CSP and allow for asymmetric effects of strengths and concerns on future institutional ownership. Literature has established a positive relationship between CSP and firm financial performance. In particular, the benefits of responsible performance have been argued to accrue in the long term, and as such could be enjoyed mainly by long-term investors. This rationale provides incentives to these investors to promote CSR practices once they become shareholders so that they reap the respective financial rewards in the long run. To empirically test this hypothesis, we estimate the following reduced-form model: 21 21 CSP𝑖,𝑡= 𝛼+ 𝛽1LIO𝑖,𝑡−1 + 𝛄𝐘𝒊,𝒕−𝟏+ 𝜀𝑖,𝑡 (5) CSP𝑖,𝑡= 𝛼+ 𝛽1LIO𝑖,𝑡−1 + 𝛄𝐘𝒊,𝒕−𝟏+ 𝜀𝑖,𝑡 CSP𝑖,𝑡= 𝛼+ 𝛽1LIO𝑖,𝑡−1 + 𝛄𝐘𝒊,𝒕−𝟏+ 𝜀𝑖,𝑡 (5) In Equation (5), the subscripts 𝑖 and 𝑡 denote firm and the time (year), respectively. 15 See Waddock and Graves (1997), Neubaum and Zahra (2006), and Cao, Liang, and Zhan (2016). 3.4. Methodology 𝐶𝑆𝑃 is either 𝐴𝐺𝐺_𝐶𝑆𝑃, 𝐴𝐺𝐺_𝑆, or 𝐴𝐺𝐺_𝐶, representing overall CSP, overall strengths and overall concerns, respectively. Our variable of interest is 𝐿𝐼𝑂. It is calculated as yearend shareholdings of long-term institutional investors relative to total shares outstanding for a given firm on a given year. 𝑌 is a vector of control variables and 𝛄 is a coefficient vector. Following the literature, we include in 𝑌, firm size (MV), book-to-market ratio (BM), leverage (DTA), and return on asset (ROA) as control variables.15 We expect larger firms and more profitable firms to have more slack resources that can be allocated to CSR projects. In contrast, leverage is expected to have a negative effect on overall CSP because as leverage increases, firms pay more interest and have fewer resources available for CSR activities. If a firm’s CSR policies reflect its culture, it is reasonable to assume that its CSP is autocorrelated and as a result the inclusion of lagged dependent variable (𝐴𝐺𝐺_𝐶𝑆𝑃𝑖,𝑡−1) in 𝑌 is warranted. We also include 𝑁𝑜𝑛−𝑙𝑜𝑛𝑔−𝑡𝑒𝑟𝑚 𝐼𝑂 (NLIO) to capture the impact of other institutional ownership (including short-term and medium-term ownership) on future CSP. In addition, we control for industry fixed effects and year fixed effects as in equations (3) and (4). Appendix 2 summarizes definitions and data sources for these control variables. In Equation (5), the subscripts 𝑖 and 𝑡 denote firm and the time (year), respectively. 𝐶𝑆𝑃 is either 𝐴𝐺𝐺_𝐶𝑆𝑃, 𝐴𝐺𝐺_𝑆, or 𝐴𝐺𝐺_𝐶, representing overall CSP, overall strengths and overall concerns, respectively. Our variable of interest is 𝐿𝐼𝑂. It is calculated as yearend shareholdings of long-term institutional investors relative to total shares outstanding for a given firm on a given year. 𝑌 is a vector of control variables and 𝛄 is a coefficient vector. Following the literature, we include in 𝑌, firm size (MV), book-to-market ratio (BM), leverage (DTA), and return on asset (ROA) as control variables.15 We expect larger firms and more profitable firms to have more slack resources that can be allocated to CSR projects. In contrast, leverage is expected to have a negative effect on overall CSP because as leverage increases, firms pay more interest and have fewer resources available for CSR activities. If a firm’s CSR policies reflect its culture, it is reasonable to assume that its CSP is autocorrelated and as a result the inclusion of lagged dependent variable (𝐴𝐺𝐺_𝐶𝑆𝑃𝑖,𝑡−1) in 𝑌 is warranted. 4.1 Descriptive statistics Table 2 reports the descriptive statistics of key variables. Panel A contains the information on CSP indicators. The overall CSP score (AGG_CSP) is negative on average, indicating a relatively higher average concern score than strength score. Indeed, this is confirmed by the lower average of AGG_S compared to AGG_C (0.05 versus 0.08). Looking at the five CSP dimensions separately, 22 five have negative (or zero) scores, ranging between -0.07 and 0. Community (COM_CSP), in contrast, has a positive average score of 0.02. Consistent with the findings in Bouslah, Kryzanowski and M’Zali (2013), the absolute mean values of all six dimensions are close to zero, revealing that the typical firm-year observation in our sample has largely equal number of strengths and concerns. Panel B of Table 2 contains institutional ownership measures and it shows that the average total institutional ownership for firms in the sample is 65%, out of which 17% is short-term and 20% is long-term, according to our churn-rate based classification. Panel C of the same table reports descriptive statistics for our control variables. Over our sample period, the average firm BETA is 1.066, which is almost the same as the beta of the market portfolio, indicating that our sample is comprehensive and representative. The typical firm in our sample has average leverage (DTA) of 0.254 and average book-to-market ratio (BM) of 0.561. 26.1% of our sample firms are included in the S&P 500 Index and the average firm age is about 22 years. In panel D, we report the mean values of our three aggregate CSP measures across tertiles of 𝐿𝐼𝑂 and 𝑆𝐼𝑂, respectively. Consistent with our prediction, both average AGG_SMC and average AGG_S increase as 𝐿𝐼𝑂 increases but decrease as 𝑆𝐼𝑂 increases (from the bottom tertile to the top tertile). It is interesting that AGG_C increases with 𝐿𝐼𝑂, which is counterintuitive and warrants a formal regression analysis, controlling for other relevant factors. The different (actually opposite) patterns of the relation between CSP measures and the two types of institutional ownership (long-term and short- term) signal the importance of examining long-term and short-term investors separately. INSERT TABLE 2 ABOUT HERE Table 3 presents the pairwise correlations among all variables used in the paper. Almost all of the correlation coefficients among control variables are quite low (less than 35%), suggesting that multicollinearity should not affect our analysis. The exception to this, expectedly so, comes from 23 the high correlations between market value, log of stock price and index membership. Iteratively dropping each of these variables from our model specifications does not change our results. 4.2 Main results This section presents our main empirical results. We first investigate the impact of a firm’s overall CSP score on its future institutional ownership. We next zoom in on specific aspects of firms’ CSR activities. More precisely, to further understand the mechanism through which CSP is associated with institutional ownership, we look at overall strengths, overall concerns, and dimension-level CSR scores (e.g. COM_CSP, DIV_CSP, EMP_CSP, ENV_CSP, and PRO_CSP), respectively. Lastly, we examine whether and how (e.g. through enhancing strengths or reducing concerns) long-term investors, as shareholders, promote future CSR activities. A. How do CSR activities affect institutional ownership? A. How do CSR activities affect institutional ownership? Table 4 contains the results, focusing on overall CSP. The insignificant coefficient of the main CSP variable (AGG_CSP) in column 1 implies that institutional investors as a whole might not factor in CSP when they make investment decisions. However, this finding might just as well be the result of the opposite attitudes toward CSR of long-term and short-term investors, as we explained in Section 2. Specifically, it is possible that mixing the two types of investors under the same umbrella buries the true effects of CSP and leads to the insignificant outcome. To disentangle the possibly differing attitudes towards CSR for long-term and short-term investors, we replace the independent variable TIO in specification 1 (representing total institutional ownership) with SIO and LIO in specifications 2 and 3 (representing long-term and short-term institutional ownership respectively). The negative and significant coefficient of AGG_CSP in column 2 24 24 indicates that short-term investors do consider CSR in their decision-making models and they tend to avoid firms with higher CSP. On the other hand, column 3 shows that long-term investors are attracted by CSR and prefer to invest in socially friendly firms. These findings are consistent with our prediction and more importantly, highlight the usefulness of recognizing the significant role investment horizon plays in determining CSR effects on institutional ownership. INSERT TABLE 4 ABOUT HERE The negative bias in the CSP-firm performance link established in the literature and discussed in Section 2 of this paper suggests asymmetric effects of strengths and concerns on future institutional ownership. To empirically test the theory, we replace overall CSP with strengths (AGG_S) and concerns (AGG_C) and re-estimate our model. Indeed, results in Table 5 show that firms’ positive and negative social actions affect investors’ preference differently. The negative coefficients of AGG_S and AGG_C in column 1 imply that institutional investors as a whole (i.e. when not categorising them according to their investment horizon) dislike both strengths and concerns, which is in stark contrast with the finding in column 1 of Table 4 that institutional investors have an indifferent attitude toward CSR. The two contradicting results are consistent with Godfrey et al. (2010)’s argument that the process of netting a firm’s social strengths and concerns “obscures more than it reveals”. More importantly, when taking into consideration investor horizon, the results in columns 2 and 3 suggest that long-term investors’ preference for firms with higher CSP as displayed in Table 4 is mainly driven by an avoidance of firms with higher social controversies, whereas the negative relationship between CSP and short-term ownership is largely caused by short-term investors’ avoidance of firms with higher social strengths. 25 INSERT TABLE 5 ABOUT HERE The overall CSP of a firm is the combination of its performance in several dimensions, including community, diversity, employee relationship, environment, and product. The aggregation of the five dimensions of CSR activities into a single measure AGG_CSP facilitates our analysis, which reveals the general relationship between CSR and institutional ownership. However, individual dimensions may offer additional informative content and enable us to investigate the difference between and relative importance of those dimensions in terms of their influence on firm performance and thus future institutional ownership. Table 6 shows that the impacts of the five dimensions are heterogeneous. Results in columns 6 through 10 indicate that, among the five dimensions, only firms with better employee relationship and higher product quality from a social perspective attract long-term investors. Short-term investors, on the other hand, seem to only pay attention to the environment and product dimensions of CSR activities, as the negative and significant coefficients of ENV_CSP and PRO_CSP in columns 4 and 5 suggest. It is worth noting that the product dimension is the only common dimension that both long-term and short-term investors consider when they select their investment. INSERT TABLE 6 ABOUT HERE B. Do long-term institutional investors promote CSR and if so, how? B. Do long-term institutional investors promote CSR and if so, how? 16 The three examples are: 1). In 2017, PGGM engaged in dialogue with Tyson to improve its wastewater management; 2). PGGM engaged in dialogue with various companies in the mining, oil and gas sectors, including Glencore and FreePort McmoRan, to improve their assessment of potential human rights violations; 3). PGGM also voted against the excessive remuneration policy of McKesson. See https://www pggm nl/english/what-we- do/Documents/Summary Responsible Investment Annual Report pdf ; ) g p y ww.pggm.nl/english/what-we- do/Documents/Summary_Responsible_Investment_Annual_Report.pdf B. Do long-term institutional investors promote CSR and if so, how? Rational long-term institutional investors would promote CSR of their invested firms if, as the literature argues, positive corporate social activities yield long-run financial benefits. Investors increasingly use engagement strategies to ensure that their portfolios incorporate CSR issues. For example, in their 2017 annual report, PGGM, the second largest pension fund in the Netherlands, 26 states: “As an active shareholder, we vote at shareholders’ meetings around the world. In 2017, we voted at 3,524 shareholder meetings. In addition, we attempt to realise ESG improvements by engaging in dialogue with companies and market parties. In 2017 we engaged in dialogue with 361 companies and 8 market parties. We achieved a total of 50 engagement results.” The three concrete examples of the engagement mentioned in the same report are all about reducing CSR controversies.16 Furthermore, studying 682 engagements across 296 firms worldwide, Hoepner et al. (2018) conclude that “the goal of most of these engagements is to engender higher standards of corporate ESG practices that serve as an insurance mechanism against harmful, risk-inducing events”. Therefore, we expect that long-term institutional investors are likely to improve CSR mainly through reducing CSR controversies. Table 7 reports the results of estimating Equation (5). More precisely, the dependent variable in specification 1 is overall CSP (AGG_CSP) one year after the investor bought shares of the firm and the dependent variable in specification 2 is AGG_CSP five years after the purchase. As shown in column 1 of Table 7, 𝐿𝐼𝑂 enters into the regression with a positive and significant coefficient, confirming the intuition that long-term investors promote overall CSP. Interestingly, comparing the results in columns 1 and 2, the positive association between 𝐿𝐼𝑂 and overall CSP is enhanced economically as we increase horizon from one year to five years. This finding may suggest that it takes time for institutional ownership to materially impact the culture of a firm and lead to higher levels of CSP. We look at the asymmetry between strengths and concerns by regressing overall strengths and concerns, separately, on 𝐿𝐼𝑂 and control variables. Columns 3 through 6 in Table 7 contain the results. We employ AGG_S (AGG_C) one year after the investor’s purchase of the firm’s shares as the dependent variable in column 3 (5), and AGG_S (AGG_C) for the respective five-year point 27 as the dependent variable in column 4 (6). 17 To corroborate our results and further show that increase in LIO leads to increase in CSR performance, we conduct regression analysis looking at the change of long-term IO and the change of CSR scores. The change regression results show that indeed long-term institutional investors increase CSR performance and they do so by immediately reducing controversies (see Appendix 3 Table A3.1). B. Do long-term institutional investors promote CSR and if so, how? Overall, it appears that as shareholders, long-term investors not only increase positive social activities but also decrease social controversies. Interestingly, our analysis further discovers certain asymmetry between the two types of activities. Specifically, the insignificant coefficient of 𝐿𝐼𝑂 in column 5 combined with the significant coefficient in column 6 indicate that long-term investors promote social strengths rather slowly. In contrast, results in column 3 suggest that long-term investors almost immediately reduce controversies after becoming shareholders. This may have to do either with the asymmetric financial effects of concerns versus strengths as we previously noted (greater for the former) or it may be that it simply takes more time, know-how and overall resources for a firm to proactively do good than to reduce its socially/environmentally harmful activities.17 INSERT TABLE 7 ABOUT HERE In Section 4.2.A, we show that long-term investors intend to invest in firms with high CSR performance. Then, if firms already investing in CSR (e.g. have already established a stable CSR policy) are more likely to keep investing, the positive relationship between LIO and CSP shown in Table 7 might simply be the result of the persistence of CSR performance. To address this concern, we divide our sample firms into two groups according to a firm’s CSP as follows: one that consists of high CSP firms—firms with a CSP that is higher than the industry average, and the other consists of low CSP firms—firms with a CSP that is lower than the industry average. We then investigate the impact of long-term IO on CSP with the two sub-samples, respectively. As shown in Appendix 3 Table A3.2, we find significant long-term IO effects for both subsamples and the 28 effect in some cases is even stronger for the low CSP group (see columns 4 and 12). This indicates that the long-term IO effect on CSP shown in Table 7 is not likely to be solely due to the persistence of CSR performance and thus alleviates the relevant concern. Having shown that long-term investors promote overall CSP, we now have a closer look at the various components of CSR. Table 8 contains the results, which are qualitatively similar and consistent with the results obtained with overall CSP, confirming a positive relationship between LIO and CSP. Specifically, we find that long-term institutional investors promote almost all dimensions of CSP. Our results are in stark contrast to those in Borghesi et al. (2014), which are derived using overall IO. This once again highlights the important role that investment horizon plays in the IO-CSP relationship.18 18 Note that NLIO (short-term + medium-term IO) has a negative impact on almost all CSP dimensions. 4.3 Accounting for endogeneity A common criticism in studies investigating market reactions to CSP is the potential endogeneity between the CSP proxies and financial metrics of interest. Our use of lagged independent variables in our baseline regressions allows us to alleviate this issue as we do not explore a contemporaneous link between institutional ownership and corporate social performance. Instead, we posit, investigate and find a bidirectional, lead-lag relationship between the two, where CSP and institutional ownership influence each other, albeit with some time needed for this feedback process to occur. This seems intuitive enough as we would not expect immediate changes of institutional ownership due to changes in CSP as this would entail significant 29 transaction costs in rebalancing the portfolios of institutional investors. We would expect even less so an immediate change in CSP given changes in the profile of the institutional owners of a firm. This is due to the sizeable upfront costs and time constraints that are frequently associated with changing the social and environmental output of a given firm. Nevertheless, it needs to be recognised that every feedback process like the one we have found is dynamic and as such there may be a part of the interaction between the two variables that occurs in a contemporaneous fashion. A further concern arises from the potential omitted variable bias. Specifically, there may be some firm characteristics beyond what we have controlled in our baseline regressions that are correlated with both the dependent variable and independent variables of interest. To address the potential endogeneity issue and reinforce the result of existence of the virtuous circle of long-term institutional ownership and responsible firm conduct, we perform several robustness tests in the context of instrumental variable (IV) estimations. We first look at the causality that goes from CSP to institutional ownership (our Hypothesis 1). Following Benlemlih and Bitar (2016), we use as instruments the initial level of the firm’s overall CSP score (𝐴𝐺𝐺_𝐶𝑆𝑃_𝐼𝑁𝐼) and the industry-year average of overall CSP scores (𝐴𝐺𝐺_𝐶𝑆𝑃_𝐼𝑌). These two instruments are likely to be correlated with the firm’s contemporaneous CSR score (the relevancy condition) and are unlikely to be endogenous to the firm’s contemporaneous institutional ownership (the exclusion restriction). Our IV approach consists of two steps. 19 For the detailed explanations of the Russell Index reconstitution, please refer to www.ftserussell.com/research-insights/russell-reconstitution. 4.3 Accounting for endogeneity 𝐴𝐺𝐺_𝐶𝑆𝑃_𝐼𝑁𝐼 and 𝐴𝐺𝐺_𝐶𝑆𝑃_𝐼𝑌 are used as instruments in the first stage regression: 𝐴𝐺𝐺_𝐶𝑆𝑃𝑖,𝑡= 𝛼+ 𝛽1AGG_CSP_INI𝑖,𝑡+ 𝛽2AGG_CSP_IY𝑖,𝑡+ 𝛄𝐗𝒊,𝒕+ 𝜀𝑖,𝑡 (6) 𝐴𝐺𝐺_𝐶𝑆𝑃𝑖,𝑡= 𝛼+ 𝛽1AGG_CSP_INI𝑖,𝑡+ 𝛽2AGG_CSP_IY𝑖,𝑡+ 𝛄𝐗𝒊,𝒕+ 𝜀𝑖,𝑡 (6) (6) where we include in 𝐗 the same control variables as in Equation (3). In the second stage regression, we re-estimate Equation (3) by replacing AGG_CSP with 𝐴𝐺𝐺_𝐶𝑆𝑃 ̂ , the predicted value of overall CSP from Equation (6). 30 30 The 2SLS regression results are contained in Table 9. We find in the first stage regression estimates that the two IVs are highly significant with expected signs (column 1). The results of the second stage regressions are presented in columns 2 through 4. The insignificant (column 2), negatively significant (column 3) and positively significant (column 4) coefficients of 𝐴𝐺𝐺_𝐶𝑆𝑃 clearly show that investment horizon matters, and short-term investors tend to avoid firms with higher CSP, whereas long-term investors tend to do the opposite, reinforcing our earlier baseline regression findings regarding Hypothesis 1. 20 Before 2003, only around 1100 firms were covered by KLD and therefore the overlap between KLD- covered firms and the Russell 2000 firms was limited. To address this concern, we re-estimate Table 10 using a subsample spanning from 2003 to 2012. The results become even stronger (see Appendix 3, table A3.3). INSERT TABLE 9 ABOUT HERE To check the robustness of our Hypothesis 3 results and ensure the path of causality that runs from long-term institutional ownership to CSP, we exploit the nature of the Russell index composition and annual reconstitution, following Fich et al. (2015), and Crane et al. (2016). The Russell 1000 and 2000 indexes are reconstituted in June every year. Based on the market capitalization of US firm common stocks as of May 31, the largest 1,000 firms are included in the Russell 1000 index and the subsequent 2,000 firms are included in the Russell 2000 index.19 Both indexes are value-weighted and no other criteria besides the market capitalization are used in the index reconstitution. Therefore, when a stock drops from the Russell 1000 to the Russell 2000 index or gets newly added in the Russell 2000 index, the index-tracking (long-term) institutional ownership of the stock will increase exogenously. On the other hand, there is a negative and exogenous shock on a firm's index-tracking (long-term) institutional ownership when a stock moves up from the Russell 2000 to the Russell 1000 index or gets excluded from the Russell 2000 index. 31 Our IV approach consists of two steps. The switch of firms between the two Russell indexes and the inclusion/exclusion of firms in the Russell 2000 index are used as the IVs in our first stage regression: 𝐿𝐼𝑂𝑖,𝑡= 𝛼+ 𝛽1R1TR2𝑖,𝑡+ 𝛽2R2TR1𝑖,𝑡+ 𝛽3R2TN𝑖,𝑡+ 𝛽4NTR2𝑖,𝑡+ 𝛄𝐘𝒊,𝒕+ 𝜀𝑖,𝑡 (7) where R1TR2𝑖,𝑡 (R2TR1𝑖,𝑡) is an indicator variable equal to 1 if firm 𝑖 switches from the Russell 1000 (2000) index to the Russell 2000 (1000) index in year 𝑡 and 0 otherwise. R2TN𝑖,𝑡 (NTR2𝑖,𝑡) is a dummy equal to 1 if firm 𝑖 leaves (enters) the Russell 2000 index and 0 otherwise. The relevancy condition of our IVs is satisfied because the index reconstitution apparently affects the long-term institutional ownership in all firms. At the same time the exclusion restriction is also satisfied because the only index assignment rule is mechanically based on the rank of stock market capitalization, i.e. firm size. Put differently, switching between the two Russell indexes should not have any direct effect on a firm’s CSR activities. We include in 𝒀 the same control variables as in Equation (5). In the second stage regression, we re-estimate Equation (5) by replacing 𝐿𝐼𝑂 with 𝐿𝐼𝑂 ̂ , the predicted value of long-term institutional ownership from Equation (7). INSERT TABLE 9 ABOUT HERE 𝐿𝐼𝑂𝑖,𝑡= 𝛼+ 𝛽1R1TR2𝑖,𝑡+ 𝛽2R2TR1𝑖,𝑡+ 𝛽3R2TN𝑖,𝑡+ 𝛽4NTR2𝑖,𝑡+ 𝛄𝐘𝒊,𝒕+ 𝜀𝑖,𝑡 (7) (7) Table 10 reports the 2SLS regression results. Looking at the first stage regression estimates in column 1, all IVs are statistically significant, confirming that the relevancy condition is satisfied. The results of the second stage regressions are presented in columns 2 through 7. It is clear that the results are consistent with those contained in Table 7, supporting our baseline analysis conclusion regarding Hypothesis 3 that long-term investors improve overall CSP of their owned firms (columns 2 and 4).20 32 INSERT TABLE 10 ABOUT HERE It is worth mentioning that MSCI ESG Research, the successor of KLD, introduced significant ratings methodology changes in 2010, following the takeover of RiskMetrics by MSCI. To investigate the potential impact of these methodology changes on our main results, we conduct further robustness tests. Specifically, we re-estimate Tables 9 and 10 using a sub-sample period of 2003-2009. The results of these robustness tests remain qualitatively the same (see Appendix 3 Tables A3.4 and A3.5). 5. Conclusions Our study investigates the impact that investment horizon has on institutional investors’ preference for corporate social performance. Unlike previous literature, we use a direct measure of institutional investors’ trading frequency and, consequently, the average duration of their holdings, in order to distinguish between long-term and short-term investors. In addition, we explore to what extent the well-established asymmetry in stakeholder perception (and financial impact) between positive and negative CSP outcomes also influences institutional demand for the associated firms. Finally, we expand our exploration in order to identify whether there is also a link running in the opposite direction, i.e. if long-term/short-term investors also attempt (and manage) to influence corporate culture and change the levels of corporate social performance of the firms in their portfolios. Our results are revealing and intuitive as they are highly aligned with the predictions that stakeholder theory makes regarding the value-relevant impacts of stronger CSP –which should manifest in the long-run (Jones, 1995). Indeed, we show that although institutional ownership as 33 a whole appears to be unrelated to the CSP of invested firms, long-term investors prefer higher CSP and short-term investors tend to avoid it. These results are also in line with the conclusions of Bushee (1998) who finds that the levels by which firms are held by long-term investors are inversely associated with “managerial myopia”. Such companies tend to be less pressed to provide immediate results to their investors and hence appreciate resources that are more likely to generate rather delayed returns (such as R&D investments or improved CSP). Additional exploratory analysis reveals that long-term investors’ preference for higher CSP is mainly driven by a significant avoidance of firms associated with more controversies whereas the negative link between short-term owners and CSP is primarily a result of their dislike for corporations with more social/environmental strengths. Lastly, long term investors seem to promote an overall betterment of the social performance of the firms they own but this improvement takes time – as results are stronger when we look at 5-year horizons. Hence, the picture that emerges is one of a “virtuous circle” between long-term institutional ownership and CSP, where one pushes the other to higher levels. The results are of tremendous importance to firm managers. 5. Conclusions KLD Database COM_CSP Community score, calculated by taking the net difference between adjusted community strength and concern scores. KLD Database DIV_CSP Diversity score, calculated by taking the net difference between adjusted diversity strength and concern scores. KLD Database EMP_CSP Employee score, calculated by taking the net difference between adjusted employee strength and concern scores. KLD Database ENV_CSP Environment score, calculated by taking the net difference between adjusted environmental strength and concern scores. KLD Database PSQ_CSP Product score, calculated by taking the net difference between adjusted product strength and concern scores. KLD Database TIO Total institutional ownership, calculated as yearend shareholdings of all institutional investors relative to total shares outstanding. 13F Database LIO Long-term institutional ownership, calculated as yearend shareholdings of long-term institutional investors relative to total shares outstanding. At each year end, institutional investors are classified as long-term or short-term based on their churn rates. 13F Database SIO Short-term institutional ownership, calculated as yearend shareholdings of long-term institutional investors relative to total shares outstanding. At each year end, institutional investors are classified as long-term or short-term based on their churn rates. 13F Database NLIO Non-long-term institutional ownership, calculated as the difference between TLO and LIO. 13F Database Appendix 1 Definitions and data sources of CSR and institutional ownership measures Variable Definition Source AGG_CSP Overall CSP score, calculated as the sum of yearly adjusted individual CSP scores of the five main qualitative issue areas: community, diversity, employee relationship, environment, and product. For each dimension, adjusted CSP is computed by taking the net difference between adjusted strength and concern scores. KLD Database AGG_S Overall Strength index, calculated as the sum of yearly adjusted individual Strength scores of the five main qualitative issue areas: community, diversity, employee relationship, environment, and product. KLD Database AGG_C Overall Concern index, calculated as the sum of yearly adjusted individual Concern scores of the five main qualitative issue areas: community, diversity, employee relationship, environment, and product. KLD Database COM_CSP Community score, calculated by taking the net difference between adjusted community strength and concern scores. KLD Database DIV_CSP Diversity score, calculated by taking the net difference between adjusted diversity strength and concern scores. KLD Database EMP_CSP Employee score, calculated by taking the net difference between adjusted employee strength and concern scores. KLD Database ENV_CSP Environment score, calculated by taking the net difference between adjusted environmental strength and concern scores. 5. Conclusions Executives which are proponents of the ethical and financial incentives for better CSP (especially via the avoidance of any controversial practices) can rest assured that their initiatives will be appreciated by long-term investors who will also, in turn, push for further improvements in this direction. Individual responsible investors can also be reassured that their interest in good social corporate performers is shared by institutional investors who will, ceteris paribus, hold these firms for longer periods of time and thus help in retaining their prices to certain levels and reducing their downside risk. Lastly, policy makers who wish to promote corporate and market sustainability will now be more definitively informed that it is long-term investing institutions who mostly appreciate such characteristics and thus, it is them who should be appropriately incentivised. 34 34 Though we make some novel contributions in the literature, more work needs to be done in this direction. KLD STATS is the most widely used database in this field, yet it is not without its limitations and drawbacks. Alternative sources of CSP data are required in order to offer convergent validity to our conclusions. Our analysis is also entirely limited to the US market. Given the increased popularity and importance of SRI in Europe as well as in other areas around the globe, our methodology could be replicated to see if our main conclusions hold or whether there is a geographic element to them. Lastly, it would be really interesting for future research to explore whether the relationships we uncover also hold outside of the equity market (particularly for bonds where there is now substantial relevant literature). 35 35 Appendix 1 Definitions and data sources of CSR and institutional ownership measures Variable Definition Source AGG_CSP Overall CSP score, calculated as the sum of yearly adjusted individual CSP scores of the five main qualitative issue areas: community, diversity, employee relationship, environment, and product. For each dimension, adjusted CSP is computed by taking the net difference between adjusted strength and concern scores. KLD Database AGG_S Overall Strength index, calculated as the sum of yearly adjusted individual Strength scores of the five main qualitative issue areas: community, diversity, employee relationship, environment, and product. KLD Database AGG_C Overall Concern index, calculated as the sum of yearly adjusted individual Concern scores of the five main qualitative issue areas: community, diversity, employee relationship, environment, and product. 5. Conclusions KLD Database PSQ_CSP Product score, calculated by taking the net difference between adjusted product strength and concern scores. KLD Database TIO Total institutional ownership, calculated as yearend shareholdings of all institutional investors relative to total shares outstanding. 13F Database LIO Long-term institutional ownership, calculated as yearend shareholdings of long-term institutional investors relative to total shares outstanding. At each year end, institutional investors are classified as long-term or short-term based on their churn rates. 13F Database SIO Short-term institutional ownership, calculated as yearend shareholdings of long-term institutional investors relative to total shares outstanding. At each year end, institutional investors are classified as long-term or short-term based on their churn rates. 13F Database NLIO Non-long-term institutional ownership, calculated as the difference between TLO and LIO. 13F Database ndix 1 Definitions and data sources of CSR and institutional ownership measures Variable 36 Appendix 2 Definitions and data sources of control variables Appendix 2 Definitions and data sources of control variables Variable Definition Source MV Market capitalization, calculated as the log of the product of the stock price and number of shares outstanding at year end. CRSP Database AGE Firm age, calculated as the log of the number of quarters since first return appears in CRSP. CRSP Database DY Dividend yield, calculated as quarterly total dividends per share divided by stock price of the previous quarter. CRSP Database & Compustat S&PIDX Dummy variable that equals one if a firm is listed in the S&P 500 index and zero otherwise. CRSP Database DTA Leverage, calculated as total debt divided by total asset. Compustat BETA Systematic risk (𝛽1,𝑖), estimated from the following regression: 𝑅𝑖,𝑡−𝑅𝑓= 𝛼𝑖+ 𝛽1,𝑖(𝑅𝑚−𝑅𝑓) + 𝛽2,𝑖𝑆𝑀𝐵+ 𝛽3,𝑖(𝐻𝑀𝐿) + 𝜖𝑖,𝑡 Using the previous 5-year monthly returns. CRSP Database pp Variable Definition Source MV Market capitalization, calculated as the log of the product of the stock price and number of shares outstanding at year end. CRSP Database AGE Firm age, calculated as the log of the number of quarters since first return appears in CRSP. CRSP Database DY Dividend yield, calculated as quarterly total dividends per share divided by stock price of the previous quarter. CRSP Database & Compustat S&PIDX Dummy variable that equals one if a firm is listed in the S&P 500 index and zero otherwise. CRSP Database DTA Leverage, calculated as total debt divided by total asset. 5. Conclusions Compustat BETA Systematic risk (𝛽1,𝑖), estimated from the following regression: 𝑅𝑖,𝑡−𝑅𝑓= 𝛼𝑖+ 𝛽1,𝑖(𝑅𝑚−𝑅𝑓) + 𝛽2,𝑖𝑆𝑀𝐵+ 𝛽3,𝑖(𝐻𝑀𝐿) + 𝜖𝑖,𝑡 Using the previous 5-year monthly returns. CRSP Database IRISK Idiosyncratic risk, calculated as √𝑣𝑎𝑟(𝜖𝑖,𝑡) ∗3, where 𝑣𝑎𝑟(𝜖𝑖,𝑡) is the variance of the error term derived from the above equation using previous 5-year monthly returns. CRSP Database PRC Share price CRSP Database TOV Turnover of stock holdings, calculated as quarterly trading volume divided by total shares outstanding. CRSP Database RET Cumulative gross stock return over the past three months. CRSP Database EPS Earnings per share. Compustat BM Book-to-market ratio, calculated as book value of equity divided by market value of equity. Compustat ROA Return on assets, calculated as net income divided by total assets. Compustat CASH The ratio of cash and short term investments to total asset. Compustat BETA Systematic risk (𝛽1,𝑖), estimated from the following regression: 𝑅𝑖,𝑡−𝑅𝑓= 𝛼𝑖+ 𝛽1,𝑖(𝑅𝑚−𝑅𝑓) + 𝛽2,𝑖𝑆𝑀𝐵+ 𝛽3,𝑖(𝐻𝑀𝐿) + 𝜖𝑖,𝑡 Using the previous 5 year monthly returns CRSP Database IRISK Idiosyncratic risk, calculated as √𝑣𝑎𝑟(𝜖𝑖,𝑡) ∗3, where 𝑣𝑎𝑟(𝜖𝑖,𝑡) is the variance of the error term derived from the above equation using previous 5-year monthly returns. CRSP Database PRC Share price CRSP Database TOV Turnover of stock holdings, calculated as quarterly trading volume divided by total shares outstanding. CRSP Database RET Cumulative gross stock return over the past three months. CRSP Database EPS Earnings per share. Compustat BM Book-to-market ratio, calculated as book value of equity divided by market value of equity. Compustat ROA Return on assets, calculated as net income divided by total assets. Compustat CASH The ratio of cash and short term investments to total asset. Compustat 37 37 Appendix 3 Additional robustness tests All independent variables are in the current year t. The variable of interest, Long term institutional ownership (LIO), is defined based on churn ratio as in Yan and Zhang (2009). Control variables include ownership of institutional investors that are not long term (NLIO), firm size (LOGMV), Book to market ratio (BM), Return on assets (ROA), and leverage ratio (DTA), Cash holding (CASH). Detailed variable definitions can be found in appendix 1 and 2. Time fixed effects (Year) and industry fixed effects (2 Digit SIC code) are included in all regressions. titutional investors’ influence on future overall CSP (High CSP firms versus Low CSP firms) f d b d b A Table A3.2 Institutional investors’ influence on future overall CSP (High CSP firms versus Low CSP firms) h i l f CSP f i i i l hi d h l i bl D d i bl AGG CS Table A3.2 Institutional investors’ influence on future overall CSP (High CSP firms versus Low CSP firms) Table A3.2 displays the regression results of CSP measures on measures of institutional ownership and other control variables. Dependent variables AGG_CSP, AGG_C and AGG_S denote standardized overall CSP score, CSP concerns score and CSP strengths score respectively. Dependent variables in column 1, 2,5,6,9 and 10 are measured at t+1 year while dependent variables in column 3, 4, 7, 8, 11 and 12 are measured at t+5 years. Column 1,3,5,7,9, and 11 are results based on firms with CSP higher than industry average. Column 2, 4, 6, 8 ,10 and 12 represent results for firms with CSP lower than industry average. All independent variables are in the current year t. The variable of interest, Long term institutional ownership (LIO), is defined based on churn ratio as in Yan and Zhang (2009). Control variables include ownership of institutional investors that are not long term (NLIO), firm size (LOGMV), Book to market ratio (BM), Return on assets (ROA), and leverage ratio (DTA), Cash holding (CASH). Detailed variable definitions can be found in appendix 1 and 2. Time fixed effects (Year) and industry fixed effects (2 Digit SIC code) are included in all regressions. Table A3.2 displays the regression results of CSP measures on measures of institutional ownership and other control variables. Dependent variables AGG_CSP, AGG_C and AGG_S denote standardized overall CSP score, CSP concerns score and CSP strengths score respectively. Appendix 3 Additional robustness tests Dependent variables in column 1, 2,5,6,9 and 10 are measured at t+1 year while dependent variables in column 3, 4, 7, 8, 11 and 12 are measured at t+5 years. Column 1,3,5,7,9, and 11 are results based on firms with CSP higher than industry average. Column 2, 4, 6, 8 ,10 and 12 represent results for firms with CSP lower than industry average. All independent variables are in the current year t. The variable of interest, Long term institutional ownership (LIO), is defined based on churn ratio as in Yan and Zhang (2009). Control variables include ownership of institutional investors that are not long term (NLIO), firm size (LOGMV), Book to market ratio (BM), Return on assets (ROA), and leverage ratio (DTA), Cash holding (CASH). Detailed variable definitions can be found in appendix 1 and 2. Time fixed effects (Year) and industry fixed effects (2 Digit SIC code) are included in all regressions. , and * denote significance at the 10%, 5%, and 1% level respectively. Standard errors are clustered at firm level and robust t-statistics are reported in brackets. Appendix 3 Additional robustness tests Table A3.1 Institutional investors’ influence on future overall CSP (change regression) Table A3.1 displays the regression results of the change of CSP measures on the change of long-term institutional ownership and other control variables. Dependent variables ΔAGG_CSP, ΔAGG_C and ΔAGG_S denote the change of standardized overall CSP score, CSP concerns score and CSP strengths score respectively. Dependent variables are measured at t+1 year. All independent variables are in the current year t. The variable of interest, the change of long-term institutional ownership (ΔLIO), is defined based on churn ratio as in Yan and Zhang (2009) and calculated as LIO𝑡−LIO𝑡−1. Control variables include ownership of institutional investors that are not long-term (NLIO), firm size (LOGMV), Book to market ratio (BM), Return on assets (ROA), and leverage ratio (DTA), Cash holding (CASH). Detailed variable definitions can be found in appendix 1 and 2. Time fixed effects (Year) and industry fixed effects (2 Digit SIC code) are included in all regressions. , * and * denote significance at the 10%, 5%, and 1% level respectively. Standard errors are clustered at firm level and robust t-statistics are reported in brackets. (1) (2) (3) VARIABLES ΔAGG_CSP(t+1) ΔAGG_C(t+1) ΔAGG_S(t+1) ΔLIO 0.022 -0.024*** -0.011 [2.45] [-3.72] [-1.26] NLIO 0.006* 0.002 0.005 [1.78] [0.88] [1.46] LOGMV 0.007* 0.000 0.007* [14.52] [0.32] [13.62] BM 0.001 0.000 0.001 [1.03] [0.42] [0.86] ROA -0.008 -0.000 -0.004 [-2.01] [-0.11] [-0.99] DTA -0.004 0.000 -0.004 [-1.28] [0.15] [-1.05] CASH -0.012* -0.001 -0.011* [-3.22] [-0.19] [-2.81] CONSTANT -0.297* 0.080* -0.204* [-24.35] [7.92] [-16.22] Observations 16,573 16,573 16,573 R-squared 0.344 0.286 0.101 Time FE YES YES YES IND FE YES YES YES 38 Table A3.2 Institutional investors’ influence on future overall CSP (High CSP firms versus Low CSP firms) Table A3.2 Institutional investors’ influence on future overall CSP (High CSP firms versus Low CSP firms) Table A3.2 displays the regression results of CSP measures on measures of institutional ownership and other control variables. Dependent variables AGG_CSP, AGG_C and AGG_S denote standardized overall CSP score, CSP concerns score and CSP strengths score respectively. Dependent variables in column 1, 2,5,6,9 and 10 are measured at t+1 year while dependent variables in column 3, 4, 7, 8, 11 and 12 are measured at t+5 years. Column 1,3,5,7,9, and 11 are results based on firms with CSP higher than industry average. Column 2, 4, 6, 8 ,10 and 12 represent results for firms with CSP lower than industry average. Appendix 3 Additional robustness tests Instrumental variables used are dummy variables indicating the stock switching from the Russell 1000 index into the Russell 2000 index (R1TR2), switching from the Russell 2000 index into the Russell 1000 index (R2TR1), dropping out of the Russell 2000 index due to a market value decrease (R2TN) and getting included in the Russell 2000 index due to a market value increase (NTR2). The fitted values of LIO from the first stage regression are then used in the second stage regressions displayed in columns 2 through 7. The dependent variables in the second stage regressions AGG_CSP, AGG_C and AGG_S denote standardized overall CSP score, CSP concerns score and CSP strengths score respectively. The dependent variables in columns 2, 4 and 6 are measured at the t+1 year while the dependent variables in columns 3, 5 and 7 are measured at the t+5 year. All independent variables are in the current year t. Control variables include ownership of institutional investors that are not long term (NLIO), firm size (LOGMV), book to market ratio (BM), return on assets (ROA), leverage ratio (DTA) and Cash Holding (CASH). Detailed variable definitions can be found in appendix 1 and 2. Time fixed effects (Year) and industry fixed effects (2 Digit SIC code) are included in all regressions. , * and *** denote significance at the 10%, 5%, and 1% level respectively. Standard errors are clustered at firm level and robust t-statistics are reported in brackets. d at firm level and robust t statistics are reported in brackets. Appendix 3 Additional robustness tests (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) VARIABLES AGG_CSP(t+1) AGG_CSP(t+1) AGG_CSP(t+5) AGG_CSP(t+5) AGG_C(t+1) AGG_C(t+1) AGG_C(t+5) AGG_C(t+5) AGG_S(t+1) AGG_S(t+1) AGG_S(t+5) AGG_S(t+5) LIO 0.009 0.009 0.012 0.034* -0.026* -0.018 -0.021** 0.011 -0.020* -0.010 0.018 0.027* [0.92] [1.09] [0.61] [1.70] [-4.09] [-2.43] [-2.10] [0.79] [-1.87] [-1.37] [1.00] [1.76] NLIO -0.039* 0.012* -0.056* -0.013 -0.017* -0.009 -0.002 0.007 -0.042* -0.013* -0.039* -0.009 [-7.51] [2.75] [-5.94] [-1.33] [-4.70] [-2.25] [-0.39] [1.04] [-7.17] [-3.16] [-4.35] [-1.03] LOGMV 0.017* 0.001 0.028* 0.018* 0.014* 0.014* 0.015* 0.016* 0.031* 0.019* 0.044* 0.036*** [19.90] [1.41] [17.46] [10.83] [24.66] [19.68] [15.69] [14.01] [30.69] [23.65] [27.87] [23.26] BM -0.000 -0.001 0.005 0.013* 0.006* 0.004 0.018* 0.009* 0.007 0.004 0.017 0.023* [-0.21] [-0.65] [0.78] [1.82] [3.31] [1.45] [5.00] [1.87] [2.17] [1.54] [2.35] [4.77] ROA 0.003 0.008* 0.039* -0.021 -0.018* -0.012 -0.017 -0.007 -0.018* -0.006 -0.023 -0.011 [0.49] [2.81] [2.58] [-1.15] [-3.57] [-2.25] [-2.11] [-0.87] [-2.62] [-1.35] [-1.59] [-1.22] DTA -0.003 -0.014* -0.028* -0.015 0.001 0.010 -0.000 0.012* -0.008 0.003 -0.040*** -0.007 [-0.54] [-3.43] [-2.66] [-1.50] [0.18] [2.55] [-0.03] [1.74] [-1.40] [0.82] [-4.23] [-0.86] CASH -0.004 0.000 -0.016 0.011 -0.004 0.000 -0.013* -0.004 -0.003 -0.001 -0.028* 0.005 [-0.58] [0.00] [-1.32] [1.05] [-0.88] [0.08] [-1.93] [-0.58] [-0.42] [-0.25] [-2.63] [0.57] CONSTANT -0.318* -0.075* -0.547* -0.430* -0.194* -0.215* -0.250* -0.244* -0.587* -0.356* -0.857* -0.735* [-17.58] [-3.84] [-16.14] [-10.39] [-15.25] [-11.11] [-11.89] [-7.89] [-26.82] [-17.68] [-24.54] [-19.98] Observations 9,832 9,990 5,176 4,733 9,605 9,812 5,171 4,682 9,605 9,812 5,171 4,682 R-squared 0.341 0.309 0.304 0.260 0.226 0.327 0.263 0.312 0.359 0.183 0.362 0.307 Time FE YES YES YES YES YES YES YES YES YES YES YES YES IND FE YES YES YES YES YES YES YES YES YES YES YES YES 39 Table A3.3 Institutional investors’ influence on future CSP: 2SLS (03-12) Table A3.3 Institutional investors influence on future CSP: 2SLS (03 12) Table A3.3 displays the 2SLS regression results of CSP measures on long-term institutional ownership and other control variables, using a sub-sample of 2003-2012. The dependent variable in the first stage regression (reported in column 1) is the variable of interest, long-term institutional ownership (LIO), defined based on churn ratio as in Yan and Zhang (2009). Appendix 3 Additional robustness tests (1) (2) (3) (4) (5) (6) (7) VARIABLES LIO AGG_CSP (t+1) AGG_CSP (t+5) AGG_C (t+1) AGG_C (t+5) AGG_S (t+1) AGG_S (t+5) R1TR2 0.053* [6.87] R2TN -0.055* [-8.08] R2TR1 0.026* [3.09] NTR2 -0.042* [-8.05] LIO 0.284* 0.624* -0.211* -0.125 0.033 0.473 [3.64] [2.75] [-4.0`2] [-1.15] [0.51] [2.37] NLIO -0.036* -0.079* -0.005 0.004 -0.038* -0.066* [-6.90] [-6.80] [-1.38] [0.66] [-8.01] [-6.47] LOGMV 0.013* 0.015* 0.027* 0.016* 0.018* 0.033* 0.046* [12.55] [10.82] [8.17] [17.67] [10.65] [25.98] [15.85] BM 0.008 -0.004* -0.001 0.006 0.020* 0.004* 0.010 [2.33] [-2.73] [-0.17] [2.44] [4.94] [1.67] [1.43] ROA 0.015* -0.005 -0.014 -0.009 -0.013 -0.019* -0.037 [3.36] [-1.05] [-0.75] [-2.00] [-1.43] [-4.00] [-2.41] DTA -0.014* -0.007 -0.015 0.005 0.004 -0.003 -0.022 [-1.84] [-1.46] [-1.36] [1.42] [0.74] [-0.72] [-2.41] CASH -0.070* 0.019** 0.034* -0.019*** -0.019* -0.003 0.009 [-8.83] [2.54] [1.79] [-3.58] [-1.92] [-0.42] [0.55] CONSTANT -0.124* -0.341* -0.636* -0.184* -0.286* -0.633* -0.925*** [-6.14] [-17.17] [-14.74] [-13.43] [-11.57] [-32.12] [-24.01] OBSERVATIONS 18,051 14,853 5,853 14,506 5,969 14,506 5,969 R-SQUARED 0.266 0.250 0.322 0.278 0.338 0.308 0.411 TIME FE YES YES YES YES YES YES YES IND FE YES YES YES YES YES YES YES 40 Table A3.4 Institutional investors’ preference of aggregate CSP: 2SLS (03-09) Table A3.4 displays the 2SLS regression results of various measures of institutional ownership on overall CSP and other control variables, using a sub-sample of 2003-2009. The first column displays the regression of AGG_CSP on the instrumental variables AGG_CSP_INT (the initial value of CSP) and AGG_CSP_IY (the average CSP of firms in the same industry at the same year) and other control variables. The fitted values of AGG_CSP from the first stage regression are then used in the second stage regressions displayed in columns 2 through 4. The dependent variables TIO, SIO, and LIO denote ownership of all institutional investors, short-term institutional investors and long-term institutional investors respectively, measured at the year t+1. Long term and short-term investors are defined following Yan and Zhang (2009) based on churn ratio. All independent variables are measured in the current year t. The variable of interest is the overall CSP score (AGG_CSP) based on the KLD database. Control variables include firm size (LOGMV), natural log of firm age (LOGAGE), natural log of stock price (LOGPRC), Book to market ratio (BM), CAPM beta of stock (BETA), idiosyncratic volatility (IRISK), quarterly stock turnover (TOV), earnings per share (EPS), index membership dummy (S&PIDX), dividend yield (DY), leverage (DTA). Appendix 3 Additional robustness tests The dependent variables in the second stage regressions AGG_CSP, AGG_C and AGG_S denote standardized overall CSP score, CSP concerns score and CSP strengths score respectively. The dependent variables in columns 2, 4 and 6 are measured at the t+1 year while the dependent variables in columns 3, 5 and 7 are measured at the t+5 year. All independent variables are in the current year t. Control variables include ownership of institutional investors that are not long term (NLIO), firm size (LOGMV), book to market ratio (BM), return on assets (ROA), leverage ratio (DTA) and Cash Holding (CASH). Detailed variable definitions can be found in appendix 1 and 2. Time fixed effects (Year) and industry fixed effects (2 Digit SIC code) are included in all regressions. , * and *** denote significance at the 10%, 5%, and 1% level respectively. Standard errors are clustered at firm level and robust t-statistics are reported in brackets. Table A3.5 Institutional investors’ influence on future CSP: 2SLS (03-09) Table A3.5 displays the 2SLS regression results of CSP measures on long-term institutional ownership and other control variables, using a sub-sample of 2003-2009. The dependent variable in the first stage regression (reported in column 1) is the variable of interest, long-term institutional ownership (LIO), defined based on churn ratio as in Yan and Zhang (2009). Instrumental variables used are dummy variables indicating the stock switching from the Russell 1000 index into the Russell 2000 index (R1TR2), switching from the Russell 2000 index into the Russell 1000 index (R2TR1), dropping out of the Russell 2000 index due to a market value decrease (R2TN) and getting included in the Russell 2000 index due to a market value increase (NTR2). The fitted values of LIO from the first stage regression are then used in the second stage regressions displayed in columns 2 through 7. The dependent variables in the second stage regressions AGG_CSP, AGG_C and AGG_S denote standardized overall CSP score, CSP concerns score and CSP strengths score respectively. The dependent variables in columns 2, 4 and 6 are measured at the t+1 year while the dependent variables in columns 3, 5 and 7 are measured at the t+5 year. All independent variables are in the current year t. Appendix 3 Additional robustness tests Detailed variable definition can be found in appendix 1 and 2. Time fixed effects (Year) and industry fixed effects (2 Digit SIC code) are included in all regressions. , * and * denote significance at the 10%, 5%, and 1% level respectively. Standard errors are clustered at firm level and robust t-statistics are reported in brackets. p (1) (2) (3) (4) VARIABLES AGG_CSP TIO (t+1) SIO (t+1) LIO (t+1) AGG_CSP_INT 0.496* [17.43] AGG_CSP_IY 0.910* [10.34] 0.054 -0.089* 0.111* AGG_CSP [0.77] [-2.80] [3.51] 0.054 -0.089* 0.111*** LOGMV -0.003* 0.033* 0.005* 0.006* [-1.96] [13.36] [4.91] [6.13] LOGAGE -0.000 -0.016* -0.010* 0.012* [-0.18] [-4.44] [-6.28] [7.89] LOGPRC -0.000 0.045* 0.003 0.016* [-0.16] [9.74] [1.19] [7.56] BM -0.006* 0.037* 0.008* 0.013* [-5.00] [4.39] [4.15] [3.19] BETA -0.004* 0.010 0.010* -0.002 [-2.85] [2.57] [5.07] [-1.36] IRISK -0.001 -0.275* -0.060* -0.079* [-0.16] [-7.92] [-3.93] [-4.69] TOV -0.005 0.261* 0.157* -0.005 [-0.73] [7.42] [7.48] [-0.76] RET -0.007 0.110* 0.094* -0.007 [-1.50] [5.22] [9.27] [-0.86] EPS -0.001* -0.007* -0.000 -0.002* [-2.97] [-3.57] [-0.16] [-3.00] S&PIDX 0.004 -0.044* -0.022* 0.005 [0.81] [-7.07] [-7.68] [1.56] DY 0.206 -1.855* -0.538* -0.110 [1.97] [-3.50] [-3.04] [-0.68] DTA -0.013 0.142* 0.057* 0.006 [-2.12] [10.33] [8.45] [1.07] CONSTANT 0.073 -0.599* -0.079* -0.112*** [2.55] [-13.41] [-4.19] [-5.73] OBSERVATIONS 10,118 10,030 10,030 10,030 R-SQUARED 0.295 0.345 0.283 0.156 TIME FE YES YES YES YES IND FE YES YES YES YES 41 Table A3.5 Institutional investors’ influence on future CSP: 2SLS (03-09) Table A3.5 displays the 2SLS regression results of CSP measures on long-term institutional ownership and other control variables, using a sub-sample of 2003-2009. The dependent variable in the first stage regression (reported in column 1) is the variable of interest, long-term institutional ownership (LIO), defined based on churn ratio as in Yan and Zhang (2009). Instrumental variables used are dummy variables indicating the stock switching from the Russell 1000 index into the Russell 2000 index (R1TR2), switching from the Russell 2000 index into the Russell 1000 index (R2TR1), dropping out of the Russell 2000 index due to a market value decrease (R2TN) and getting included in the Russell 2000 index due to a market value increase (NTR2). The fitted values of LIO from the first stage regression are then used in the second stage regressions displayed in columns 2 through 7. Appendix 3 Additional robustness tests Control variables include ownership of institutional investors that are not long term (NLIO), firm size (LOGMV), book to market ratio (BM), return on assets (ROA), leverage ratio (DTA) and Cash Holding (CASH). Detailed variable definitions can be found in appendix 1 and 2. Time fixed effects (Year) and industry fixed effects (2 Digit SIC code) are included in all regressions. , * and * denote significance at the 10%, 5%, and 1% level respectively. Standard errors are clustered at firm level and robust t-statistics are reported in brackets. Table A3.5 Institutional investors’ influence on future CSP: 2SLS (03-09) d at firm level and robust t statistics are reported in brackets. (1) (2) (3) (4) (5) (6) (7) VARIABLES LIO AGG_CSP (t+1) AGG_CSP (t+5) AGG_C (t+1) AGG_C (t+5) AGG_S (t+1) AGG_S (t+5) R1TR2 0.051* [6.15] R2TN -0.048* [-6.30] R2TR1 0.020 [2.14] NTR2 -0.043* [-6.83] LIO 0.327* 0.647 -0.252* -0.195 -0.002 0.424* [4.01] [2.48] [-3.77] [-1.58] [-0.03] [1.85] NLIO -0.028* -0.072* 0.000 0.004 -0.026* -0.059* [-6.13] [-6.85] [0.03] [0.65] [-6.43] [-6.47] LOGMV 0.013* 0.002 0.027* 0.018* 0.019* 0.022* 0.047* [12.52] [1.23] [7.60] [17.09] [10.43] [18.83] [14.95] BM 0.008 -0.006* -0.001 0.005 0.020* -0.001 0.011 [2.12] [-3.66] [-0.16] [2.04] [5.04] [-0.73] [1.51] ROA 0.011*** 0.001 -0.017 -0.007 -0.011 -0.006* -0.037 [2.77] [0.31] [-0.85] [-1.43] [-1.22] [-1.68] [-2.38] DTA -0.002 -0.009 -0.015 0.004 0.004 -0.007* -0.023 [-0.20] [-2.10] [-1.42] [0.98] [0.64] [-1.78] [-2.54] CASH -0.062* 0.027*** 0.037* -0.026* -0.024 -0.004 0.006 [-7.44] [3.66] [1.71] [-4.14] [-2.22] [-0.69] [0.34] CONSTANT -0.141* -0.092* -0.640* -0.221* -0.293* -0.422* -0.937* [-6.67] [-5.16] [-14.42] [-14.34] [-11.61] [-24.11] [-23.59] OBSERVATIONS 12,457 11,448 5,853 11,448 5,853 11,448 5,853 R-SQUARED 0.187 0.090 0.322 0.220 0.338 0.216 0.411 TIME FE YES YES YES YES YES YES YES IND FE YES YES YES YES YES YES YES 42 References Barber, Brad M., and Terrance Odean. 2008. ‘All That Glitters: The Effect of Attention and News on the Buying Behavior of Individual and Institutional Investors’. 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Switzer, L.N., J., Wang. 2017. ‘Institutional Investment Horizon, the Information Environment and Firm Credit Risk’. Journal of Financial Stability 29: 57-71. Waddock, Sandra A., and Samuel B. Graves. 1997. ‘The Corporate Social Performance-Financial Performance Link’. Strategic Management Journal 18 (4): 303–319. Yan, Xuemin, and Zhe Zhang. 2009. ‘Institutional Investors and Equity Returns: Are Short-term Institutions Better Informed’? Review of Financial Studies 22 (2): 893-924. 46 Table 1 Investment horizon measures (churn rate based versus other classifications) Table 1 Investment horizon measures (churn rate based versus other classifications) Table 1 reports the proportion of each investor type under the fiduciary duty classification (Panel A) and under the Bushee classification (Panel B) that is categorised as long-term, short-term and other (i.e. medium term) investors based on churn-rate. References The fiduciary duty classification and Bushee classifications are provided by Professor Brian Bushee.21 The churn-rate based classification (Long-term, Short-term, and Other) is created using the churn rate (see Appendix 1 for details) ) g ( pp ) (1) Long Term (2) Short Term (3) Other Panel A: Investors classified by fiduciary duties: Banks 51.58% 36.07% 12.36% Corporate Pension Funds 48.53% 25.23% 26.23% Independent Investment advisors 25.67% 44.66% 29.67% Insurance Companies 33.89% 48.52% 17.59% Investment Companies 26.54% 51.47% 21.99% Public Pension Funds 25.49% 43.15% 31.36% University Endowment 49.09% 36.63% 14.28% Miscellaneous 38.60% 21.92% 39.47% Panel B: Investors classified by Bushee (2001): Dedicated Investors 30.93% 45.77% 23.30% Quasi Indexer 43.34% 48.81% 7.86% Transient Investors 7.98% 36.96% 55.06% 47 Table 2 Summary Statistics VARIABLE N MEAN STD SKEW KURT Min 25% 50% 75% MAX Panel A: CSR AGG_CSP 22801 -0.023 0.105 1.520 7.318 -0.542 -0.083 -0.028 0.021 0.919 AGG_S 22800 0.054 0.096 3.386 14.921 0.000 0.000 0.021 0.065 0.919 AGG_C 22801 0.077 0.073 1.633 4.720 0.000 0.028 0.056 0.111 0.722 COM_CSP 21924 0.015 0.174 0.651 15.512 -1.000 0.000 0.000 0.000 1.000 DIV_CSP 22799 -0.072 0.290 0.064 0.784 -1.000 -0.333 0.000 0.125 1.000 EMP_CSP 22794 -0.020 0.167 0.155 3.334 -1.000 -0.033 0.000 0.000 1.000 ENV_CSP 22800 0.003 0.142 0.903 9.929 -0.833 0.000 0.000 0.000 1.000 PSQ_CSP 22184 -0.027 0.194 0.500 8.900 -1.000 0.000 0.000 0.000 1.000 Panel B: IO TIO 22795 0.646 0.221 -0.549 -0.348 0.000 0.501 0.675 0.819 1.000 SIO 22795 0.165 0.101 0.923 1.370 0.000 0.090 0.150 0.225 0.852 LIO 22795 0.201 0.102 1.214 3.982 0.000 0.130 0.187 0.257 0.943 NLIO 22795 0.445 0.188 -0.154 -0.531 0.000 0.312 0.455 0.582 0.946 Panel C: Control MV 22801 6395.07 20416.20 9.80 139.23 8.03 458.50 1400.21 4338.72 519815.79 BM 22801 0.561 0.607 -27.689 1879.880 -43.685 0.302 0.488 0.740 3.342 AGE 22801 22.168 15.908 0.733 -0.481 1.000 9.000 18.000 34.000 63.000 BETA 19752 1.066 0.637 1.016 2.354 -1.230 0.623 0.987 1.409 5.151 IRISK 19752 0.113 0.084 3.331 26.464 0.001 0.059 0.091 0.141 1.565 TOV 22801 0.170 0.175 6.544 118.455 0.001 0.071 0.125 0.211 6.196 PRC 22801 32.839 45.713 23.227 891.966 -5.059 14.863 26.430 41.783 2351.950 RET 22801 0.034 0.120 0.371 6.301 -0.828 -0.021 0.034 0.088 0.965 EPS 22644 0.380 1.309 24.027 1031.960 -19.130 0.083 0.328 0.605 71.160 S&PIDX 22801 0.261 0.439 1.091 -0.811 0.000 0.000 0.000 1.000 1.000 DY 22765 0.004 0.008 10.885 253.131 0.000 0.000 0.002 0.006 0.309 DTA 22801 0.254 0.196 0.986 0.943 0.000 0.098 0.225 0.361 1.000 ROA 22614 0.020 0.173 -22.028 1260.600 -12.331 0.008 0.032 0.070 2.170 CASH 22801 0.127 0.166 2.333 5.982 0.000 0.022 0.061 0.161 0.989 Panel D: LIOP TERCILE AGG_CSP AGG_S AGG_C 1 -0.032 0.038 0.070 2 -0.020 0.062 0.081 3 -0.018 0.061 0.078 DIFF 0.014* 0.024* 0.008* T [8.7] [16.44] [7.21] SIOP TERCILE AGG_CSP AGG_S AGG_C 1 -0.016 0.056 0.074 2 -0.022 0.061 0.081 3 -0.032 0.045 0.075 DIFF -0.016* -0.010* 0.000 T [-9.52] [-7.06] [0.14] 48 Table 3 Correlation Matrix Variables (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) 1) AGG_CSR 1.000 2) AGG_S 0.651 1.000 3) AGG_C -0.405 0.191 1.000 4) TIO -0.031 0.047 0.065 1.000 5) SIO -0.102 -0.073 0.019 0.574 1.000 6) LIO 0.096 0.159 0.055 0.532 -0.059 1.000 7) NLIO -0.090 -0.029 0.050 0.888 0.700 0.084 1.000 8) LOGMV 0.138 0.358 0.243 0.216 0.054 0.151 0.174 1.000 9) BM -0.021 -0.024 -0.002 -0.022 -0.076 0.073 -0.071 -0.160 1.000 10) LOGAGE 0.094 0.193 0.115 0.039 -0.123 0.233 -0.089 0.369 -0.009 1.000 11) BETA -0.061 -0.004 0.055 0.171 0.225 -0.014 0.212 -0.013 -0.037 -0.102 1.000 12) IRISK -0.091 -0.110 -0.026 -0.034 0.119 -0.143 0.042 -0.283 0.028 -0.207 0.285 1.000 13) TOV -0.056 0.027 0.093 0.293 0.326 0.020 0.368 0.040 -0.035 -0.090 0.297 0.353 1.000 14) LOGPRC 0.104 0.154 0.060 0.198 0.049 0.149 0.133 0.632 -0.158 0.293 -0.227 -0.378 -0.070 1.000 15) RET 0.010 -0.009 -0.021 0.015 0.158 -0.072 0.054 0.029 -0.159 -0.012 0.055 -0.059 -0.050 0.099 1.000 16) EPS 0.021 0.048 0.040 0.008 -0.018 0.040 -0.021 0.187 0.037 0.111 -0.075 -0.158 -0.098 0.391 0.101 1.000 17) S&PIDX 0.108 0.280 0.189 0.053 -0.007 0.088 0.022 0.618 -0.070 0.355 0.036 -0.142 -0.040 0.337 -0.001 0.089 1.000 18) DY 0.056 0.057 0.008 -0.144 -0.146 0.017 -0.206 0.085 0.061 0.139 -0.180 -0.163 -0.112 0.073 -0.026 0.031 0.075 1.000 19) DTA -0.042 -0.003 0.048 0.056 0.080 -0.013 0.074 0.047 -0.078 -0.005 0.088 0.056 0.039 -0.092 -0.030 -0.083 0.022 0.174 1.000 20) ROA 0.034 0.054 0.030 0.111 0.034 0.092 0.083 0.202 0.007 0.123 -0.100 -0.239 -0.061 0.313 0.136 0.236 0.094 0.069 -0.092 1.000 21) CASH -0.020 -0.045 -0.030 0.041 0.136 -0.098 0.166 -0.145 -0.145 -0.215 0.191 0.215 0.228 -0.198 0.037 -0.065 -0.132 -0.186 -0.231 -0.217 1.00 Table 3 Correlation Matrix Table 3 Correlation Matrix 2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) 49 Table 4 Institutional investors’ preference of aggregate CSP Table 4 Institutional investors’ preference of aggregate CSP Table 4 displays the regression results of various measures of institutional ownership on aggregate CSP and other control variables. Dependent variables TIO, SIO, and LIO denote ownership of all institutional investors, short-term institutional investors and long-term institutional investors respectively, measured at year t+1. Long term and short-term investors are defined following Yan and Zhang (2009) based on churn ratio. All independent variables are in the current year t. Main variable of interest is the overall CSP score based on the KLD database. Control variables include firm size (LOGMV), natural log of firm age (LOGAGE), natural log of stock price (LOGPRC), Book to market ratio (BM), CAPM beta of stock (BETA), idiosyncratic volatility (IRISK), quarterly stock turnover (TOV), earnings per share (EPS), index membership dummy (S&PIDX), dividend yield (DY), leverage (DTA). Detailed variable definition can be found in appendix 1 and 2. Time fixed effects (Year) and industry fixed effects (2 Digit SIC code) are included in all regressions. , * and * denote significance at the 10%, 5%, and 1% level respectively. Standard errors are clustered at firm level and robust t-statistics are reported in brackets. are reported in brackets. (1) (2) (3) VARIABLES TIO(t+1) SIO(t+1) LIO(t+1) AGG_CSP -0.010 -0.020* 0.012 [-1.34] [-4.36] [2.40] TIO 0.882* [152.19] SIO 0.590* [55.24] LIO 0.710* [45.50] LOGMV 0.004* 0.000 0.003* [5.31] [0.25] [5.53] LOGAGE -0.004* -0.004* 0.001 [-3.22] [-4.00] [1.33] LOGPRC 0.001 -0.002* 0.007*** [0.39] [-1.80] [6.44] BM 0.005* 0.004* 0.005* [1.79] [4.28] [6.34] BETA 0.001 0.006* -0.001 [0.70] [4.95] [-0.79] IRISK -0.052* -0.027* -0.038* [-3.64] [-3.03] [-4.01] TOV -0.012 0.047* -0.004 [-1.14] [6.74] [-1.00] RET 0.072* 0.036* 0.004 [7.02] [5.54] [0.80] EPS 0.001 0.000 -0.001 [0.86] [0.46] [-1.07] S&PIDX -0.005 0.000 -0.002 [-2.30] [0.01] [-1.36] DY -0.105 -0.079 -0.066 [-0.76] [-0.90] [-0.81] DTA 0.004 0.023* -0.005 [0.72] [6.07] [-1.42] CONSTANT -0.100* 0.061* -0.080* [-6.14] [5.56] [-7.73] OBSERVATIONS 19,504 19,504 19,504 R-SQUARED 0.786 0.508 0.607 TIME FE YES YES YES IND FE YES YES YES 50 Table 5 Institutional investors’ preference of CSP strengths and concerns Table 5 displays the regression results of various measures of institutional ownership on CSP Strengths, CSP Concerns and other control variables. Dependent variables TIO, SIO, and LIO denote ownership of all institutional investors, short-term institutional investors and long-term institutional investors respectively, measured at year t+1. Table 4 Institutional investors’ preference of aggregate CSP Variables of interest are COM_CSP, DIV_CSP, EMP_CSP, ENV_CSP, PSQ_CSP, representing the standardized CSP scores of Community, Diversity, Employee, Environment and Product, from KLD database. Control variables include firm size (LOGMV), natural log of firm age (LOGAGE), natural log of stock price (LOGPRC), Book to market ratio (BM), CAPM beta of stock (BETA), idiosyncratic volatility (IRISK), quarterly stock turnover (TOV), earnings per share (EPS), index membership dummy (S&PIDX), dividend yield (DY), leverage (DTA). Detailed variable definitions can be found in appendix 1 and 2. Time fixed effects (Year) and industry fixed effects (2 Digit SIC code) are included in all regressions. , * and *** denote significance at the 10%, 5%, and 1% level respectively. Standard errors are clustered at firm level and robust t-statistics are reported in brackets Table 6 Institutional investors preference of specific CSP dimensions Table 6 displays the regression results of various measures of institutional ownership on measures of specific CSP dimensions and other control variables. Dependent variables SIO and LIO denote ownership of short-term institutional investors and long-term institutional investors respectively, measured at year t+1. Long-term and short-term investors are defined based on churn ratio as in Yan and Zhang (2009). All independent variables are in the current year t. Variables of interest are COM_CSP, DIV_CSP, EMP_CSP, ENV_CSP, PSQ_CSP, representing the standardized CSP scores of Community, Diversity, Employee, Environment and Product, from KLD database. Control variables include firm size (LOGMV), natural log of firm age (LOGAGE), natural log of stock price (LOGPRC), Book to market ratio (BM), CAPM beta of stock (BETA), idiosyncratic volatility (IRISK), quarterly stock turnover (TOV), earnings per share (EPS), index membership dummy (S&PIDX), dividend yield (DY), leverage (DTA). Detailed variable definitions can be found in appendix 1 and 2. Time fixed effects (Year) and industry fixed effects (2 Digit SIC code) are included in all regressions. , * and *** denote significance at the 10%, 5%, and 1% level respectively. Standard errors are clustered at firm level and robust t-statistics are reported in brackets. in brackets. Table 4 Institutional investors’ preference of aggregate CSP Long term and short-term investors are defined following Yan and Zhang (2009) based on churn ratio. All independent variables are in the current year t. AGG_S and AGG_C are the variables of interest and are measured as the standardized CSP Strengths score and Concerns score from the KLD database, respectively. Control variables include firm size (LOGMV), natural log of firm age (LOGAGE), natural log of stock price (LOGPRC), Book to market ratio (BM), CAPM beta of stock (BETA), idiosyncratic volatility (IRISK), quarterly stock turnover (TOV), earnings per share (EPS), index membership dummy (S&PIDX), dividend yield (DY), leverage (DTA). Detailed variable definitions can be found in appendix 1 and 2. Time fixed effects (Year) and industry fixed effects (2 Digit SIC code) are included in all regressions. , * and * denote significance at the 10%, 5%, and 1% level respectively. Standard errors are clustered at firm level and robust t-statistics are reported in brackets are reported in brackets. (1) (2) (3) VARIABLES TIO(t+1) SIO(t+1) LIO(t+1) AGG_S -0.017 -0.022* 0.006 [-2.13] [-4.68] [1.33] AGG_C -0.027 0.000 -0.033* [-2.45] [0.07] [-4.90] TIO 0.881* [151.62] SIO 0.590* [55.35] LIO 0.709* [45.52] LOGMV 0.005* 0.000 0.003* [5.79] [0.78] [6.00] LOGAGE -0.004* -0.004* 0.001 [-3.12] [-3.89] [1.43] LOGPRC 0.000 -0.002 0.006* [0.17] [-2.01] [6.21] BM 0.005* 0.004* 0.005* [1.91] [4.34] [6.57] BETA 0.001 0.006* -0.001 [0.78] [5.04] [-0.74] IRISK -0.052* -0.026* -0.038* [-3.61] [-2.96] [-4.00] TOV -0.011 0.047* -0.004 [-1.11] [6.78] [-0.98] RET 0.072* 0.037*** 0.004 [7.04] [5.57] [0.79] EPS 0.001 0.000 -0.001 [0.88] [0.51] [-1.02] S&PIDX -0.004* 0.001 -0.002 [-1.89] [0.34] [-1.07] DY -0.104 -0.080 -0.063 [-0.76] [-0.91] [-0.78] DTA 0.004 0.023* -0.005 [0.76] [6.07] [-1.42] CONSTANT -0.110* 0.054* -0.084* [-6.56] [4.76] [-7.83] OBSERVATIONS 19,503 19,503 19,503 R-SQUARED 0.786 0.508 0.607 TIME FE YES YES YES IND FE YES YES YES 51 Table 6 Institutional investors’ preference of specific CSP dimensions Table 6 Institutional investors’ preference of specific CSP dimensions Table 6 displays the regression results of various measures of institutional ownership on measures of specific CSP dimensions and other control variables. Dependent variables SIO and LIO denote ownership of short-term institutional investors and long-term institutional investors respectively, measured at year t+1. Long-term and short-term investors are defined based on churn ratio as in Yan and Zhang (2009). All independent variables are in the current year t. Table 4 Institutional investors’ preference of aggregate CSP (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) VARIABLES SIO(t+1) SIO(t+1) SIO(t+1) SIO(t+1) SIO(t+1) LIO(t+1) LIO(t+1) LIO(t+1) LIO(t+1) LIO(t+1) COM_CSP -0.004 0.003 [-1.53] [1.32] DIV_CSP 0.001 -0.002 [0.33] [-1.32] EMP_CSP -0.003 0.005** [-1.00] [2.04] ENV_CSP -0.006* 0.001 [-1.93] [0.28] PSQ_CSP -0.008* 0.008* [-3.11] [2.85] LOGMV -0.000 -0.000 -0.000 0.000 -0.000 0.003* 0.003* 0.003* 0.003* 0.003* [-0.04] [-0.19] [-0.05] [0.01] [-0.14] [4.96] [5.88] [5.67] [5.76] [5.72] BM 0.004* 0.004* 0.004* 0.004* 0.004* 0.005* 0.005* 0.005* 0.005* 0.005* [4.20] [4.31] [4.30] [4.29] [4.05] [6.26] [6.31] [6.38] [6.29] [5.91] LOGAGE -0.003* -0.004* -0.004* -0.004* -0.004* 0.002* 0.001 0.001 0.001 0.002* [-3.63] [-4.01] [-4.03] [-4.07] [-3.85] [1.72] [1.42] [1.39] [1.35] [1.77] BETA 0.005* 0.006* 0.006* 0.006* 0.006* -0.001 -0.001 -0.001 -0.001 -0.001 [4.48] [5.06] [5.03] [5.05] [4.89] [-0.77] [-0.90] [-0.79] [-0.88] [-0.66] IRISK -0.028* -0.026* -0.026* -0.027* -0.027* -0.037* -0.038* -0.038* -0.038* -0.037* [-3.14] [-2.99] [-2.99] [-3.01] [-3.07] [-3.88] [-4.04] [-4.04] [-4.03] [-3.88] TOV 0.049* 0.047* 0.047* 0.047* 0.047* -0.004 -0.004 -0.004 -0.004 -0.005 [6.86] [6.75] [6.75] [6.76] [6.73] [-1.04] [-0.96] [-0.96] [-0.98] [-1.19] LOGPRC -0.002* -0.002* -0.002* -0.002* -0.002* 0.007* 0.007* 0.007* 0.007* 0.007* [-1.66] [-1.77] [-1.81] [-1.80] [-1.78] [6.41] [6.32] [6.44] [6.43] [6.26] RET 0.038* 0.037* 0.037* 0.037* 0.037* 0.005 0.004 0.004 0.004 0.005 [5.77] [5.60] [5.59] [5.58] [5.59] [1.03] [0.71] [0.78] [0.76] [1.04] EPS 0.000 0.000 0.000 0.000 0.000 -0.001 -0.001 -0.001 -0.001 -0.001 [0.58] [0.53] [0.52] [0.50] [0.69] [-1.06] [-1.10] [-1.08] [-1.08] [-0.99] S&PIDX -0.000 -0.000 -0.000 0.000 0.000 -0.002 -0.002 -0.002 -0.002 -0.003* [-0.06] [-0.05] [-0.05] [0.02] [0.01] [-1.49] [-1.29] [-1.33] [-1.32] [-1.82] DY -0.124 -0.082 -0.081 -0.083 -0.113 -0.049 -0.063 -0.066 -0.064 -0.051 [-1.35] [-0.93] [-0.92] [-0.94] [-1.25] [-0.57] [-0.78] [-0.81] [-0.79] [-0.63] DTA 0.025* 0.024* 0.024* 0.024* 0.025*** -0.006* -0.005 -0.005 -0.005 -0.005* [6.22] [6.12] [6.10] [6.12] [6.33] [-1.86] [-1.47] [-1.44] [-1.48] [-1.67] CONSTANT 0.062* 0.065* 0.063* 0.063* 0.064* -0.077* -0.085* -0.080* -0.081* -0.083* [5.61] [5.62] [5.74] [5.74] [5.76] [-7.48] [-7.97] [-7.74] [-7.92] [-8.18] OBSERVATIONS 18,738 19,502 19,497 19,503 18,934 18,738 19,502 19,497 19,503 18,934 R-SQUARED 0.508 0.508 0.508 0.508 0.507 0.612 0.607 0.607 0.607 0.607 TIME FE YES YES YES YES YES YES YES YES YES YES IND FE YES YES YES YES YES YES YES YES YES YES 52 Table 7 Institutional investors’ influence on future overall CSP l f f l h d Table 7 displays the regression results of CSP measures on measures of institutional ownership and other control variables. Table 4 Institutional investors’ preference of aggregate CSP Dependent variables AGG_CSP, AGG_C and AGG_S denote standardized overall CSP score, CSP concerns score and CSP strengths score respectively. Dependent variables in column 1, 3 and 5 are measured at t+1 year while dependent variables in column 2, 4 and 6 are measured at t+5 years. All independent variables are in the current year t. The variable of interest, Long term institutional ownership (LIO), is defined based on churn ratio as in Yan and Zhang (2009). Control variables include ownership of institutional investors that are not long-term (NLIO), firm size (LOGMV), Book to market ratio (BM), Return on assets (ROA), and leverage ratio (DTA), Cash holding (CASH). Detailed variable definitions can be found in appendix 1 and 2. Time fixed effects (Year) and industry fixed effects (2 Digit SIC code) are included in all regressions. , * and * denote significance at the 10%, 5%, and 1% level respectively. Standard errors are clustered at firm level and robust t-statistics are reported in brackets in brackets. (1) (2) (3) (4) (5) (6) VARIABLES AGG_CSP(t+1) AGG_CSP(t+5) AGG_C(t+1) AGG_C(t+5) AGG_S(t+1) AGG_S(t+5) LIO 0.020 0.031 -0.027* -0.007 -0.008 0.027 [2.57] [2.11] [-4.99] [-0.80] [-1.23] [2.25] NLIO -0.022* -0.046* -0.010* 0.006 -0.029* -0.028* [-5.26] [-6.40] [-3.32] [1.41] [-7.72] [-4.46] LOGMV 0.012* 0.021* 0.014* 0.017* 0.028* 0.040* [17.01] [17.66] [28.87] [22.67] [38.49] [35.27] BM -0.002* 0.006 0.006 0.016* 0.005 0.019* [-1.94] [1.33] [2.32] [5.42] [2.26] [4.25] ROA 0.009* 0.028 -0.017* -0.013 -0.012* -0.011 [2.71] [2.33] [-3.81] [-2.40] [-2.88] [-1.38] DTA -0.014* -0.023* 0.008* 0.007 -0.004 -0.026*** [-3.57] [-3.09] [2.78] [1.47] [-0.98] [-4.03] CASH 0.003 0.001 -0.003 -0.009* -0.001 -0.010 [0.66] [0.16] [-0.78] [-1.84] [0.16] [-1.40] CONSTANT -0.229* -0.424* -0.192* -0.296* -0.538* -0.778* [-14.91] [-16.73] [-17.57] [-18.42] [-34.47] [-32.16] Observations 19,842 9,919 19,436 9,863 19,436 9,863 R-squared 0.208 0.246 0.246 0.274 0.251 0.319 Time FE YES YES YES YES YES YES IND FE YES YES YES YES YES YES 53 53 Table 8 Institutional investors’ influence on specific future CSP dimensions Table 8 displays the regression results of different CSR dimensions on long term institutional ownership. Dependent variables are COM_CSP, DIV_CSP, EMP_CSP, ENV_CSP, PSQ_CSP, representing the standardized CSP scores of Community, Diversity, Employee, Environment and Product, from KLD database, measured at year t+1. Our interested variable Long-term institutional ownership is defined based on churn ratio as in Yan and Zhang (2009). All independent variables are in the current year t. Table 9 Institutional investors’ preference of aggregate CSP: 2SLS Table 9 Institutional investors’ preference of aggregate CSP: 2SLS Table 9 displays the 2SLS regression results of various measures of institutional ownership on overall CSP and other control variables. The first column displays the regression of AGG_CSP on the instrumental variables AGG_CSP_INT (the initial value of CSP) and AGG_CSP_IY (the average CSP of firms in the same industry at the same year) and other control variables. The fitted values of AGG_CSP from the first stage regression are then used in the second stage regressions displayed in columns 2 through 4. The dependent variables TIO, SIO, and LIO denote ownership of all institutional investors, short-term institutional investors and long-term institutional investors respectively, measured at the year t+1. Long term and short-term investors are defined following Yan and Zhang (2009) based on churn ratio. All independent variables are measured in the current year t. The variable of interest is the overall CSP score (AGG_CSP) based on the KLD database. Control variables include firm size (LOGMV), natural log of firm age (LOGAGE), natural log of stock price (LOGPRC), Book to market ratio (BM), CAPM beta of stock (BETA), idiosyncratic volatility (IRISK), quarterly stock turnover (TOV), earnings per share (EPS), index membership dummy (S&PIDX), dividend yield (DY), leverage (DTA). Detailed variable definitions can be found in appendix 1 and 2. Time fixed effects (Year) and industry fixed effects (2 Digit SIC code) are included in all regressions. , * and *** denote significance at the 10%, 5%, and 1% level respectively. g g Standard errors are clustered at firm level and robust t-statistics are reported in brackets. dard errors are clustered at firm level and robust t statistics are reported in brackets. Table 4 Institutional investors’ preference of aggregate CSP Control variables include ownership of institutional investors that are not long-term (NLIO), firm size (LOGMV), natural log of firm age (LOGAGE), natural log of stock price (LOGPRC), Book to market ratio (BM), CAPM beta of stock (BETA), idiosyncratic volatility (IRISK), quarterly stock turnover (TOV), earnings per share (EPS), index membership dummy (S&PIDX), dividend yield (DY), leverage (DTA), cash holding (CASH). Detailed variable definitions can be found in appendix 1 and 2. Time fixed effects (Year) and industry fixed effects (2 Digit SIC code) are included in all regressions. , * and *** denote significance at the 10%, 5%, and 1% level respectively. Standard errors are clustered at firm level and robust t-statistics are reported in brackets. Table 8 Institutional investors’ influence on specific future CSP dimensions d d b p (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) VARIABLES COM_CSP(t+1) COM_CSP(t+5) DIV_CSP(t+1) DIV_CSP(t+5) EMP_CSP(t+1) EMP_CSP(t+5) ENV_CSP(t+1) ENV_CSP(t+5) PSQ_CSP(t+1) PSQ_CSP(t+5) LIO 0.024* -0.003 0.071* 0.089 0.027 -0.075* 0.001 0.030* -0.004 0.078* [1.91] [-0.11] [3.20] [2.53] [2.01] [-3.55] [0.10] [1.66] [-0.27] [2.69] NLIO -0.015 -0.018 -0.028** -0.033* -0.039* -0.042* -0.003 -0.029* 0.024* -0.030 [-2.13] [-1.40] [-2.49] [-1.81] [-5.60] [-3.87] [-0.59] [-3.15] [2.99] [-2.19] LOGMV 0.014* 0.018* 0.078* 0.098* 0.006* 0.009* 0.012* 0.025* -0.023* -0.027* [11.20] [8.34] [52.78] [43.47] [5.64] [5.43] [11.33] [14.87] [-15.01] [-11.17] BM 0.007* 0.010 0.012* 0.035* -0.008*** -0.013* -0.003* 0.004 -0.017* -0.047* [2.91] [1.13] [2.90] [3.27] [-3.11] [-1.69] [-1.78] [0.70] [-3.04] [-5.16] ROA -0.008 -0.008 -0.050* -0.020 0.065* 0.073* 0.016* 0.021* 0.034*** 0.016 [-1.29] [-0.60] [-3.05] [-0.79] [5.71] [4.91] [2.62] [1.83] [3.08] [0.85] DTA 0.012* -0.009 0.019* -0.000 -0.016 -0.036* -0.003 -0.023 -0.025* -0.026* [1.87] [-0.72] [1.71] [-0.02] [-2.30] [-3.24] [-0.61] [-2.30] [-2.85] [-1.68] CASH 0.023* 0.006 -0.007 0.017 0.038* 0.093* 0.018* 0.007 0.007 -0.046* [3.09] [0.40] [-0.55] [0.77] [4.49] [7.18] [3.03] [0.71] [0.73] [-2.74] CONSTANT -0.193* -0.269* -1.502* -1.925* -0.253* -0.251* -0.252* -0.493* 0.497* 0.590* [-7.23] [-5.83] [-47.74] [-38.98] [-10.70] [-6.76] [-10.89] [-13.60] [15.02] [11.57] OBSERVATIONS 18,587 9,281 19,434 9,863 19,428 9,858 19,436 9,863 18,829 9,380 R-SQUARED 0.119 0.139 0.291 0.337 0.118 0.140 0.149 0.190 0.155 0.194 TIME FE YES YES YES YES YES YES YES YES YES YES IND FE YES YES YES YES YES YES YES YES YES YES 54 Table 9 Institutional investors’ preference of aggregate CSP: 2SLS (1) (2) (3) (4) VARIABLES AGG_CSP TIO (t+1) SIO (t+1) LIO (t+1) AGG_CSP_INT 0.544* [19.04] AGG_CSP_IY 0.887* [16.82] AGG_CSP -0.008 -0.027 0.036* [-0.40] [-1.99] [2.87] TIO 0.882* [151.15] SIO 0.591* [55.44] LIO 0.709* [45.38] LOGMV 0.008* 0.004* 0.000 0.003* [5.11] [5.24] [0.33] [5.20] LOGAGE 0.002 -0.004* -0.004*** 0.001 [0.79] [-3.07] [-3.96] [1.36] LOGPRC -0.002 0.000 -0.002* 0.007*** [-0.94] [0.20] [-1.86] [6.05] BM -0.002 0.005* 0.004* 0.005* [-1.52] [1.85] [4.26] [6.49] BETA -0.006* 0.001 0.005* -0.001 [-3.22] [0.66] [4.87] [-0.68] IRISK -0.017* -0.053* -0.027* -0.038* [-1.80] [-3.71] [-3.05] [-4.05] TOV 0.007 -0.011 0.047* -0.004 [0.85] [-1.08] [6.77] [-0.99] RET -0.014* 0.073* 0.037*** 0.005 [-2.58] [7.19] [5.59] [1.00] EPS -0.001 0.001 0.000 -0.001 [-0.72] [0.86] [0.50] [-0.97] S&PIDX 0.009* -0.005** 0.000 -0.002 [1.94] [-2.33] [0.09] [-1.64] DY 0.209* -0.105 -0.079 -0.068 [1.73] [-0.76] [-0.90] [-0.84] DTA -0.009 0.004 0.023* -0.005 [-1.43] [0.75] [6.04] [-1.40] CONSTANT -0.166* -0.101* 0.060* -0.080*** [-5.47] [-6.21] [5.44] [-7.56] OBSERVATIONS 19,704 19,526 19,526 19,526 R-SQUARED 0.325 0.784 0.508 0.606 TIME FE YES YES YES YES IND FE YES YES YES YES 55 Table 10 Institutional investors’ influence on future CSP: 2SLS ble 10 displays the 2SLS regression results of CSP measures on long-term institutional ownership and other control variables. The dependent variable in the fir ge regression (reported in column 1) is the variable of interest, long-term institutional ownership (LIO), defined based on churn ratio as in Yan and Zhang (2009 trumental variables used are dummy variables indicating the stock switching from the Russell 1000 index into the Russell 2000 index (R1TR2), switching from Russell 2000 index into the Russell 1000 index (R2TR1), dropping out of the Russell 2000 index due to a market value decrease (R2TN) and getting included Russell 2000 index due to a market value increase (NTR2). The fitted values of LIO from the first stage regression are then used in the second stage regression played in columns 2 through 7. The dependent variables in the second stage regressions AGG_CSP, AGG_C and AGG_S denote standardized overall CSP scor P concerns score and CSP strengths score respectively. The dependent variables in columns 2, 4 and 6 are measured at the t+1 year while the dependent variabl columns 3, 5 and 7 are measured at the t+5 year. All independent variables are in the current year t. Table 9 Institutional investors’ preference of aggregate CSP: 2SLS institutional investors that are not long term (NLIO), fir e (LOGMV), book to market ratio (BM), return on assets (ROA), leverage ratio (DTA) and Cash Holding (CASH). Detailed variable definitions can be found pendix 1 and 2. Time fixed effects (Year) and industry fixed effects (2 Digit SIC code) are included in all regressions. , * and * denote significance at the 10% , and 1% level respectively. Standard errors are clustered at firm level and robust t-statistics are reported in brackets. (1) (2) (3) (4) (5) (6) (7) VARIABLES LIO AGG_CSP (t+1) AGG_CSP (t+5) AGG_C (t+1) AGG_C (t+5) AGG_S (t+1) AGG_S (t+5) R1TR2 0.051* [7.42] R2TN -0.056* [-8.49] R2TR1 0.034* [4.21] NTR2 -0.035* [-7.43] LIO 0.230* 0.277 -0.278* -0.160 -0.077 0.144 [2.68] [1.29] [-5.19] [-1.46] [-1.05] [0.76] NLIO -0.026* -0.051*** -0.004 0.009* -0.028* -0.031* [-5.91] [-6.32] [-1.44] [1.90] [-6.97] [-4.34] LOGMV 0.012* 0.010* 0.018* 0.017* 0.018* 0.029* 0.038* [13.15] [7.29] [6.24] [19.94] [11.82] [23.93] [14.92] BM 0.008 -0.004* 0.005 0.008* 0.017* 0.005 0.019* [2.54] [-2.87] [0.93] [3.12] [5.60] [2.42] [3.89] ROA 0.019* 0.005 0.024* -0.011** -0.010* -0.010** -0.013 [4.35] [1.31] [1.87] [-2.57] [-1.76] [-2.36] [-1.47] DTA -0.012* -0.011* -0.020 0.005* 0.005 -0.004 -0.024* [-1.69] [-2.78] [-2.53] [1.67] [1.05] [-1.19] [-3.56] CASH -0.071* 0.018 0.019 -0.021* -0.020 -0.006 -0.001 [-9.98] [2.37] [1.12] [-4.09] [-2.17] [-0.84] [-0.07] CONSTANT -0.069* -0.211* -0.407* -0.213* -0.307* -0.544* -0.771* [-3.60] [-12.36] [-13.54] [-17.75] [-16.72] [-31.91] [-27.56] OBSERVATIONS 23,269 19,842 9,919 19,436 9,863 19,436 9,863 R-SQUARED 0.266 0.208 0.246 0.245 0.274 0.251 0.318 TIME FE YES YES YES YES YES YES YES IND FE YES YES YES YES YES YES YES Table 10 Institutional investors’ influence on future CSP: 2SLS d 1% level respectively. Standard errors are clustered at firm level and robust t statistics are reported in brackets. Table 9 Institutional investors’ preference of aggregate CSP: 2SLS (1) (2) (3) (4) (5) (6) (7) VARIABLES LIO AGG_CSP (t+1) AGG_CSP (t+5) AGG_C (t+1) AGG_C (t+5) AGG_S (t+1) AGG_S (t+5) R1TR2 0.051* [7.42] R2TN -0.056* [-8.49] R2TR1 0.034* [4.21] NTR2 -0.035* [-7.43] LIO 0.230* 0.277 -0.278* -0.160 -0.077 0.144 [2.68] [1.29] [-5.19] [-1.46] [-1.05] [0.76] NLIO -0.026* -0.051* -0.004 0.009* -0.028* -0.031* [-5.91] [-6.32] [-1.44] [1.90] [-6.97] [-4.34] LOGMV 0.012* 0.010* 0.018* 0.017* 0.018* 0.029* 0.038* [13.15] [7.29] [6.24] [19.94] [11.82] [23.93] [14.92] BM 0.008 -0.004* 0.005 0.008* 0.017* 0.005 0.019* [2.54] [-2.87] [0.93] [3.12] [5.60] [2.42] [3.89] ROA 0.019* 0.005 0.024* -0.011** -0.010* -0.010** -0.013 [4.35] [1.31] [1.87] [-2.57] [-1.76] [-2.36] [-1.47] DTA -0.012* -0.011* -0.020 0.005* 0.005 -0.004 -0.024* [-1.69] [-2.78] [-2.53] [1.67] [1.05] [-1.19] [-3.56] CASH -0.071* 0.018 0.019 -0.021* -0.020 -0.006 -0.001 [-9.98] [2.37] [1.12] [-4.09] [-2.17] [-0.84] [-0.07] CONSTANT -0.069* -0.211* -0.407* -0.213* -0.307* -0.544* -0.771* [-3.60] [-12.36] [-13.54] [-17.75] [-16.72] [-31.91] [-27.56] OBSERVATIONS 23,269 19,842 9,919 19,436 9,863 19,436 9,863 R-SQUARED 0.266 0.208 0.246 0.245 0.274 0.251 0.318 TIME FE YES YES YES YES YES YES YES IND FE YES YES YES YES YES YES YES 56
https://openalex.org/W2154051090	https://bmcgenomics.biomedcentral.com/counter/pdf/10.1186/1471-2164-11-300	English	null	Proteomics-based confirmation of protein expression and correction of annotation errors in the Brucella abortus genome	BMC genomics	2,010	cc-by	8,663	RESEARCH ARTICLE Open Access BioMed Central © 2010 Lamontagne et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Com- mons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduc- tion in any medium, provided the original work is properly cited. Abstract Background: Brucellosis is a major bacterial zoonosis affecting domestic livestock and wild mammals, as well as humans around the globe. While conducting proteomics studies to better understand Brucella abortus virulence, we consolidated the proteomic data collected and compared it to publically available genomic data. Results: The proteomic data was compiled from several independent comparative studies of Brucella abortus that used either outer membrane blebs, cytosols, or whole bacteria grown in media, as well as intracellular bacteria recovered at different times following macrophage infection. We identified a total of 621 bacterial proteins that were differentially expressed in a condition-specific manner. For 305 of these proteins we provide the first experimental evidence of their expression. Using a custom-built protein sequence database, we uncovered 7 annotation errors. We provide experimental evidence of expression of 5 genes that were originally annotated as non-expressed pseudogenes, as well as start site annotation errors for 2 other genes. Conclusions: An essential element for ensuring correct functional studies is the correspondence between reported genome sequences and subsequent proteomics studies. In this study, we have used proteomics evidence to confirm expression of multiple proteins previously considered to be putative, as well as correct annotation errors in the genome of Brucella abortus strain 2308. cella ovis, Brucella canis, Brucella neotomae and Brucella microti which infect goats, cattle, pigs, sheep, dogs, desert wood rats and common voles, respectively [1,4]. Two Brucella species infecting marine mammals such as dol- phins, whales, seals, sea lions and walrus have also been defined as Brucella ceti and Brucella pinnipedialis [5-7]. With the exception of B. suis biovar 3, the Brucella genome is encoded on two chromosomes, containing in total approximately 3,500 genes. Genome sequences from 32 different Brucella strains, representing all species, have been published either as complete genomes (10 strains) or as draft assemblies in NCBI (22 strains) [8-14]. The raw genome sequencing data of 78 other strains is also available in the Sequence Read Archive of NCBI. The genome sequences were very highly homologous, although regions of unique genetic material were also observed. It is possible that these regions are involved in establishing the distinct host preferences and biological * Correspondence: eparamithiotis@caprion.com 1 Caprion Proteomics Inc., 7150 Alexander-Fleming, Montreal, Quebec, Canada Full list of author information is available at the end of the article Research article Proteomics-based confirmation of protein expression and correction of annotation errors in the Brucella abortus genome Julie Lamontagne1, Maxime Béland1, Anik Forest1, Alexandra Côté-Martin1, Najib Nassif1, Fadi Tomaki1, Ignacio Moriyón2, Edgardo Moreno3 and Eustache Paramithiotis1 Julie Lamontagne1, Maxime Béland1, Anik Forest1, Alexandra Côté-Martin1, Najib Nassif1, Fadi Tomaki1, Ignacio Moriyón2, Edgardo Moreno3 and Eustache Paramithiotis1 Lamontagne et al. BMC Genomics 2010, 11:300 http://www.biomedcentral.com/1471-2164/11/300 Lamontagne et al. BMC Genomics 2010, 11:300 http://www.biomedcentral.com/1471-2164/11/300 * Correspondence: eparamithiotis@caprion.com Background However, each experiment was a separate comparative study designed to identify differen- tially expressed bacterial proteins under specific condi- tions per experiment. Proteins that were not sufficiently differentially expressed under the experimental condi- tions used would have not been identified. Thus, while our results can be used to confirm that the proteins reported were expressed, they may underestimate under what conditions they can become expressed. Correction of fi e pse dogene annotations Unlike other pathogenic bacteria, Brucella virulence does not appear to be the result of relatively few virulence genes that can be transferred horizontally via plasmids, phages, or assembled in pathogenicity islands. Brucella also lack typical virulence factors such as exotoxins, fla- gella, capsules, and type III secretion systems. Rather, the pathogen's virulence appears to be an integrated aspect of its physiology. Therefore, to better understand Brucella virulence, we will need to better understand the Brucella proteome, including how it changes during the different stages of the intracellular and extracellular Brucella life- cycles, and how it interacts with host proteins and pro- cesses. Indeed, we have previously demonstrated that Brucella bacteria are capable of extensive, reversible, remodeling of their cell envelopes [16]. Furthermore, dur- ing the establishment of an intracellular infection, Bru- cella bacteria also appear able to carry out extensive, and reversible, modifications to their biosynthetic pathways and respiration in order to adapt to the changing microenvironments encountered in infected host cells [17]. This suggests that the Brucella proteome is consid- erably more dynamic than previously suspected, and that in depth proteomic analysis of the pathogen, as well as integration of these data with the available genomic infor- mation, will result in novel mechanistic and possibly therapeutic insights. In this work we have generated a synthesis of the pro- teomic datasets we produced from multiple independent comparisons of Brucella strains either grown in media or retrieved from infected host cells. Some of this data is currently publicly available [[16,17];http://proteomicsre- source.org/Default.aspx] with the remainder becoming available as part of this work. These studies were origi- nally designed to identify experimental condition-specific differences in the Brucella proteome. We compiled the experimental evidence for any Brucella protein detected and compared the proteomic data to the available genomic data. Correction of five pseudogene annotations Correction of five pseudogene annotations In previous studies using B. abortus 2308, we used the genome databases available on NCBI for B. abortus, B. melitensis and B. suis for protein identification. More than once, we obtained peptides which matched proteins supposedly expressed only by the latter two species. Upon verification, those peptides were manually assigned to ORFs of previously annotated pseudogenes of B. abor- tus strain 2308 (NCBI taxonomy ID 359391). We there- fore assembled a custom protein database which included the predicted translation sequence of all B. abortus 2308 ORFs annotated as pseudogenes. Using this database, we were able to confirm the protein expression of five of Background Brucella species bacteria are gram negative alpha pro- teobacteria superbly adapted for survival in intracellular environments. They infect a wide range of mammals, including essentially all economically important domestic mammals, many wild species, and humans. Brucellosis is the largest bacterial zoonosis in the world [1-3]. In humans, untreated brucellosis is a long lasting disease characterized by recurrent fever episodes and clinical manifestations that include spondylitis, severe head- aches, joint or abdominal pain, endocarditis, and menin- goencephalitis. In severe non-treated cases brucellosis can cause death [1-3]. Seven terrestrial Brucella species have been defined: Brucella melitensis, Brucella abortus, Brucella suis, Bru- Lamontagne et al. BMC Genomics 2010, 11:300 http://www.biomedcentral.com/1471-2164/11/300 Page 2 of 10 behavior of the different Brucella species sequenced to date [15]. tus isolated at different time points post-infection from RAW264.7 macrophages [17] and of phagosomes isolated from infected murine phagocytic cells. We obtained 1704 peptides representing 621 different proteins, correspond- ing to approximately 20% of the predicted proteome. For 305 proteins, we are reporting the first experimental evi- dence of their expression in B. abortus 2308 (Table 1). We also report genome annotation errors for two proteins, expression of ORFs annotated pseudogenes for four pro- teins and one correction to the sequence of another pre- viously annotated pseudogene which allows for its full length expression. Peptide sequences corresponding to these 312 proteins are listed in Additional File 1. The pep- tide coverage for the 305 newly demonstrated proteins varied from 1 to 20, with an average of three peptides per protein. In order to confirm the expression of proteins identified by a single peptide, we manually validated all MSMS spectra that had a sequence assignment score smaller than 45. Forty-four of the 305 proteins were described previously as hypothetical with no putative function. When subcellular localizations were predicted using three publicly available tools [18-20], 226 proteins were predicted to be cytosolic, ten were inner membrane proteins, 25 were periplasmic, three were outer mem- brane proteins and the localization of 48 proteins could not be predicted (Table 1). Experimental evidence for the expression of the other 309 of the 620 proteins has been demonstrated previously by our group [16,17] and others [21-31]. It is important to note that we are reporting an analysis of the combined results of several independent experiments using the same bacterial strain and technol- ogy to acquire the data. Background We provide the first direct experimental evidence for the expression of 305 Brucella proteins, but also identified experimental evidence for the expression of five genes previously annotated as pseudogenes, and of start site errors in two other genes. Results and Discussion First experimental evidence of the expression of 305 proteins in B. abortus 2308 Samples used for the proteomic analysis came from B. abortus either grown extracellularly in media or isolated from infected RAW264.7 macrophages. The extracellular samples included whole bacteria grown directly in tryptic soy broth, outer membrane preparations (blebs) [16] and cytosols. Intracellular samples consisted of viable B. abor- Lamontagne et al. BMC Genomics 2010, 11:300 http://www.biomedcentral.com/1471-2164/11/300 Page 3 of 10 Table 1: B. Results and Discussion First experimental evidence of the expression of 305 proteins in B. abortus 2308 abortus 2308 proteins for which the expression was demonstrated for the first time Cytoplasm BAB1_0002 DnaN BAB1_0855 GRX family BAB1_1449 UDP-N- BAB1_2149 PepS BAB1_0022 Unknown BAB1_0856 BolA-related acetylmuramate BAB1_2168 RpsO; S15 BAB1_0023 AroA BAB1_0857 FGAM synthase II L-alanine ligase BAB1_2173 FabB BAB1_0035 KdsB BAB1_0861 PurS BAB1_1508 CarB BAB2_0083 Eda2 BAB1_0063 Unknown BAB1_0864 HpcH/HpaI BAB1_1512 CspA BAB2_0090 GCN5-related BAB1_0071 ArgG BAB1_0874 AcpP BAB1_1523 GreA N-acetyltransferase BAB1_0100 Putative AsnC family BAB1_0880 HAD-like BAB1_1528 SseA-1 BAB2_0109 Gnd BAB1_0107 Trs-ABC (P-loop) BAB1_0886 NN:DBI PRT BAB1_1538 OmpR BAB2_0160 Unknown BAB1_0118 Unknown BAB1_0896 ArgS BAB1_1547 PepQ BAB2_0162 L-carnitine BAB1_0122 GyrB BAB1_0898 NagZ BAB1_1549 PrsA dehydratase BAB1_0139 NifU BAB1_0918 GatB/Yqey BAB1_1553 YchF BAB2_0177 YafB BAB1_0159 S30EA BAB1_0924 AccC BAB1_1613 Unknown BAB2_0186 Fumarate hydratase BAB1_0160 PtsN-like BAB1_0933 PCRF 2 BAB1_1645 DhaK-1 BAB2_0187 Unknown BAB1_0191 GABAtrnsam BAB1_0943 TyrS BAB1_1646 DhaK-2 BAB2_0191 HAD-like, BAB1_0204 AdhP BAB1_0949 SufC BAB1_1655 GabD subfamily IIA BAB1_0215 ThiE BAB1_0955 DeaD BAB1_1669 PAS domain BAB2_0198 Pseudouridine BAB1_0216 ThiG BAB1_0960 Trs heavy metal BAB1_1671 TcaR synthase BAB1_0242 ManR BAB1_1014 MetG BAB1_1687 Dut BAB2_0216 3-hydroxybutyryl-CoA BAB1_0285 HisD BAB1_1030 Gor BAB1_1695 PurA dehydrogenase BAB1_0317 Trs arginine/ ornithine BAB1_1037 Mandelate racemase; BAB1_1702 Phosphoglucosa mine BAB2_0246 P47K BAB1_0331 ArgD muconate lactonizing mutase BAB2_0293 Gal BAB1_0344 Pip BAB1_1043 Unknown BAB1_1719 ThiE BAB2_0295 DgoK BAB1_0353 Unknown BAB1_1050 FolB BAB1_1722 Efp BAB2_0296 KdgA dehydrogenase BAB1_1077 Ach1p BAB1_1751 Unknown BAB2_0335 NADH:flavin oxidore- BAB1_0416 DUF85 BAB1_1096 NifU-like BAB1_1761 PyK ductase/NADH oxidase BAB1_0429 Polyprenyl synthetase BAB1_1098 PRA-CH BAB1_1778 FdxA BAB2_0337 RocF BAB1_0446 DnaJ BAB1_1121 DNA gyrase subunit A BAB1_1781 Unknown BAB2_0343 Trx-2 BAB1_0447 FabI-1 BAB1_1130 ClpA/B BAB1_1804 MarR family BAB2_0358 Dcp BAB1_0482 FabD BAB1_1132 ClpP BAB1_1810 AtpH BAB2_0361 TypA BAB1_0484 AcpP BAB1_1156 KdsA BAB1_1813 Transaldolase BAB2_0365 FbaA BAB1_0489 Guanylate kinase BAB1_1157 PyrG BAB1_1815 LeuS BAB2_0366 RpiB/LacA/LacB BAB1_0510 ThrC BAB1_1161 TpiA BAB1_1819 ACAT BAB2_0367 TIM 2 BAB1_0525 PpdK BAB1_1164 TrpC BAB1_1824 PurH BAB2_0370 EryC BAB1_0532 Transthyretin BAB1_1169 GltX BAB1_1837 CynT BAB2_0448 Unknown BAB1_0540 Formyl transferase, BAB1_1170 GltA BAB1_1840 MmsA BAB2_0457 FolD N-terminal BAB1_1174 FabZ BAB1_1872 PrfA BAB2_0459 Pgl BAB1_0544 DegT/DnrJ/EryC1/ StrS BAB1_1187 Endoribonuclease BAB1_1874 LysC BAB2_0460 Zwf BAB1_0561 Man-6-P isomerase L-PSP BAB1_1879 GrxC BAB2_0483 ShuT type II BAB1_1188 GDPD BAB1_1887 HemC BAB2_0513 GcvT BAB1_0570 XylA BAB1_1205 ElaB-domain BAB1_1895 FtsK-gamma BAB2_0518 PutA BAB1_0587 Unknown BAB1_1212 BhbA BAB1_1918 LpdA-2 BAB2_0566 AldA BAB1_0588 ATP/GTP-binding BAB1_1213 Unknown; conserved BAB1_1926 SucC BAB2_0568 Unknown Table 1: B. abortus 2308 proteins for which the expression was demonstrated for the first time Lamontagne et al. Results and Discussion First experimental evidence of the expression of 305 proteins in B. abortus 2308 BMC Genomics 2010, 11:300 http://www.biomedcentral.com/1471-2164/11/300 Page 4 of 10 BAB1_0641 Alanine aminopep- BAB1_1223 AlaS BAB1_1936 GloB BAB2_0572 IlvE tidase: Neutral zinc BAB1_1224 RecA BAB1_1946 SecA BAB2_0620 Unknown metallopeptidase, BAB1_1233 RpsM; S13 BAB1_1970 FadB BAB2_0642 Acyl-CoA zinc-binding region BAB1_1234 Adk BAB1_1971 EtfA dehydrogenase BAB1_0666 DapA BAB1_1241 RpsH; S8 BAB1_1988 HisC BAB2_0644 Metal-dependent BAB1_0671 RpoZ BAB1_1242 RpsN; S14 BAB1_1993 Ppa hydrolase BAB1_0688 PyrC-1 BAB1_1244 RplX; L24 BAB1_2006 RegA BAB2_0645 GatC BAB1_0697 CysS BAB1_1245 RplN; L14 BAB1_2016 RpmB; L28 BAB2_0646 GatA BAB1_0718 MoaD BAB1_1248 RplP; L16 BAB1_2023 ClpA/clpB BAB2_0851 GuaB BAB1_0740 Unknown BAB1_1249 RpsC; S3 BAB1_2059 ParB BAB2_0961 DapA BAB1_0775 AspS BAB1_1256 RpsJ; S10 BAB1_2080 HslU BAB2_0976 AldB BAB1_0780 HemB BAB1_1266 RplJ; L10 BAB1_2081 HslV BAB2_0988 ArgB BAB1_0787 GlyA BAB1_1280 Unknown BAB1_2087 HisE BAB2_0990 Unknown BAB1_0789 RibD BAB1_1286 GloA BAB1_2096 PTS system IIA BAB2_0991 DapD BAB1_0790 RibE BAB1_1294 Aminotransferase subunit BAB2_0993 DapE BAB1_0813 CysD BAB1_1297 Unknown BAB1_2109 AccD BAB2_1009 MgsA BAB1_0817 Unknown; conserved BAB1_1376 UreA BAB1_2133 Unknown BAB2_1012 DapB BAB1_0826 NuoE BAB1_1408 IlvB BAB1_2134 SMP-30 BAB2_1013 Gpm BAB1_0842 ProS BAB1_2135 Glutathione synthetase Inner membrane BAB1_0400 Unknown BAB1_1283 DUF192 BAB2_0261 RecA BAB2_0877 Binding-protein- BAB1_0425 NhaA BAB1_1703 FtsH BAB2_0709 FtsK-alpha dependent transport BAB1_0542 WbkC BAB1_1712 MotA; TolQ; ExbB BAB2_0728 CydA system inner membrane component Periplasm BAB1_0010 Trs-ABC oligopeptide BAB1_1118 PpiB-1 BAB2_0427 Trs-ABC spermidine/ putrescine BAB2_0697 Unknown; conserved BAB1_0155 OstA-like BAB1_1362 LacI BAB2_0812 Trs-ABC oligopeptide BAB1_0404 Unknown BAB1_1413 DegP BAB2_0451 Trs-ABC oligopeptide AppA family BAB1_0444 PdxH BAB1_1890 YciI-like protein AppA family BAB2_0879 Trs-ABC spermidine/ putrescine BAB1_0739 ETC complex I BAB1_1919 Unknown BAB2_0593 Trs-ABC amino acid BAB1_0776 Unknown BAB1_1981 TlpA BAB2_0611 Trs-ABC amino acid BAB2_0880 Unknown BAB1_0881 Trs-ABC amino acid BAB2_0374 Unknown BAB2_0664 Trs-ABC peptide BAB2_1109 XylF BAB1_1117 PpiB-2 Outer membrane BAB1 0659 Omp2a BAB1 0707 OstA BAB1 0963 TolC Table 1: B. Results and Discussion First experimental evidence of the expression of 305 proteins in B. abortus 2308 abortus 2308 proteins for which the expression was demonstrated for the first time (Continued) BAB1_0641 Alanine aminopep- BAB1_1223 AlaS BAB1_1936 GloB BAB2_0572 IlvE tidase: Neutral zinc BAB1_1224 RecA BAB1_1946 SecA BAB2_0620 Unknown metallopeptidase, BAB1_1233 RpsM; S13 BAB1_1970 FadB BAB2_0642 Acyl-CoA zinc-binding region BAB1_1234 Adk BAB1_1971 EtfA dehydrogenase BAB1_0666 DapA BAB1_1241 RpsH; S8 BAB1_1988 HisC BAB2_0644 Metal-dependent BAB1_0671 RpoZ BAB1_1242 RpsN; S14 BAB1_1993 Ppa hydrolase BAB1_0688 PyrC-1 BAB1_1244 RplX; L24 BAB1_2006 RegA BAB2_0645 GatC BAB1_0697 CysS BAB1_1245 RplN; L14 BAB1_2016 RpmB; L28 BAB2_0646 GatA BAB1_0718 MoaD BAB1_1248 RplP; L16 BAB1_2023 ClpA/clpB BAB2_0851 GuaB BAB1_0740 Unknown BAB1_1249 RpsC; S3 BAB1_2059 ParB BAB2_0961 DapA BAB1_0775 AspS BAB1_1256 RpsJ; S10 BAB1_2080 HslU BAB2_0976 AldB BAB1_0780 HemB BAB1_1266 RplJ; L10 BAB1_2081 HslV BAB2_0988 ArgB BAB1_0787 GlyA BAB1_1280 Unknown BAB1_2087 HisE BAB2_0990 Unknown BAB1_0789 RibD BAB1_1286 GloA BAB1_2096 PTS system IIA BAB2_0991 DapD BAB1_0790 RibE BAB1_1294 Aminotransferase subunit BAB2_0993 DapE BAB1_0813 CysD BAB1_1297 Unknown BAB1_2109 AccD BAB2_1009 MgsA BAB1_0817 Unknown; conserved BAB1_1376 UreA BAB1_2133 Unknown BAB2_1012 DapB BAB1_0826 NuoE BAB1_1408 IlvB BAB1_2134 SMP-30 BAB2_1013 Gpm BAB1_0842 ProS BAB1_2135 Glutathione synthetase Table 1: B. abortus 2308 proteins for which the expression was demonstrated for the first time (Continued) tus 2308 proteins for which the expression was demonstrated for the first time (Continued) Lamontagne et al. BMC Genomics 2010, 11:300 http://www.biomedcentral.com/1471-2164/11/300 Page 5 of 10 Unknown localization BAB1_0030 Unknown BAB1_0991 Unknown BAB1_1543 DUF526 BAB1_2123 RpmI; L35 BAB1_0170 GrpE BAB1_1070 WrbA BAB1_1559 FbcF BAB1_2176 YaeC/NLPA lipoprotein BAB1_0389 CcoP BAB1_1113 Unknown; conserved BAB1_1641 Unknown BAB1_2186 RpsT; S20 BAB1_0413 AtpB BAB1_1152 PdhA BAB1_1647 FabG domain BAB2_0207 Unknown BAB1_0418 Unknown BAB1_1230 RplQ; L17 BAB1_1693 bZIP BAB2_0243 YedY BAB1_0420 Unknown BAB1_1232 RpsK; S11 BAB1_1728 RpmE; L31 BAB2_0269 RpsU; S21 BAB1_0453 Unknown BAB1_1240 PplF; L6 BAB1_1749 Unknown BAB2_0351 OsmC-like protein BAB1_0479 RpsR, S18 BAB1_1260 RpsL; S12 BAB1_1768 Unknown BAB2_0356 Unknown BAB1_0627 Unknown BAB1_1270 SecE BAB1_1784 DUF336 BAB2_0677 Unknown BAB1_0650 Unknown BAB1_1341 Unknown BAB1_1814 Unknown BAB2_0726 YbgT BAB1_0810 RpsI; S9 BAB1_1384 Cibk BAB1_1858 RplU; L21 BAB2_0869 HlyD BAB1_0830 NDH-1 subunit I BAB1_1514 AspC BAB1_1984 LysA BAB2_1002 NqoB Locus tags and descriptions of proteins are indicated and proteins are organized by predicted subcellular localization. Table 1: B. abortus 2308 proteins for which the expression was demonstrated for the first time (Continued) Table 1: B. abortus 2308 proteins for which the expression was demonstrated for the first gated the reasons for which these genes had been anno- tated as pseudogenes. Results and Discussion First experimental evidence of the expression of 305 proteins in B. abortus 2308 abortus 2308, a fusion of the two genes would generate a much larger protein. However, the start codon in the corresponding ORF of vaccine B. abortus S19 (BAbS19_II02060) is different from BMEII1020, and even more different from the start codon and carboxyl terminal sequence of the counterparts in B. suis (BSUIS_B0227), B. ovis (BOV_A0203), B. canis (BCAN_B0224) and B. ceti (BCETI_6000534). As a con- sequence, the lengths of B. abortus and B. melitensis pro- teins differ considerably from those of other Brucella. Since the BAB2_0216 peptide that we found is located in the N-terminal section of the protein (Figure 1), we are able to confirm the expression of this originally annotated pseudogene, but were unable to confirm the expression of the full length protein. The second peptide, "TDLLPIMK", was found to match the cytoplasmic B. melitensis keto-hydroxyglutarate- aldolase (BMEII0009) and then assigned to BAB2_0083 in B. abortus 2308. This peptide overlaps the region upstream to the currently annotated translation start site and the first three amino acids based on the annotated translation start site (Figure 2B). Alignment of the cur- rent B. abortus 2308 protein sequence with its counter- parts in other Brucella strains and species indicates that the 2308 protein sequence is falsely truncated. Other start sites lead to proteins having N-terminals longer by 11, 26 or 44 amino acids. Although we cannot clearly indicate the actual start site of BAB1_1926 or BAB2_0083, we can confirm that their N-terminals are longer than currently annotated. Based on the homology of the B. abortus 2308 genome being highest with that of other B. abortus strains, one can speculate that the start sites would be identical to those mapped in these strains. g The sequence of the BAB1_1768 pseudogene was found to be misannotated in B. abortus 2308. The peptide sequence "TAGYGVGGAALGALAGGAIGGNGR" could not be found in the B. abortus 2308 nucleotide-derived proteome but matched the B. melitensis locus tag BMEI0287. In fact, except for 1 nucleotide, the corre- sponding 2308 genomic sequence is identical to that of BMEI0287 (Figure 2C). In B. abortus 2308, a single nucle- otide insertion in BAB1_1768 modifies the reading frame, hence its original annotation as a pseudogene. The manu- ally validated peptide matches B. abortus 2308 only when the additional nucleotide is removed, indicating that the sequence for locus BAB1_1768 should be corrected (Fig- ure 1). Also to note is the earlier start site in B. Results and Discussion First experimental evidence of the expression of 305 proteins in B. abortus 2308 The genomic sequence of the cytoplasmic protein with a conserved DUF 883 domain BAB1_1205 was found to be identical to BMEI0805, its B. gated the reasons for which these genes had been anno- tated as pseudogenes. The genomic sequence of the cytoplasmic protein with a conserved DUF 883 domain BAB1_1205 was found to be identical to BMEI0805, its B. these ORFs (Figure 1): BAB1_1205, BAB1_1645, BAB1_1646, BAB1_1768 and BAB2_0216. The MSMS spectra of the 18 peptides representing these former pseudogenes were manually validated. We thus investi- these ORFs (Figure 1): BAB1_1205, BAB1_1645, BAB1_1646, BAB1_1768 and BAB2_0216. The MSMS spectra of the 18 peptides representing these former pseudogenes were manually validated. We thus investi- Figure 1 B. abortus 2308 former pseudogenes. Peptide sequences identified by mass spectrometry are highlighted in grey. Corresponding B. me- litensis 16 M locus tags are indicated between parentheses. BAB2_0216 VSVERHADGVATVRINRPEARNALNLTTRQQLAEHFRALSGDESVRAIVLTGGETCFVAG 60 ADVREFASAGPIEMYLRHTEYLWDAIASCAKPVIAAVNGYALGGGCELAMHCDIIVAGEG 120 AVFGQPEVKLGLMPGAGGTQRLIRAVGKFQAMRIALTGCMVPAAEALSIGMISEMTANER 180 TLPRAHELAVEIARLPALAVAQIKEVMLVGADLPLDGALALERKAFQLLFDSKDQKRAQP 240 LSSKNANLPITDARTMERSINHIAIVGAGVMGTGIAQIAAQAGLVTQIFDAREGAAAASR 300 DRLASTLAKLAEKGKISAEDAQTAVSRIEICSSIQELADCDLVVEAIVEKLDAKQALFLE 360 LEAVVSGNCILATNTSSLSVTSIARVCRHPERVAGFHFFNPVPLMKVVEVIDGLTTDPAV 420 GDALLVLAKRMGHHGIRAKDMPGFIINHAGRAYGTEALKILGECVAPRGDIDRILRESAG 480 FRMGPLELFDLTGLDVSHPVMESIYNQFYQEPRYSPSALTRQMLEGGYVGRKVGQGFYRY 540 EDGKMVAPPVPQPVPAVDIMPSVWISADCDEDKEQLYALLRSLGATVETGALPSAEALCL 600 LAPYGYDATTACELAGSDPARTVCIDMLPGLDRHRTLMMTPATSPAFRDAAHALLARDGV 660 NVTVIRDSVGFVAQRTLAAIVNLACDIAQQGIATADDIDQAVRLGLGYPQGPLAWGGFSD 720 PENIVAYAGINRRPPLQAKPMVAPQGRAGPFPALRGAGYRLEHFRAKSAKRLRGEISPLD 780 Enoyl-CoA hydratase (BMEII1021) MKSRLTMIAVAGLLAFSTAACTTNEQRTAGYGVGGAALGALAGGAIGGNGRGALTGAAIG 60 AVAGTLLGAAQTRNGTQYCRYRDPYGRIYEAPCQ 94 BAB1_1768 Hypothetical protein (BMEI0287) BAB1_1646 Dak phosphatase domain (BMEI0396) LSPQLIHITGDTMQRFINNPDEVVEDTVRGFVKAHSDIIRLAENPRVIAAKDAPVAGKVG 60 VVTGGGSGHEPAFIGYTGKNMLDAVAVGELFSSPTAKSFHDAIREANGGKGVVVLYGNYA 120 GDNMNVKMATKLAAKDGIDVATVVANDDVCSAPAAEREKRRGVAGEIFMWKVGGAKAATG 180 ATLEEVRATAQKAIDNCRSIGVGLGPCTLPAVGHPNFEIAPGTMEVGIGHHGEPGVRVEP 240 LKSAAEVARDMCQIVLDDHGLAEGTEVAVLVSGLGATPLNELYILNDTIETEIRARGLKI 300 HRTYIGNYFTSLEMVGATLTVMALDSELKELLDVEVRCTTIL 342 VRKRPVQTLNNAKAGDIVLTMAERIVENRAYLSEIDGKIGDGDHGVNMAKGFNMAAERLQ 60 GKNETLAASLDTLGTVLMTEIGGSMGPLYGVMFTEFAEKIDGVDNIDAAAFSHMLHAGLE 120 GIQSIGSAKVGDKTLLDTLVPAVEAFDEANAAGKSFAEALEALVAAAEKGRDSTINLVAR 180 IGRASHLGERSLGVLDAGATSCAIILKVLGEGARERLQ 218 Dak phosphatase domain (BMEI0397) BAB1_1645 Hypothetical protein (BMEI0805) BAB1_1205 MAEANINDIQQALEKQIAEMRTELKRMSRSLASHSDDLKARAEDAMDEASGRLRHAAQTV 60 RERGQVVAEAVRENPGTATTLFGTAGIIGILIGVAIGCALSERR 104 AEANINDIQQALEK AEDAMDEASGR AEDAMDEASGR + m5\|1 Oxidation (M) ARAEDAMDEADGR ARAEDAMDEASGR + m5\|1 Oxidation (M) GQVVAEAVR SLASHSDDLK Locus tag Protein description Peptide sequence Protein sequence QQLAEHFR TAGYGVGGAALGALAGGAIGGNGR + m22\|1 Deamidation (N) AATGATLEEVR FINNPDEVVEDTVR NMLDAVAVGELFSSPTAK NMLDAVAVGELFSSPTAK + m2\|1 Oxidation (M) VGVVTGGGSGHEPAFIGYTGK AGDIVLTMAER AYLSEIDGK SFAEALEALVAAAEK TLLDTLVPAVEAFDANAAGK Locus tag Protein description Peptide sequence Protein sequence mer pseudogenes. Peptide sequences identified by mass spectrometry are highlighted in grey. Corresponding B. me- cated between parentheses. Lamontagne et al. BMC Genomics 2010, 11:300 http://www.biomedcentral.com/1471-2164/11/300 Page 6 of 10 melitensis counterpart. Apart from the short length of this protein, there was no apparent reason for its pseudo- gene annotation (Figure 1). For BAB1_1645 and BAB1_1646 (Figure 1), the nucleotidic sequence was 99% identical to their BMEI0397 and BMEI0396 counterparts, leading to two cytoplasmic B. abortus 2308 dihydroxyace- tone kinases involved in glycerolipid metabolism that are 98% and 100% identical to the B. melitensis proteins, respectively. The case of BAB2_0216, which seems to be a 3-hydroxybutyryl-CoA dehydrogenase, was more com- plex and confusing, having a single nucleotide deletion when compared to B. melitensis. Conclusions Mass spectrometry has proven to be a valuable tool to identify and correct genomic annotation errors in the study of microorganisms [33-37]. We performed a pro- teomics analysis of B. abortus 2308 proteins expressed upon extracellular and intracellular growth conditions to validate existing gene predictions at the protein level, to Operons Since genes that are part of an operon are usually co-tran- scribed, it is possible that these genes might also be co- translated [32]. Considering all proteins identified by our studies, we were able to almost fully reconstitute one of the two ribosomal RNA operons, with all but BAB1_1237 found. Additionally, the previously mentioned BAB1_1645 and BAB1_1646 genes are predicted to be part of an operon containing 6 genes, BAB1_1645 to BAB1_1650 http://www.microbesonline.org/operons/ gnc359391.html. Four of these proteins were detected in our studies, although only BAB1_1645, -46 and -48 were found in the same experimental condition. Results and Discussion First experimental evidence of the expression of 305 proteins in B. abortus 2308 abortus 2308, and all other species sequenced to date, when com- pared to B. melitensis 16 M. We believe that the B. abor- tus 2308 start site was correctly assigned in the publicly available genome given the clear presence of a ribosome binding site in position -8 of the B. abortus sequence. Results and Discussion First experimental evidence of the expression of 305 proteins in B. abortus 2308 This deletion would lead to the silencing of the stop codon which creates two sepa- rate proteins in B. melitensis, BMEII1020 and BMEII1021. In B. abortus 2308, a fusion of the two genes would generate a much larger protein. However, the start codon in the corresponding ORF of vaccine B. abortus S19 (BAbS19_II02060) is different from BMEII1020, and even more different from the start codon and carboxyl terminal sequence of the counterparts in B. suis (BSUIS_B0227), B. ovis (BOV_A0203), B. canis (BCAN_B0224) and B. ceti (BCETI_6000534). As a con- sequence, the lengths of B. abortus and B. melitensis pro- teins differ considerably from those of other Brucella. Since the BAB2_0216 peptide that we found is located in the N-terminal section of the protein (Figure 1), we are able to confirm the expression of this originally annotated pseudogene, but were unable to confirm the expression of the full length protein. to the sequence found upstream of their currently anno- tated start sites (Figure 2). The peptide sequence "MNI- HEYQAK" was first found to match the cytoplasmic B. melitensis succinyl-CoA synthetase subunit beta protein (BMEI0138) and then assigned manually to BAB1_1926. Sequence comparison with other Brucella species and strains shows that the B. abortus 2308 protein start site is not shared with any of the subject sequences (Figure 2A). In fact, all homologues of this protein in other Brucella strains or species share the same start site, which is found 22 amino acids upstream of the B. abortus 2308 site. Moreover, a ribosome binding site can clearly be mapped to position -8 of the proposed new translation start site. We therefore believe this new start site to be accurate. melitensis counterpart. Apart from the short length of this protein, there was no apparent reason for its pseudo- gene annotation (Figure 1). For BAB1_1645 and BAB1_1646 (Figure 1), the nucleotidic sequence was 99% identical to their BMEI0397 and BMEI0396 counterparts, leading to two cytoplasmic B. abortus 2308 dihydroxyace- tone kinases involved in glycerolipid metabolism that are 98% and 100% identical to the B. melitensis proteins, respectively. The case of BAB2_0216, which seems to be a 3-hydroxybutyryl-CoA dehydrogenase, was more com- plex and confusing, having a single nucleotide deletion when compared to B. melitensis. This deletion would lead to the silencing of the stop codon which creates two sepa- rate proteins in B. melitensis, BMEII1020 and BMEII1021. In B. Correction of two start site annotations errors abortus 2308 M A V Y S V E BAB1_1926 1874421 1874594 1874450 1873291 BAB1_1927 NC_007618 1874400 A * ** BAB1_1768 M K S R L T M I A V A G L L A F S T A A C T T N E Q R T bab1_1768 ttgtaggagatgtttcATGAAATCTCGTCTTACGATGATTGCTGTTGCTGGCTTGCTGGCGTTCTCGACCGCCGCGTGCACGACGAACGAACAGCGTACG bmeI0287 ttgtaggagatgtttcatgaaatctcgtcttacgATGATTGCTGTTGCTGGCTTGCTGGCGTTCTCGACCGCCGCGTGCACGACGAACGAACAGCGTACG BMEI0287 ----------------------------------M I A V A G L L A F S T A A C T T N E Q R T * * * * * * * * * * * * * * * * * * * * * * BAB1_1768 A W L R R W W C G S R C S C R W R N W R Q W P W C S D G C C D R C G bab1_1768 GCTTGGTTACGGCGTTGGTGGTGCGGCTCTCGGTGCTCTTGCCGGTGGCGCAATTGGCGGCAATGGCCGTGGTGCTCTGACGGGTGCTGCGATCGGTGCGGT bmeI0287 GCT-GGTTACGGCGTTGGTGGTGCGGCTCTCGGTGCTCTTGCCGGTGGCGCAATTGGCGGCAATGGCCGTGGTGCTCTGACGGGTGCTGCGATCGGTGCGGT BMEI0287 A G Y G V G G A A L G A L A G G A I G G N G R G A L T G A A I G A V * BAB1_1768 C R H T S W C S P D A STOP--------------------------------------------------------------- bab1_1768 TGCAGGCACACTTCTTGGTGCAGCCCAGACGCGTAA---------------------------------------------------------------- bmeI0287 TGCAGGCACACTTCTTGGTGCAGCCCAGACGCGTAATGGCACGCAATATTGCCGTTACCGCGATCCGTATGGCCGCATCTACGAAGCGCCTTGCCAGTAA BMEI0287 A G T L L G A A Q T R N G T Q Y C R Y R D P Y G R I Y E A P C Q STOP C BAB2_0083 86363 tttgttctgaagccttgcgtatttcatcaaggggatgcgcccaaccgtcgagcgagccgatgtcgcagaaaaccgatcttcttcttcccatcATGAAAGGC 86263 B. melitensis 16M M R I S S R G C A Q P S S E P M S Q K T D L L L P I M K G B. abortus 9-941 M S Q K T D L L L P I M K G B. ovis M S Q K T D L L L P I M K G B. abortus 2308 M K G NC_007624 86271 86463 86375 85666 86263 BAB2_0084 B ** re 2 Annotation errors in the B. abortus 2308 genome. (A, B) The original start codon annotation in the publicly available genome (NCB my ID 359391) of the succinyl-CoA synthetase subunit beta (BAB1 1926, panel A) and of the KHG aldolase (BAB2 0083, panel B) are indicate 1874450 agaggacaaccagatgaatattcacgaataccaggccaagcgcctgcttcacacctacggcgcgccgatcgccaatggtGTGGCTGTCTATTCCGTCGAA 1874400 B. abortus 9-941 M N I H E Y Q A K R L L H T Y G A P I A N G V A V Y S V E B. Correction of two start site annotations errors melitensis 16M M N I H E Y Q A K R L L H T Y G A P I A N G V A V Y S V E B. ovis M N I H E Y Q A K R L L H T Y G A P I A N G V A V Y S V E B. suis M N I H E Y Q A K R L L H T Y G A P I A N G V A V Y S V E B. abortus 2308 M A V Y S V E BAB1_1926 1874421 1874594 1874450 1873291 BAB1_1927 NC_007618 1874400 A * ** BAB1_1768 M K S R L T M I A V A G L L A F S T A A C T T N E Q R T bab1_1768 ttgtaggagatgtttcATGAAATCTCGTCTTACGATGATTGCTGTTGCTGGCTTGCTGGCGTTCTCGACCGCCGCGTGCACGACGAACGAACAGCGTACG bmeI0287 ttgtaggagatgtttcatgaaatctcgtcttacgATGATTGCTGTTGCTGGCTTGCTGGCGTTCTCGACCGCCGCGTGCACGACGAACGAACAGCGTACG BMEI0287 ----------------------------------M I A V A G L L A F S T A A C T T N E Q R T * * * * * * * * * * * * * * * * * * * * * * BAB1_1768 A W L R R W W C G S R C S C R W R N W R Q W P W C S D G C C D R C G bab1_1768 GCTTGGTTACGGCGTTGGTGGTGCGGCTCTCGGTGCTCTTGCCGGTGGCGCAATTGGCGGCAATGGCCGTGGTGCTCTGACGGGTGCTGCGATCGGTGCGGT bmeI0287 GCT-GGTTACGGCGTTGGTGGTGCGGCTCTCGGTGCTCTTGCCGGTGGCGCAATTGGCGGCAATGGCCGTGGTGCTCTGACGGGTGCTGCGATCGGTGCGGT BMEI0287 A G Y G V G G A A L G A L A G G A I G G N G R G A L T G A A I G A V * BAB1_1768 C R H T S W C S P D A STOP--------------------------------------------------------------- bab1_1768 TGCAGGCACACTTCTTGGTGCAGCCCAGACGCGTAA---------------------------------------------------------------- bmeI0287 TGCAGGCACACTTCTTGGTGCAGCCCAGACGCGTAATGGCACGCAATATTGCCGTTACCGCGATCCGTATGGCCGCATCTACGAAGCGCCTTGCCAGTAA BMEI0287 A G T L L G A A Q T R N G T Q Y C R Y R D P Y G R I Y E A P C Q STOP C BAB2_0083 86363 tttgttctgaagccttgcgtatttcatcaaggggatgcgcccaaccgtcgagcgagccgatgtcgcagaaaaccgatcttcttcttcccatcATGAAAGGC 86263 B. melitensis 16M M R I S S R G C A Q P S S E P M S Q K T D L L L P I M K G B. abortus 9-941 M S Q K T D L L L P I M K G B. ovis M S Q K T D L L L P I M K G B. Correction of two start site annotations errors abortus 2308 M K G NC_007624 86271 86463 86375 85666 86263 BAB2_0084 B ** B BAB1_1768 M K S R L T M I A V A G L L A F S T A A C T T N E Q R T bab1_1768 ttgtaggagatgtttcATGAAATCTCGTCTTACGATGATTGCTGTTGCTGGCTTGCTGGCGTTCTCGACCGCCGCGTGCACGACGAACGAACAGCGTACG bmeI0287 ttgtaggagatgtttcatgaaatctcgtcttacgATGATTGCTGTTGCTGGCTTGCTGGCGTTCTCGACCGCCGCGTGCACGACGAACGAACAGCGTACG BMEI0287 ----------------------------------M I A V A G L L A F S T A A C T T N E Q R T * * * * * * * * * * * * * * * * * * * * * * BAB1_1768 A W L R R W W C G S R C S C R W R N W R Q W P W C S D G C C D R C G bab1_1768 GCTTGGTTACGGCGTTGGTGGTGCGGCTCTCGGTGCTCTTGCCGGTGGCGCAATTGGCGGCAATGGCCGTGGTGCTCTGACGGGTGCTGCGATCGGTGCGGT bmeI0287 GCT-GGTTACGGCGTTGGTGGTGCGGCTCTCGGTGCTCTTGCCGGTGGCGCAATTGGCGGCAATGGCCGTGGTGCTCTGACGGGTGCTGCGATCGGTGCGGT BMEI0287 A G Y G V G G A A L G A L A G G A I G G N G R G A L T G A A I G A V * BAB1_1768 C R H T S W C S P D A STOP--------------------------------------------------------------- bab1_1768 TGCAGGCACACTTCTTGGTGCAGCCCAGACGCGTAA---------------------------------------------------------------- bmeI0287 TGCAGGCACACTTCTTGGTGCAGCCCAGACGCGTAATGGCACGCAATATTGCCGTTACCGCGATCCGTATGGCCGCATCTACGAAGCGCCTTGCCAGTAA BMEI0287 A G T L L G A A Q T R N G T Q Y C R Y R D P Y G R I Y E A P C Q STOP C Figure 2 Annotation errors in the B. abortus 2308 genome. (A, B) The original start codon annotation in the publicly available genome (NCBI tax- onomy ID 359391) of the succinyl-CoA synthetase subunit beta (BAB1_1926, panel A) and of the KHG aldolase (BAB2_0083, panel B) are indicated by double asterisks whereas the corrected start site is indicated by a single asterisk (BAB1_1926 only). The peptides sequenced by mass spectrometry are highlighted in grey. The 5'-end of the CDS, as currently annotated, are underlined. The predicted sequence of the RBS found in proximity of the cor- rected start site of BAB1_1926 is boxed. Numbers next to the nucleotide sequence and the schematic gene representation indicate the position in the genome sequence (NC_007618 or NC_007624). (C) Genomic and amino acid sequences of BAB1_1768, as currently found in the publicly available genome, were aligned to their counterparts in B. melitensis 16 M (BMEI0287). The sequence of the peptide detected by mass spectrometry is high- lighted in grey. Matching nucleotides are indicated by vertical bars and matching amino acids are indicated by asterisks. Correction of two start site annotations errors abortus 2308 M K G NC_007624 86271 86463 86375 85666 86263 BAB2_0084 B ** 1874450 agaggacaaccagatgaatattcacgaataccaggccaagcgcctgcttcacacctacggcgcgccgatcgccaatggtGTGGCTGTCTATTCCGTCGAA 1874400 B. abortus 9-941 M N I H E Y Q A K R L L H T Y G A P I A N G V A V Y S V E B. melitensis 16M M N I H E Y Q A K R L L H T Y G A P I A N G V A V Y S V E B. ovis M N I H E Y Q A K R L L H T Y G A P I A N G V A V Y S V E B. suis M N I H E Y Q A K R L L H T Y G A P I A N G V A V Y S V E B. abortus 2308 M A V Y S V E BAB1_1926 1874421 1874594 1874450 1873291 BAB1_1927 NC_007618 1874400 A * ** B BAB2_0083 86363 tttgttctgaagccttgcgtatttcatcaaggggatgcgcccaaccgtcgagcgagccgatgtcgcagaaaaccgatcttcttcttcccatcATGAAAGGC 86263 B. melitensis 16M M R I S S R G C A Q P S S E P M S Q K T D L L L P I M K G B. abortus 9-941 M S Q K T D L L L P I M K G B. ovis M S Q K T D L L L P I M K G B. Correction of two start site annotations errors Another type of annotation error identified in our studies was the erroneous assignment of gene translation start sites. For 2 proteins of B. abortus 2308, we report the expression of manually validated peptides corresponding Lamontagne et al. BMC Genomics 2010, 11:300 http://www.biomedcentral.com/1471-2164/11/300 Page 7 of 10 Figure 2 Annotation errors in the B. abortus 2308 genome. (A, B) The original start codon annotation in the publicly available genome (NCB onomy ID 359391) of the succinyl-CoA synthetase subunit beta (BAB1_1926, panel A) and of the KHG aldolase (BAB2_0083, panel B) are indicate double asterisks whereas the corrected start site is indicated by a single asterisk (BAB1_1926 only). The peptides sequenced by mass spectrometr highlighted in grey. The 5'-end of the CDS, as currently annotated, are underlined. The predicted sequence of the RBS found in proximity of the rected start site of BAB1_1926 is boxed. Numbers next to the nucleotide sequence and the schematic gene representation indicate the positio the genome sequence (NC_007618 or NC_007624). (C) Genomic and amino acid sequences of BAB1_1768, as currently found in the publicly ava 1874450 agaggacaaccagatgaatattcacgaataccaggccaagcgcctgcttcacacctacggcgcgccgatcgccaatggtGTGGCTGTCTATTCCGTCGAA 1874400 B. abortus 9-941 M N I H E Y Q A K R L L H T Y G A P I A N G V A V Y S V E B. melitensis 16M M N I H E Y Q A K R L L H T Y G A P I A N G V A V Y S V E B. ovis M N I H E Y Q A K R L L H T Y G A P I A N G V A V Y S V E B. suis M N I H E Y Q A K R L L H T Y G A P I A N G V A V Y S V E B. Prediction of protein localization The localization of newly demonstrated proteins was pre- dicted using PSORTb version 2.0.4 http://www.psort.org/ psortb/index.html, CELLO version 2.5 http://cello.life. nctu.edu.tw/ and PSLpred http://www.imtech.res.in/ raghava/pslpred/index.html. For a localization to be assigned, a minimum of 2 of the 3 predictions had to match. Protein databases Four types of B. abortus 2308 samples were prepared: outer membranes, cytosols, intracellular bacteria isolated from infected RAW264.7 macrophages and extracellular bacteria from overnight cultures. Outer membrane sam- ples were prepared and processed for mass spectrometry analysis as previously described [16]. Cytoplasmic frac- tions were prepared as described previously [38]. Briefly, bacteria grown in tryptic soy broth (Difco) in 2-liter flasks on an orbital shaker and harvested by centrifuga- tion in sealed cups at 7,000 × g for 20 min. The thick slurry of bacteria were suspended in 10 mM phosphate- buffered saline (pH 7.2) was passed twice through a French press (Pressure Cell 40 K, Aminco; SLM Instru- ments Inc., Urbana, Ill.) at an internal pressure of 35,000 lb/in2. The homogenate was digested with 50 mg of DNase II type V and RNase A per ml (Sigma) for 18 h at 37°C and fractionated by ultracentrifugation. The cell envelopes in the bottom of the tube removed and the cytoplasmic fractions in the supernatant, filtered, lyo- philized and characterized as described previously [39]. Intracellular bacteria were isolated from RAW264.7 mac- rophages 3, 20 and 44 hours post-infection as previously described [17]. Proteins were extracted from intracellular and extracellular bacteria using the same method and digested for mass spectrometry as previously described [17]. The databases were composed of protein sequences obtained from the National Center for Biotechnology Information (NCBI) protein database (for B. abortus 2308, NC_007618 and NC_007624; for B. melitensis 16 M, NC_003317 and NC_003318; for Mus musculus, all protein sequences contained under taxonomy ID 10090) and of B. abortus 2308 "pseudoproteins" corresponding to the custom translation of pseudogenes. Genomic regions corresponding to the 316 entries annotated as pseudo- genes in NCBI were directly translated and added to the database. Additionally, the ORF Finder tool from NCBI was used to determine other possible protein sequences corresponding to the pseudogenes. The ORF search was done by including 0 to 200 bp upstream or downstream from these regions. All resulting ORFs spanning the entire pseudogene sequence were kept. Ribosome bind- ing sites were mapped when possible according to the sequence described in reference [41]. A total of 471 trans- lated protein sequences were added to the NCBI data- bases. Protein identification Protein identification was done by submitting LC-MS/ MS spectra to Mascot software (MatrixScience, Boston, MA) and searching against custom protein databases (see below). The parameters used for the Mascot search and protein homology clustering were previously detailed [16]. No multidimensional fingerprinting method was used. Annotation for each protein was performed using ExPASy Proteomics tools http://us.expasy.org/tools/ #proteome, Kegg GenomeNet Database Service http:// Liquid Chromatography - Mass Spectrometry (LC-MS) Liquid Chromatography Mass Spectrometry (LC MS) Peptide digests were analyzed by liquid chromatography coupled to mass spectrometry (LC-MS) as described [40]. Briefly, the samples were injected onto a reversed-phase column (Jupiter C18, Phenomenex, Torrance, CA) for HPLC separation. For LC-MS survey scans, the mass spectra were acquired over 400-1600 Da at a rate of 1 spectrum/second. Peptide sequencing was achieved by targeted and shotgun LC-MS/MS. For MS/MS scans, the mass range was 50-2000 Da, and each spectrum was acquired in 2 seconds. For LC-MS/MS, the duty cycle was one survey scan followed by one product ion scan (MS/ MS). Additional material Additional file 1 Proteins newly demonstrated in B. abortus 2308. Each entry is represented by a gene locus tag, description of the protein and the sequences of the peptides measured. Proteins are organized by predicted subcellular localization. Correction of two start site annotations errors The predicted sequence of the RBS found in proximity of the B. abortus start site is boxed. acquire useful information on B. abortus 2308 expressed proteins and to identify and correct inaccurately anno- tated ORFs. We were able to confirm the expression of over 300 previously unreported proteins and five pseudo- genes, and corrected two wrongly assigned translation start sites. Taken together, these findings further demon- strate that computational genomic annotation errors can be corrected using proteomics. This will lead to improved databases and thus better protein identification and func- tional annotation. Lamontagne et al. BMC Genomics 2010, 11:300 http://www.biomedcentral.com/1471-2164/11/300 Page 8 of 10 Lamontagne et al. BMC Genomics 2010, 11:300 http://www.biomedcentral.com/1471-2164/11/300 www.genome.jp/ and literature mining of orthologous genes and proteins. Validation of mass spectrometry results Sequences assigned to MS/MS spectra of peptides, which were mapped to pseudogenes or to genomic regions annotated as untranslated regions, were manually vali- dated. 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Received: 18 November 2009 Accepted: 12 May 2010 Published: 12 May 2010 This article is available from: http://www biomedcentral com/1471-2164/11/300 © 2010 Lamontagne 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 reprod BMC Genomics 2010 11:300 Authors' contributions JL designed and coordinated the study, analyzed the data and wrote the man- uscript. MB participated in the data analysis and manuscript writing. AF per- formed the mass spectrometry experiments and peptide validations. ACM participated in the data analysis. NN performed the protein identification steps. FT participated in the protein identification steps. IM participated in the data analysis and manuscript writing. EM participated in the data analysis and man- uscript writing. EP conceived of the study and participated in manuscript writ- Page 9 of 10 Lamontagne et al. BMC Genomics 2010, 11:300 http://www.biomedcentral.com/1471-2164/11/300 Lamontagne et al. BMC Genomics 2010, 11:300 http://www.biomedcentral.com/1471-2164/11/300 Lamontagne et al. BMC Genomics 2010, 11:300 http://www.biomedcentral.com/1471-2164/11/300 ing and study coordination. All authors read and approved the final manuscript. 20. Gardy JL, Laird MR, Chen F, Rey S, Walsh CJ, Ester M, Brinkman FSL: PSORTb v.2.0: expanded prediction of bacterial protein subcellular localization and insights gained from comparative proteome analysis. Bioinformatics 2005, 21:617-623. Received: 18 November 2009 Accepted: 12 May 2010 Published: 12 May 2010 This article is available from: http://www.biomedcentral.com/1471-2164/11/300 © 2010 Lamontagne 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 repro BMC Genomics 2010, 11:300 24. Robertson GT, Roop RM Jr: The Brucella abortus host factor I (HF-I) protein contributes to stress resistance during stationary phase and is a major determinant of virulence in mice. Mol Microbiol 1999, 34:690-700. Acknowledgements 21. Connolly JP, Comerci D, Alefantis TG, et al.: Proteomic analysis of Brucella abortus cell envelope and identification of immunogenic candidate proteins for vaccine development. Proteomics 2006, 6:3767-3780. This work was funded by the NIAID/NIH contract HHSN266200400056C. References Crasta OR, Folkerts O, Fei Z, Mane SP, Evans C, Martino-Catt S, Bricker B, Yu G, Du L, Sobral BW: Genome sequence of Brucella abortus vaccine strain S19 compared to virulent strains yields candidate virulence genes. PLoS One 2008, 3:e2193. 35. 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Moriyon I, Berman DT: Effects of nonionic, ionic, and dipolar ionic detergents and EDTA on the Brucella cell envelope. J Bacteriol 1982, 152:822. 40. Lucero NE, Jacob NO, Ayala SM, Escobar GI, Tuccillo P, Jacques I: Unusual clinical presentation of brucellosis caused by Brucella canis. J Med Microbiol 2005, 54:505-508. 41. Dricot A, Rual JF, Lamesch P, Bertin N, Dupuy D, Hao T, Lambert C, Hallez R, Delroisse JM, Vandenhaute J, Lopez-Goñi I, Moriyon I, Garcia-Lobo JM, Sangari FJ, Macmillan AP, Cutler SJ, Whatmore AM, Bozak S, Sequerra R, Doucette-Stamm L, Vidal M, Hill DE, Letesson JJ, De Bolle X: Generation of the Brucella melitensis ORFeome version 1.1. Genome Res 2004, 14:2201. doi: 10.1186/1471-2164-11-300 Cite this article as: Lamontagne et al., Proteomics-based confirmation of protein expression and correction of annotation errors in the Brucella abor- tus genome BMC Genomics 2010, 11:300 39. Moriyon I, Berman DT: Effects of nonionic, ionic, and dipolar ionic detergents and EDTA on the Brucella cell envelope. J Bacteriol 1982, 152:822. 40. Lucero NE, Jacob NO, Ayala SM, Escobar GI, Tuccillo P, Jacques I: Unusual clinical presentation of brucellosis caused by Brucella canis. J Med Microbiol 2005, 54:505-508. 41. Dricot A, Rual JF, Lamesch P, Bertin N, Dupuy D, Hao T, Lambert C, Hallez R, Delroisse JM, Vandenhaute J, Lopez-Goñi I, Moriyon I, Garcia-Lobo JM, Sangari FJ, Macmillan AP, Cutler SJ, Whatmore AM, Bozak S, Sequerra R, Doucette-Stamm L, Vidal M, Hill DE, Letesson JJ, De Bolle X: Generation of the Brucella melitensis ORFeome version 1.1. Genome Res 2004, 14:2201. 41. Lamontagne et al. 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https://openalex.org/W2903867232	https://www.frontiersin.org/articles/10.3389/fimmu.2018.02955/pdf	English	null	The Role of Histone Methyltransferases and Long Non-coding RNAs in the Regulation of T Cell Fate Decisions	Frontiers in immunology	2,018	cc-by	9,490	The Role of Histone Methyltransferases and Long Non-coding RNAs in the Regulation of T Cell Fate Decisions Joseph M. Gaballa, Manuel Bonﬁm Braga Neto, Guilherme Piovezani Ramos, Adebowale O. Bamidele, Michelle M. Gonzalez, Mary R. Sagstetter, Olga F. Sarmento and William A. Faubion Jr.* Joseph M. Gaballa, Manuel Bonﬁm Braga Neto, Guilherme Piovezani Ramos, Adebowale O. Bamidele, Michelle M. Gonzalez, Mary R. Sagstetter, Olga F. Sarmento and William A. Faubion Jr.* Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, United States T cell lineage decisions are critical for the development of proper immune responses to pathogens as well as important for the resolution of inﬂammatory responses. This differentiation process relies on a combination of intrinsic and extrinsic factors converging upon epigenetic regulation of transcriptional networks relevant to speciﬁc T cell lineages. As these biochemical modiﬁcations represent therapeutic opportunities in cancer biology and autoimmunity, implications of writers and readers of epigenetic marks to immune cell differentiation and function are highly relevant. Given the ready adoption of histone methyltransferase inhibitors in the clinic, we focus this review on the role of three histone modifying complexes: PRC-1, PRC-2, and G9A in modulating T cell fate decisions. Furthermore, we explore the role of long non-coding RNAs in regulating these processes, and discuss recent advances and challenges of implementing epigenetic therapies into clinical practice. Edited by: Keiko Ozato, National Institutes of Health (NIH), United States Edited by: Keiko Ozato, National Institutes of Health (NIH), United States Reviewed by: Avinash Bhandoola, National Institutes of Health (NIH), United States Jonathan Kaye, Cedars-Sinai Medical Center, United States Remy Bosselut, National Cancer Institute (NCI), United States Vishal Nehru, National Institutes of Health (NIH), United States Keywords: epigenetics, EZH2, G9a, long non-coding RNAs, PRC1, PRC2, T cell Correspondence: William A. Faubion Jr faubion.william@mayo.edu Correspondence: William A. Faubion Jr faubion.william@mayo.edu BACKGROUND Specialty section: This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology Received: 06 September 2018 Accepted: 30 November 2018 Published: 13 December 2018 Specialty section: This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology The immune system comprises a large number of cell types that have the ability to respond to external environmental cues and adopt a wide variety of cell fates. These lineage decisions are critical for the development of proper immune responses to pathogens as well as resolution of inﬂammatory responses. As part of the adaptive immune system, T cells have the capacity to respond to the external environment by modulating the expression of lineage speciﬁc factors which are critical for protecting against a wide variety of pathogens. For the development of distinct T cell lineages, naive CD4+ T cells must convert the extrinsic instructions provided by encounters with antigen-presenting cells into cell-intrinsic changes (1). These intrinsic changes are largely facilitated by transcription factors that directly induce or repress gene networks and drive T cell diﬀerentiation (2). Emerging data demonstrates that lineage speciﬁc transcription factors recruit epigenetic complexes to regulate gene expression over multiple rounds of cell division, and their roles are indispensable for maintaining T cell homeostasis. Received: 06 September 2018 Accepted: 30 November 2018 Published: 13 December 2018 Keywords: epigenetics, EZH2, G9a, long non-coding RNAs, PRC1, PRC2, T cell REVIEW published: 13 December 2018 doi: 10.3389/ﬁmmu.2018.02955 REVIEW published: 13 December 2018 doi: 10.3389/ﬁmmu.2018.02955 1GENCODE, v27 Release. Available online at: https://www.gencodegenes.org/ human/release_27.html 2Long Non-coding RNA Database v2.,0 (lncRNAdb). Available online at: http:// www.lncrnadb.org/ G9a The histone methyltransferase G9a and the related G9a-like protein (GLP) form a heterodimeric complex to catalyze mono and di-methylation of lysine 9 on histone 3 (H3K9me1 & H3K9me2) at euchromatin in vivo (39). G9a and GLP are encoded by the EHMT2 and EHMT1 genes, respectively, both of which contain a SET domain necessary for the methylation of lysine residues. G9a has been shown to play a larger role in H3K9me2 methylation in vivo, but levels of H3K9me1 and H3K9me2 are severely reduced in both G9a and GLP knockout models (39). Furthermore, G9a has been shown to promote gene activation through a methyltransferase- independent fashion in diﬀerent settings, including type II cytokine production in helper T cells, possibly by acting as a scaﬀold to recruit transcriptional machinery (40, 41). G9a/GLP- mediated H3K9me2 has been associated with cognition and adaptive behavior, germ cell development and meiosis, embryo development, cocaine-induced plasticity, tumor cell growth and metastasis, and more recently the immune response reviewed below (39, 42). PRC1 The Polycomb-Group proteins, Polycomb Repressive Complex 1 (PRC1) and 2 (PRC2), mediate post-translational modiﬁcations (PTMs) of histones required for cell diﬀerentiation and development through the regulation of chromatin structure and gene expression. PRC1 is a multimeric protein complex containing the core proteins RING1A/B, and Polycomb-group ring ﬁnger (PCGF) proteins such as Bmi-1 (PCGF4) and Mel- 18 (PCGF2). PRC1 functions to mono-ubiquitinate lysine 119 on histone H2A (H2AKub119), an epigenetic mark that is associated with transcriptional repression (9). Bmi-1 speciﬁcally is highly enriched in pericentric heterochromatin which is required for chromatin compaction and silencing (10). Although Ring1A/B is the catalytic subunit of PRC1, knockdown of Bmi-1 results in a signiﬁcant loss of H2A ubiquitylation, demonstrating the important role that it plays in facilitating the enzymatic function of PRC1 (11). In the canonical or hierarchical model of Polycomb (PcG)-mediated transcription regulation, PRC1 is primarily described as the maintenance complex which silences target genes previously marked by the initiator complex, PRC2. More recently, a histone-independent role of Bmi-1 in driving NF-κB signaling has been reported (12). An interesting story is also evolving related to a PRC2-independent role for PRC1 in the maintenance of 3D genome structure through association with super-enhancers (13, 14). No immune cell speciﬁc data has yet emerged related to these exciting areas of investigation. Citation: Gaballa JM, Braga Neto MB, Ramos GP, Bamidele AO, Gonzalez MM, Sagstetter MR, Sarmento OF and Faubion WA Jr (2018) The Role of Histone Methyltransferases and Long Non-coding RNAs in the Regulation of T Cell Fate Decisions. Front. Immunol. 9:2955. doi: 10.3389/ﬁmmu.2018.02955 Deregulation of epigenetic pathways is a feature of many cancers, autoimmune diseases, and neurodegenerative disorders (3–5). The reversible nature of epigenetic modiﬁcations makes them attractive targets for pharmacological intervention, and indeed drugs targeting histone-modifying complexes, such as Enhancer of Zeste Homolog 2 (EZH2), are currently being evaluated in patients December 2018 \| Volume 9 \| Article 2955 1 Frontiers in Immunology \| www.frontiersin.org Epigenetic Regulation of T-Cell Fates Gaballa et al. While EZH2 has a role in normal cellular and tissue function, studies involving EZH2 overexpression or genetic mutations show that EZH2 is critical in the development and progression of a variety of cancers (21–29). EZH2 is most frequently associated with the silencing of tumor suppressor genes, and decreased expression of PRC-target genes are associated with poor prognosis (30, 31). Thus, derepression of these genes using selective EZH2 enzymatic inhibitors or disruptors of PRC2 stability are likely to improve clinical outcomes, and are currently being explored in preclinical or clinical studies for cancer therapy (32–38). for treatment of malignancy (6) and immune-mediated conditions (7, 8). While recent clinical trials have demonstrated a favorable safety proﬁle of selective inhibition of EZH2 (6), a comprehensive understanding of the role that epigenetic modifying complexes play in the development and function of diﬀerent immune cell types is relevant to the development and safety of epigenetic therapeutics. Here we review the role of three histone modifying complexes: PRC-1, PRC-2, and G9A in modulating T cell fate decisions. Furthermore, we explore the role of long non-coding RNAs in regulating these processes, and discuss recent advances and challenges associated with implementing epigenetic therapies in clinical practice. Long Non-coding RNAs g g Non-coding RNA’s have emerged as an exciting new frontier of gene regulation in the immune system. It is now known that 75–90% of the human genome transcriptome is comprised of non-coding RNAs (43, 44). Long non-coding RNAs are deﬁned as transcripts with minimal coding potential that are composed of more than 200 nucleotides; an arbitrary cutoﬀ that distinguishes them from microRNAs (<200 nucleotides). Over 15,000 lncRNA genes have been annotated, although only 159 lncRNAs have known function1,2 (45), highlighting a critical gap in knowledge in the ﬁeld. They can be classiﬁed based on their position relative to protein coding genes as intergenic, intronic and antisense (46). Like mRNAs, long non- coding RNA’s undergo transcription by RNA polymerase II, are 5′ capped, spliced and polyadenylated. However, distinct from mRNA, they lack canonical ORFs (and, therefore have minimal protein-coding potential), tend to be shorter in size, have lower expression levels, fewer exons and can localize to the nucleosome, chromatin or cytoplasm. For example, long intergenic non- coding RNAs localize primarily in the nucleus, in contrast to Frontiers in Immunology \| www.frontiersin.org PRC2 PRC2 modulates chromatin dynamics via the tri-methylation of lysine 27 on histone 3 (H3K27Me3), which is associated with transcriptional repression. EZH2, ubiquitously expressed by many mammalian cell-types, is the enzymatic subunit of PRC2 which contains other supporting non-catalytic proteins namely Suppressor of Zeste (SUZ12), embryonic ectoderm development (EED), Adipocyte Binding Protein 2 (AEBP2) and Retinoblastoma protein Associated protein 46 and 48 (RpAp46/48) (15). H3K27me3 recruits protein complexes involved in chromatin compaction and is associated with inactive genes (16). Histone-independent functions of PRC2 have also been reported to play important roles in regulating transcription factor stability and T cell receptor-mediated signaling (17–20). December 2018 \| Volume 9 \| Article 2955 Frontiers in Immunology \| www.frontiersin.org 2 Epigenetic Regulation of T-Cell Fates Gaballa et al. mRNAs which are primarily localized in the cytoplasm where they undergo translation (47). Furthermore, lncRNAs function by interacting with DNA, RNA, or proteins and the majority modulate transcription in cis (aﬀecting nearby genes), although they can also modulate in trans (targeting distant genes), acting as scaﬀolds, molecular decoys and guides for epigenetic modifying complexes. Interestingly, lncRNAs can both activate and suppress target genes by a variety of mechanisms and are expressed in a cell-type and stage-speciﬁc manner (48, 49). They have been shown to play key roles in autoimmunity, cancer and infection (50–52). A recent comprehensive transcriptomic proﬁling of T cells demonstrated unique lncRNA signatures for speciﬁc T cell phenotypes signifying the relevance of lncRNA to cell and stage speciﬁc function (49). Thus, lncRNAs may represent exciting precise therapeutic targets. ultimate derivation of Th17 cells (79–81). Ultimately, lineage- speciﬁc transcription factors (FOXP3 and RORγt) drive the Treg or Th17 transcriptional program, respectively. FOXP3 and RORγt are known to reciprocally regulate one another, and the delicate balance between suppressive Tregs and eﬀector Th17 cells has proven critical for maintaining immune homeostasis (78). Epigenetic modifying complexes, namely PRC2 and G9a, play key roles in orchestrating the Treg and Th17 transcriptional programs, and disruption of these epigenetic networks are characterized by the development of autoimmunity in murine models of human disease and human inﬂammatory bowel disease (66, 82, 83). We and others have demonstrated that mice lacking EZH2 in natural FOXP3+ Tregs developed spontaneous multi-organ inﬂammation and were more susceptible to experimental models of autoimmunity (65, 66). In addition to decreased frequency of EZH2-depleted Tregs observed in certain murine tissues, DuPage et al. PRC1, PRC2, G9A, AND LNCRNAS IN THE ADAPTIVE IMMUNE SYSTEM The development of T cells, an integral component of the adaptive immune system, occurs in the thymus where thymocytes mature into distinct T cell lineages deﬁned by either CD4 or CD8 co-receptor expression. CD4+ T cells and CD8+ T cells are known to possess conventional alpha beta (αβ) T cell receptors (TCR), which recognize antigen-derived peptides bound by major histocompatibility complex (MHC) class II or I molecules, respectively. Upon antigen recognition and inﬂammatory environmental cues, naïve CD4+ T cells diﬀerentiate into distinct eﬀector T helper (Th) subsets by expressing lineage-speciﬁc transcriptional programs. Th1, Th2, and Th17 cells mediate protective anti-pathogenic responses against bacteria and viruses via the secretion of distinct IFN- γ, IL-4, and IL-17 eﬀector cytokines, respectively (53). Post- infection, Tregs, a regulatory component of the immune system, are recruited to inhibit eﬀector T cell functions and reestablish homeostasis. Tregs can be generated from the thymus (natural Tregs) or induced in the periphery (pTreg) or in vitro (iTreg) from naïve CD4+ T cells via a FOXP3-driven transcriptome (54–56). Nonetheless, persistent activation of these eﬀector T cell subsets has been associated with the pathogenesis of autoimmune disorders such as inﬂammatory bowel disease (IBD), rheumatoid arthritis (RA) and psoriasis (57). In G9a deﬁcient CD4+ T cells stimulated under Treg or Th17 promoting conditions, a signiﬁcant increase in FOXP3-expressing and IL-17A-expressing cells is observed. In undiﬀerentiated T cells, G9a normally functions as a mediator of H3K9me2 on loci associated with driving Treg and Th17 phenotypes (42). Loss of G9a-mediated H3K9me2 increases chromatin accessibility to transactivating factors and increases responsiveness to TGFβ (42). Much more work is required to deﬁne the molecular underpinnings of G9a’s eﬀects on Treg development, but some consistency is emerging regarding Th17 biology. G9a was shown to be recruited by RelB, a non- canonical NF-κB family member, to silence the IL17A locus and prevent Th17-mediated autoimmunity in an in vivo model of experimental autoimmune encephalomyelitis (EAE) (67). This work is consistent with eﬀects seen in other T cell subsets, namely Th2 cells, in which loss of G9a leads to abnormal IL-17 expression (42). How these eﬀects inﬂuence the balance between Treg and Th17 phenotypes is yet to be determined. Thus, G9a may become a viable target for therapeutic intervention of human Th17 mediated diseases. PRC2 showed that EZH2 was required to promote the FOXP3-mediated gene repression program upon TCR activation as a number of FOXP3-bound genes were de-repressed in the absence of EZH2 (65). In support of the failure of EZH2-deleted Tregs to maintain the expression of Treg-speciﬁc signature genes, EZH2-deleted Tregs displayed impaired suppression of eﬀector T cells in vitro (65, 66). Translating these ﬁndings from mice to human relevance, Crohn’s disease (CD)-lamina propria CD4+ T cells were transcriptionally diﬀerent from healthy controls (66). Speciﬁcally, normally repressed FOXP3-target genes were upregulated in CD CD4+ T cells and approximately 50% of these diﬀerentially expressed genes (DEGs) were EZH2 targets. Moreover, CD4+ T cells displayed a Th1/Th17 eﬀector-like phenotype in contrast to that of healthy controls. Thus, loss of EZH2 function and consequently Treg dysfunction may drive pathophysiological mechanisms of particular autoimmune disorders. PRC1, PRC2, G9A, AND LNCRNAS IN THE ADAPTIVE IMMUNE SYSTEM PRC1, PRC2, G9a, and a variety of lncRNAs inﬂuence T helper cell diﬀerentiation and maintenance by epigenetically regulating transcriptional programs associated with diﬀerent T cell subsets. Given their signiﬁcant inﬂuence in the pathogenicity of diseases as stated above, we focus here on the role of these molecules in the diﬀerentiation and maintenance of Th1, Th2, Treg, and Th17 phenotypes (Figure 1, Table 1). Frontiers in Immunology \| www.frontiersin.org Treg/Th17 Treg and Th17 cells appear to share precursor lineage as demonstrated by in vitro study and murine lineage tracing experiments (77, 78). While TGFβ signaling is required for both eﬀector cell types, IL-6 appears principally responsible for Three lncRNAs (Flicr, Lnc-Smad-3, and LncEGFR) have been shown to inﬂuence Treg function. Flicr is selectively expressed in both human and mouse T regulatory cells and negatively December 2018 \| Volume 9 \| Article 2955 Frontiers in Immunology \| www.frontiersin.org 3 Gaballa et al. Epigenetic Regulation of T-Cell Fates FIGURE 1 \| PRC1, PRC2, G9a, and lncRNAs regulate T cell differentiation and function. FIGURE 1 \| PRC1, PRC2, G9a, and lncRNAs regulate T cell differentiation and function. decreased expression of RORγt and IL-17 in Th17 cells, although the precise mechanism is yet to be known (76). These ﬁndings suggest a potential role for this long non-coding RNA in the pathogenesis of multiple sclerosis. regulates FOXP3 in cis leading to decreased Treg function and heightened autoimmunity (74). Mechanistically, Flicr modiﬁes chromatin accessibility in the FOXP3 locus, speciﬁcally non- coding sequence 3 (CNS3) and accessible region 5 (AR5), leading to decreased expression of FOXP3. In vivo, knockdown of Flicr decreased the incidence of autoimmune diabetes in mice (74). Th1/Th2 Lnc-Smad-3 was recently shown to modulate TGFβ-mediated Treg polarization both in human and murine assays (75). Mechanistically, lnc-Smad3 prevents the histone deacetylase HDAC1 to bind to the SMAD3 promoter region, which renders the chromatin compact and inaccessible to Ash1l, an H3K4 methyltransferase that promotes SMAD3 activation and transcription. From a disease relevance standpoint, these results suggest a potential role for this long non-coding RNA in the pathogenesis of autoimmune diseases, such as rheumatoid arthritis (75). Studies investigating the impact of G9a on Th1 biology have shown that the absence of G9a has little eﬀect on Th1 responses in vitro nor in vivo, however, it is a critical component of the Th2 regulatory machinery (40). Lehnertz et al. demonstrated G9a to be necessary for expression of lineage-speciﬁc Th2-associated cytokines such as IL-4, and that loss of G9a in CD4+ T cells prevents Th2 cell diﬀerentiation. Mice with targeted CD4+ T cell deletions of G9a were susceptible to helminth infection by Trichuris muris due to the inability to express Th2-associated cytokines. Consistent with previous work (42), the absence of G9a in CD4+ T cells also resulted in the upregulation of IL- 17A in vivo. Interestingly, whereas repression of IL-17A appears to be associated with G9a methyltransferase activity (42), Th2 gene regulation by G9a is independent of enzymatic activity, and thought to be related to G9a functioning as a scaﬀolding protein (40, 41). Lnc-EGFR was shown to stimulate Treg diﬀerentiation by a forward-feedback loop (51). Mechanistically, lnc-EGFR binds to EGFR using its R1 domain, preventing interaction with c-CBL and ubiquitination. In turn, EGFR activates ERK1/2 and AP- 1, which then leads to increased expression of lnc-EGFR and FOXP3, perpetuating increased Treg diﬀerentiation. The authors found this to be a critical pathway for hepatocellular carcinoma (51). The role of PRC1 in regulating T cell lineage fate decisions is best illustrated by the inﬂuence it has on the Th1/Th2 axis of development. Both Bmi1 (PCGF 4) and Mel-18 (PCGF 2) have been shown to physically interact with GATA3, a lineage speciﬁc transcription factor for Th2 diﬀerentiation, in a Ring ﬁnger dependent manner (59, 60). Mel-18 has been shown to LncRNA-1700040D17Rik was found to be deregulated in CD4+ cells derived from a mouse model of autoimmune encephalitis and have been shown to play a role in diﬀerentiation of Th17 cells. Th1/Th2 Additionally, OVA-speciﬁc EZH2-deﬁcient Th2 cells were pathogenic in a mouse model of allergic asthma due to an accumulated and exaggerated immune response from memory Th2 cells (64). Taken together, EZH2 inhibits eﬀector cytokine production in naïve CD4+ T cells, and loss of EZH2 enhances diﬀerentiation to eﬀector Th cells as well as eﬀector Th cell plasticity. Based on evidence from in vivo studies in mice in the context of EZH2 deletion in T cells, eﬀector Th cell dysfunction is consistent across all disease models, evidently through impaired clearance of pathogens or aggravated autoimmunity (potentiated tissue destruction). Additionally, H3K27me3-independent functions of EZH2 have been reported in T cells expressing conventional αβ-TCRs (17, 18). Vasanthakumar et al. demonstrated that EZH2 prevents NKT cell expansion through methylation, ubiquitination and subsequent degradation of the transcription factor promyelocytic leukemia zinc ﬁnger (PLZF) (17). In vivo studies have demonstrated that an increase in the frequency of NKT cells in the thymus and spleen occurs as a result of CD4+ T-speciﬁc EZH2 deletion, which may contribute to the perturbed immunity seen in murine studies previously mentioned (63, 64, 84). regulate GATA3 transcription, and knockout of mel-18 severely impacts Th2 diﬀerentiation in vivo (60). Bmi-1 regulates Th2 cell diﬀerentiation by acting as an inhibitor of GATA3 degradation and regulator of its stability. Bmi-1 overexpression in itself leads to an increase in GATA3 expression and an increase in Th2 cell diﬀerentiation under a Th2 speciﬁc cytokine milieu. Comparatively little data exist regarding the role of PRC1 in Th1 cell development/function; however adoptive transfer of CD4+ T cells from Bmi1−/−mice into nude mice showed impaired generation and maintenance of memory Th1 cells through Bmi1- mediated repression of Noxa, a pro-apoptotic gene (58). p p p p g ( ) The role of EZH2 in modulating eﬀector T function was recently illuminated by Yang et al. who showed that EZH2- deﬁcient naïve CD4+ T cells stimulated under Th1, Th2 or Th17 polarizing conditions displayed enhanced production of IFN- γ, IL-13 or IL-17 cytokines, respectively (63). Moreover, Tumes et al. also showed that EZH2 deﬁciency in naïve CD4+ T cells led to the upregulation of Th1 and Th2-associated cytokines with concomitant increase in lineage-speciﬁc transcription factors T-bet and Gata3, respectively (64). However, in vivo studies have revealed that EZH2 plays a dichotomous role in the diﬀerentiation and senescence of CD4+ T cells (63). Absence of G9a in CD4+ T cells is associated with increased IL-17A expression in vivo and in vitro (42). Recruited by RelB to silence IL17A locus in mouse model of EAE (67). EZH2-deﬁcient naïve CD4+ T cells stimulated under Th17 polarizing conditions displayed enhanced production of IL-17 (63). Th1/Th2 Overexpression of LncRNA-1700040D17Rik was associated with decreased expression of RORγt and IL-17 in Th17 cells (76). Maintains Treg signature gene expression (61). Inactivation leads to systemic immune mediated disease (61). Knockdown of Mel-18 leads to decreased expression of IL17A, IL17F, and RORC (62). EZH2-deﬁcient naïve CD4+ T cells stimulated under Th17 polarizing conditions displayed enhanced production of IL-17 (63). EZH2 is required to promote the FOXP3-mediated gene repression program following TCR stimulation (65). Loss of EZH2 in Tregs in vivo leads to multi-organ inﬂammation and increases susceptibility to experimental models of autoimmunity (65, 66). Required for Th2-speciﬁc cytokine expression (40). Loss of G9a prevents Th2 differentiation and increases IL-17A expression (40, 42). Absence of G9a in CD4+ T cells is associated with increased FOXP3 expression (42). G9a expression in CD4+ T cells is necessary for development of colitis in mice (42). Overexpression of LncRNA-1700040D17Rik was associated with decreased expression of RORγt and IL-17 in Th17 cells (76). Overexpression of LncRNA-1700040D17Rik was associated with decreased expression of RORγt and IL-17 in Th17 cells (76). displayed impaired clearance of infection due to decreased survival of memory Th1 cells (84). Additionally, OVA-speciﬁc EZH2-deﬁcient Th2 cells were pathogenic in a mouse model of allergic asthma due to an accumulated and exaggerated immune response from memory Th2 cells (64). Taken together, EZH2 inhibits eﬀector cytokine production in naïve CD4+ T cells, and loss of EZH2 enhances diﬀerentiation to eﬀector Th cells as well as eﬀector Th cell plasticity. Based on evidence from in vivo studies in mice in the context of EZH2 deletion in T cells, eﬀector Th cell dysfunction is consistent across all disease models, evidently through impaired clearance of pathogens or aggravated autoimmunity (potentiated tissue destruction). Additionally, H3K27me3-independent functions of EZH2 have been reported in T cells expressing conventional αβ-TCRs (17, 18). Vasanthakumar et al. demonstrated that EZH2 prevents NKT cell expansion through methylation, ubiquitination and subsequent degradation of the transcription factor promyelocytic leukemia zinc ﬁnger (PLZF) (17). In vivo studies have demonstrated that an increase in the frequency of NKT cells in the thymus and spleen occurs as a result of CD4+ T-speciﬁc EZH2 deletion, which may contribute to the perturbed immunity seen in murine studies previously mentioned (63, 64, 84). displayed impaired clearance of infection due to decreased survival of memory Th1 cells (84). Th2-LCR-lncRNA recruits WDR5-containing complexes to Th2-speciﬁc cytokine loci facilitating their expression (72). LincR-CcR2-5′ AS interacts with GATA-3 to upregulate chemokine genes necessary for Th2 migration (73). Frontiers in Immunology \| www.frontiersin.org Overexpression of LncRNA-1700040D17Rik was associated with decreased expression of RORγt and IL-17 in Th17 cells (76). Flicr negatively regulates FOXP3 leading to decreased Treg function (74). Lnc-Smad-3 regulates TGFβ mediated Treg differentiation by interacting with HDAC1 (75). Lnc-EGFR promotes Treg differentiation through interactions with EGFR (51). Th1/Th2 In vitro, overexpression of lncRNA-1700040D1Rik December 2018 \| Volume 9 \| Article 2955 Frontiers in Immunology \| www.frontiersin.org 4 Epigenetic Regulation of T-Cell Fates Gaballa et al. TABLE 1 \| Roles of PRC1, PRC2, G9a, and annotated lncRNAs in the development and function of Th1, Th2, Treg, and Th17 cells. Th1 Th2 Treg Th17 PRC1 Absence of Bmi1 impacts Th1 generation and maintenance (58). Regulates Th2 differentiation and cytokine expression (59, 60). Overexpression of Bmi-1 increases GATA3 expression and stability (59). Loss of Mel-18 impacts Th2 differentiation in vivo (60). Maintains Treg signature gene expression (61). Inactivation leads to systemic immune mediated disease (61). Knockdown of Mel-18 leads to decreased expression of IL17A, IL17F, and RORC (62). PRC2 Inhibits Th1 differentiation and cytokine production (63, 64). EZH2 deﬁciency enhances production of Th1 cytokines and increased T-bet expression (63, 64). Inhibits Th2 differentiation and cytokine production (63, 64). EZH2 deﬁciency enhances production of Th2 cytokines and increased GATA3 expression (63, 64). EZH2 is required to promote the FOXP3-mediated gene repression program following TCR stimulation (65). Loss of EZH2 in Tregs in vivo leads to multi-organ inﬂammation and increases susceptibility to experimental models of autoimmunity (65, 66). EZH2-deﬁcient naïve CD4+ T cells stimulated under Th17 polarizing conditions displayed enhanced production of IL-17 (63). G9a No evidence supports a role for G9a in Th1 biology. Required for Th2-speciﬁc cytokine expression (40). Loss of G9a prevents Th2 differentiation and increases IL-17A expression (40, 42). Absence of G9a in CD4+ T cells is associated with increased FOXP3 expression (42). G9a expression in CD4+ T cells is necessary for development of colitis in mice (42). Absence of G9a in CD4+ T cells is associated with increased IL-17A expression in vivo and in vitro (42). Recruited by RelB to silence IL17A locus in mouse model of EAE (67). LncRNA Linc-MAF-4 promotes Th1 differentiation through silencing of Th2 transcription factor MAF (49, 68). IFNG-AS1 recruits H3-K4-methyltransferase to Ifng locus and is upregulated in response to Th1-polarizing cytokines (52, 69–71). Th2-LCR-lncRNA recruits WDR5-containing complexes to Th2-speciﬁc cytokine loci facilitating their expression (72). LincR-CcR2-5′ AS interacts with GATA-3 to upregulate chemokine genes necessary for Th2 migration (73). Flicr negatively regulates FOXP3 leading to decreased Treg function (74). Lnc-Smad-3 regulates TGFβ mediated Treg differentiation by interacting with HDAC1 (75). Lnc-EGFR promotes Treg differentiation through interactions with EGFR (51). Th1/Th2 Distinct DNA methylation proﬁles have been demonstrated in CD8+ and CD4+ T cells isolated from patients experiencing autoimmune diseases (88–90). Epigenetic based therapeutics currently being employed for the clinic for non-inﬂammatory conditions, such as arrhythmias (procainamide), hypertension (hydralazine), and neoplasia (5-azacytidine), have been shown to induce auto-reactive pathology (7, 8). However, the 5- azacytidine derivative 5-aza-2’deoxycytidine, which is also a DNA methyltransferase inhibitor used in hematological malignancies, has been shown to have a positive outcome when administered in animal models of diabetes (91), colitis (92), multiple sclerosis (93), and graft-versus-host- disease (GvHD) (94). We need a better understanding of the implications of DNA methylation, the pharmokinetics of available compounds, and synergistic eﬀects of combination therapy with immunomodulatory drugs already in practice for autoimmune diseases to allow us to develop and implement novel therapies. As of now, we are lacking a therapeutic arsenal to target global hypomethylation, which is most often associated with lymphocytes recovered from patients experiencing some of the most common autoimmune diseases. Two lncRNAs, Th2-LCR-lncRNA and lincR-CcR2-5′AS, have been shown to inﬂuence the development and function of Th2 cells. Th2-LCR-lncRNA is selectively expressed in human Th2 cells and is transcribed in the RAD50 locus and epigenetically regulates expression of IL-4, IL-5 and IL-13 (72). Mechanistically, Th2-LCR-lncRNA recruits WDR5-containing complexes to targeted cytokine loci, enhancing transcription. Knockdown of human Th2-LCR-lncRNA in vitro causes major loss of expression of IL-4, IL-5 and IL-13 in Th2 cells through loss of H3K4me3 activating marks (72). Unfortunately, Th2-LCR-lncRNA is not conserved in mice, complicating in vivo studies. LincR-CcR2-5′AS is selectively expressed in mouse Th2 cells and upregulates CCR1, CCR2, CCR3 and CCR5 chemokine genes in a GATA3-dependent fashion (73). Interestingly, knockdown of this lincRNA not only aﬀected neighboring genes CCR2 and CCR3, but also aﬀected nearly 1,200 genes some of which were located in distant loci, suggesting it can act in both cis and trans. Although the precise mechanism is yet to be fully understood, in vitro knock down of lincR-CcR2-5′AS did not result in chromatin accessibility or modiﬁcation of H3K4me3, suggesting that it does not act through recruitment of histone-modifying enzymes or chromatin structure modiﬁcations. The ubiquitous expression of EZH2 and the opposing role it plays in diﬀerent cell-types makes EZH2 a delicate therapeutic target. Recent identiﬁcation of PRC2- and H3K27me3- independent EZH2 functions in oncogenesis indicates that a complete suppression of all oncogenic functions of EZH2 is required to combat cancer. Th1/Th2 For example, in an in vivo model of Listeria monocytogenes infection known to induce a Th1 response, CD4+ T-speciﬁc EZH2 deleted mice Two lncRNAs, MAF-4, and IFNG-AS1 (also called NeST or Tmevpg1), have been shown to inﬂuence Th1 biology by December 2018 \| Volume 9 \| Article 2955 Frontiers in Immunology \| www.frontiersin.org 5 Epigenetic Regulation of T-Cell Fates Gaballa et al. recruiting diﬀerent epigenetic modifying complexes. Linc- MAF-4 is selectively expressed in Th1 cells and promotes Th1 diﬀerentiation through epigenetic silencing of the Th2 transcription factor MAF. Downregulation of linc-MAF- 4 in human CD4+ cells skewed diﬀerentiation toward a Th2 phenotype. Mechanistically, linc-MAF-4 promotes a cis chromatin looping conformation, leading to the recruitment of chromatin remodelers EZH2 and LSD1 that place repressive H3K27me3 marks on the promoter region of MAF-4 silencing its expression (49). Recently, linc-MAF-4 was shown to be involved in the pathogenesis of multiple sclerosis by promoting Th1 cell diﬀerentiation (68). Thus, far, linc-MAF-4 has not been studied in vivo. treatment. Persistence of epigenetic maintenance of engineered modiﬁcations has been shown to be stable up to 40 days post modiﬁcation induction in vivo (86). Most epigenetic drugs currently in use inhibit DNA methyltransferase and histone deacetylase activity, and have been shown to reverse immune suppression and thus sensitize the host immune system in combination with anti-cancer therapies. Several anti-cancer mechanisms have been reported, such as enhancing antigen processing and presenting machinery pathways, inhibiting immune checkpoints, and enhancing chemokine production. For patients, there are three treatment options available: therapies reported to aﬀect DNA methylation, inhibitors of histone post-translational modiﬁcations, and compounds interfering with non-coding RNA regulation (87). Repurposing drugs and screening for new compounds that display converse eﬀects to treatment autoimmune disease is an exciting new option for autoimmune illnesses. IFNG-AS1 is expressed in CD4+ Th1, CD8+, and natural killer cells (52, 69). It is upregulated in CD4+ cells in response to Th1-diﬀerentiating cytokine stimuli and plays a critical role in transcription of Ifng. This has been demonstrated both in vitro and in vivo. Mechanistically, it has been shown to recruit the H3K4-methyltranferase complex to the Ifng locus, leading to placement of activating marks at the promoter region. It has been associated with the pathogenesis of Hashimoto’s thyroiditis (70), ulcerative colitis (71), and the immune response to viral infections in vivo (52). Th1/Th2 Anti-EZH2 therapy inhibits methylation at key repression/silencing associated histone marks, and these compounds have emerged as a promising therapy for cancer treatment, especially for B cell non-Hodgkin’s lymphoma. However, we have observed that systemic anti- EZH2 therapy leads to mucosal hypersensitivity in mice. One complicating factor is that EZH2 is also utilized by PRC1 in the nucleus, therefore more study needs to be undertaken to dissect the speciﬁc roles these complexes play in inﬂammation before on can determine whether histone methyltransferase inhibitors can be co-opted for anti-inﬂammatory therapy. Of note, cytosolic forms of PRC2 have been shown in murine models to be necessary for TCR-mediated activation of signaling pathways that drive T cell proliferation and autoimmunity. Thus, FUTURE PERSPECTIVES: EPIGENETIC MODULATION OF T CELLS IN CLINICAL PRACTICE Epigenetic mechanisms of disease are in theory inducible and reversible through environmental manipulation, however, some epigenetic features have been shown to be maintained after cellular division as a result of self-enforcing feedback mechanisms (85). The heritable, yet reversible nature of epigenetic therapy makes this a promising option for December 2018 \| Volume 9 \| Article 2955 Frontiers in Immunology \| www.frontiersin.org 6 Epigenetic Regulation of T-Cell Fates Gaballa et al. pharmacologic targeting of cytosolic PRC2 may represent a more precise therapeutic approach to suppressing autoimmunity caused by excessive T cell activation (19, 20). Precision medicine has brought about the advent of using CRISPR/Cas9 to target this gene editing tool to target epigenetic modifying enzymes to precise locus speciﬁc locations on the genome instead of the DNA endonucleases the technology originally utilized (105). This technique can be exploited to recruit enzymes that impact the methylation of the DNA, enzymes that post-translationally modify the histones, and proteins which interfere with non-coding RNA regulation. Further, it has been recently reported CRISPR/Cas9 technology can be rapidly delivered via a non-viral delivery technique capable of integrating large DNA sequences (106). These new developments will allow us ﬂexible and precise epigenetic manipulation toward creating therapeutically epi-engineered primary human immune cells without the oﬀ-target eﬀects associated with systemic epigenetic therapies. y From a translational standpoint, several studies have demonstrated that long non-coding RNAs can be used as biomarkers in malignancy and autoimmune diseases (95–97). Potential lncRNA-targeted therapeutic approaches include silencing by antisense base pairing (e.g., targeting lncUBE3ATS, which silences paternal UBE3A in Angelman’s syndrome) or by targeting molecules that are necessary for lncRNA transcription, such as transcription factors (98, 99). The cell type speciﬁc expression of lncRNAs makes them excellent targets for therapeutic intervention, as oﬀ-target eﬀects are minimized. 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https://openalex.org/W3187573488	https://www.researchsquare.com/article/rs-756411/latest.pdf	English	null	Association Between Acute Colonic Pseudo-Obstruction (Ogilvie Syndrome) And Nimodipine Use. Case Series And Comprehensive Review	Research Square (Research Square)	2,021	cc-by	4,222	Association Between Acute Colonic Pseudo- Obstruction (Ogilvie Syndrome) And Nimodipine Use. Case Series And Comprehensive Review Orlando De Jesus (  drodejesus@aol.com ) University of Puerto Rico Medical Sciences Campus: Universidad de Puerto Rico Recinto de Ciencias Medicas https://orcid.org/0000-0001-8078-3882 Orlando De Jesus (  drodejesus@aol.com ) University of Puerto Rico Medical Sciences Campus: Universidad de Puerto Rico Recinto de Ciencias Medicas https://orcid.org/0000-0001-8078-3882 José Sánchez Jiménez University of Puerto Rico Juan C. Vicenty University of Puerto Rico Juan C. Vicenty University of Puerto Rico Research Article Keywords: aneurysm, colon, nimodipine, pseudo-obstruction, subarachnoid hemorrhage Posted Date: August 10th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-756411/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Conclusion: This report linking the oral use of nimodipine with Ogilvie syndrome may further support the association of this disease with nimodipine use during the treatment of patients with aneurysmal SAH. DOI: https://doi.org/10.21203/rs.3.rs-756411/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/9 Page 1/9 Page 1/9 Background: Nimodipine is a calcium channel blocker indicated for the management of patients with aneurysmal subarachnoid hemorrhage (SAH). Oral nimodipine has rarely been implicated in the development of acute colonic pseudo-obstruction in patients treated for SAH. Nimodipine acts by inhibiting the transmembrane influx of calcium ions essential for the excitation-contraction coupling process of smooth muscle cells. We thought that its mechanism of action could predispose patients to develop acute colonic pseudo- obstruction (Ogilvie syndrome). The purpose of this study was to revise the existing literature concerning the association between acute colonic pseudo-obstruction and nimodipine use in patients with SAH. Introduction Ogilvie syndrome, also known as acute colonic pseudo-obstruction, is the distention of the colon caused by decreased motility in the absence of mechanical obstruction [1, 5, 25]. The most common risk factors associated with its development include severe infection, severe disease process, myocardial infarction, congestive heart failure, surgical procedures, metabolic disorders, electrolyte disorders, renal insufficiency, systemic lupus erythematosus, gastrointestinal carcinoma, severe trauma, spinal cord injury, cerebral stroke, Guillain-Barre, Parkinson disease, some medications, and alcohol abuse. Nimodipine, a second-generation dihydropyridine type calcium channel antagonist that blocks the influx of extracellular calcium through L-type, voltage-gated calcium channels, is indicated to manage patients with aneurysmal SAH. Nimodipine is considered a safe drug with only minor side effects, including a minimal drop of the systemic blood pressure or reversible increases in the liver enzymes [13, 18, 29, 31]. Very rarely, nimodipine had been associated with the development of acute colonic pseudo-obstruction. This report presented two patients who had aneurysmal SAH and developed acute colonic pseudo- obstruction while receiving oral nimodipine. The available literature was reviewed to determine the possible association between nimodipine use in aneurysmal SAH and Ogilvie syndrome. Methods: Two patients with aneurysmal SAH who received oral nimodipine and developed Ogilvie syndrome were discussed. All previously published cases of aneurysmal SAH associated with acute colonic pseudo- obstruction were reviewed. Case 1: Page 2/9 Page 2/9 Page 2/9 Presentation: A (age redacted) female with a BMI of 32.6 kg/m2 and a history of hypertension not using the prescribed medication for the last four years presented to the local health center after a sudden severe worst headache. She was alert and oriented with mild neck rigidity. A head computed tomographic (CT) scan showed a SAH and was intubated due to the findings before being transferred to our institution. On arrival at our institution, she had a blood pressure of 174/97 mm Hg with a Glasgow coma scale (GCS) of 10 intubated. A repeat head CT scan showed a moderate diffuse cisternal SAH with a small amount of intraventricular hemorrhage. The patient was started on oral nimodipine 60 mg orally every 4 hours and a stool softener (docusate calcium 240 mg daily). A cerebral digital subtraction angiogram (DSA) was performed the next morning, which showed a small anterior communicating aneurysm filling primarily through the left side. During the procedure, she was embolized with the complete occlusion of the aneurysm and then transferred to the intensive care unit and extubated. Post extubation, the patient had a GCS of 15. Investigations: Two days after admission, her abdomen was distended, and a kidney, ureter, and bladder (KUB) X-ray film showed a distended large bowel. She was tolerating diet and passing gas and stools; however, the distention persisted for the next two days. An abdominal CT scan showed a 10 cm dilatation of the ascending and transverse colon with mildly dilated jejunal loops. At this time, it was suspected that she had developed acute colonic pseudo-obstruction. By this day, she had been on nimodipine for four days. Treatment: The diet was suspended, and a nasogastric tube was placed with intermittent suction for five days; however, the distention did not improve. Neostigmine 2 mg was given intravenously over 3 to 5 minutes and repeated the next day. Atropine was available at the bedside but not required. A brain CT angiography showed moderate vasospasm with a 50% reduction in the diameter of the anterior cerebral vessels. Although asymptomatic, she had been five days without nimodipine; therefore, it was restarted given the possibility of delayed ischemic complications. A repeat abdominal CT scan was performed, which showed interval reduction of the dilatation, now 7 cm at the ascending and transverse colon with a normal descending colon, sigmoid, and rectum. Case 1: Diet was restarted two days later, but she had significant abdominal distention, so the diet was again stopped. A repeat cerebral DSA showed no evidence of vasospasm. It was decided not to continue the nimodipine as she was already close to 21 days post hemorrhage. As the distention had not improved, a colonoscopy for decompression was attempted, but decompression was unachieved; thus, a rectal tube was placed with an output of 850 ml in 24 hours. The abdominal distention decreased, and the tube was removed the next day, and the diet was restarted. Outcome: A KUB X-ray performed six days later showed resolution of the colon distention. The patient was discharged five days after the rectal tube was removed, and 28 days after the initiation of the pseudo-obstruction. Case 2: Page 3/9 Page 3/9 Presentation: A (age redacted) female with a BMI of 26.8 kg/m2 and a history of bipolar disease and heart arrhythmias with a pacemaker had a sudden severe headache and arrived at our emergency department for evaluation. Neurological examination showed a GCS 15. The head CT scan showed a small anterior interhemispheric SAH, and the CT angiography showed an anterior communicating artery aneurysm. The patient was started on oral nimodipine 60 mg orally every 4 hours and a stool softener (docusate calcium 240 mg daily). A cerebral DSA was performed the next morning, which confirmed the presence of an anterior communicating artery aneurysm filling primarily through the left side. She was taken to the operating room for a craniotomy and clipping of the aneurysm. Just after the opening of the dura mater, the patient had an episode of rebleeding. A ventriculostomy was placed, which relaxed the brain, and the aneurysm was successfully clipped. Postoperative, she remained alert and oriented. The head CT scan the next morning showed an increased amount of SAH. Investigations: Five days after admission, she developed a distended abdomen, and a KUB X-ray film showed a distended large bowel. She was tolerating diet and passing gas and stools; however, the distention persisted. The next day, an abdominal CT scan showed an 8 cm dilatation of the ascending and transverse colon. At this time, it was suspected that she developed acute colonic pseudo-obstruction. By this day, she had been using nimodipine for nine days. Treatment: Diet was immediately stopped, and a nasogastric tube was placed with intermittent suction. However, the distention did not improve, and a rectal tube was placed two days later with a large output in 24 hours. The abdominal distention noticeable decreased. The tube was removed the next day and started again on a diet. Nimodipine was restarted to complete the 21 days of treatment. Outcome: During the following days, the KUB X-ray showed progressive reduction of the distention of the colon, and the patient was discharged seven days after the rectal tube was removed and twelve days after the initiation of the pseudo-obstruction. Six months later, a ventriculoperitoneal shunt was placed because of the development of hydrocephalus. Discussion In 1948, Ogilvie reported two patients with signs of colonic obstruction in the absence of any organic disease of the colon [25]. He attributed its development to the interruption of the sympathetic supply to the large intestine. However, after surgical exploration, both patients were found to have disseminated abdominal metastatic disease. Ogilvie syndrome is now recognized as a colonic pseudo-obstruction caused by decreased motility in the absence of mechanical obstruction; still, the eponym was attributed to his idea of the interruption of the sympathetic supply. Ogilvie syndrome is associated with a 25-30% mortality in severe cases, which increases to 50% if the patient develops ischemia and perforation [19]. Early recognition and management are essential. The specific pathophysiology of acute colonic pseudo obstruction is still unclear. It may include many etiologies, risk factors, pathological conditions, and multiple associations leading to altered autonomic regulation of colonic motility [8,34]. Excessive parasympathetic suppression, sympathetic stimulation, or both are thought to produce an imbalance in the colonic autonomic innervation. All these factors produce temporary suppression of intestinal motili inducing acute dilatation of the colon. Nimodipine has rarely been associated with the development of Ogilvie syndrome in aneurysmal SAH. In this report, we presented two cases in which its use could have been associated with the development of acute colonic pseudo-obstruction. In 1984, Bullock and Thomas were the first to report acute colonic pseudo-obstruction in patients with a SAH [6]. Their series included two such cases; however, there is no description if nimodipine was used. Nimodipine was probably not used in those two cases as it was approved for medical use in 1985. In 1987, Torrealba et al were the first to describe a patient with acute colonic pseudo-obstruction who received intravenous nimodipine for a SAH whose condition improved after a rectal tube was inserted and the nimodipine was discontinued [32]. However, no cerebral aneurysm was identified in the angiographic studies. Hund et al. reported in 1990 a similar complication after the use of intravenous nimodipine in a patient with SAH secondary to an aneurysm of the anterior communicating artery [15]. They also mentioned four previous cases of SAH in which the patients developed abdominal distention after intravenous nimodipine. Fahy described in 1996 a case of pseudo-obstruction of the colon in a patient receiving oral nimodipine therapy for a SAH secondary to an aneurysm of the posterior communicating artery [11]. Discussion In the early 1980s, nimodipine was considered the drug of choice for preventing and treating cerebral vasospasm following SAH due to its preferential cerebrovascular action [2,3,4,22,26]. It is now recognized that oral nimodipine improves the clinical outcome in patients with delayed cerebral ischemia after SAH secondary to intracranial arterial spasm [10,24,26,27]. Nimodipine blocks the influx of extracellular calcium. The transmembrane influx of calcium ions is essential for the excitation-contraction coupling process of smooth muscle cells. Specific antagonists of this calcium influx can therefore inhibit smooth muscle contraction without regard to the exciting stimulus. Experimental evidence of inhibitory effects of calcium antagonists on intestinal smooth muscle contraction had been previously published [7,17,21,33]. These experimental data provided evidence that calcium antagonists exert inhibitory effects on the gastrointestinal tract due to their intrinsic mechanism of action. It has been demonstrated that nifedipine Page 4/9 Page 4/9 inhibits humans’ colonic electric spike activity induced by eating [21]. Side effects such as abdominal pain or constipation may occur in patients treated with oral nimodipine for extended periods [12,23,30]. In 1948, Ogilvie reported two patients with signs of colonic obstruction in the absence of any organic disease of the colon [25]. He attributed its development to the interruption of the sympathetic supply to the large intestine. However, after surgical exploration, both patients were found to have disseminated abdominal metastatic disease. Ogilvie syndrome is now recognized as a colonic pseudo-obstruction caused by decreased motility in the absence of mechanical obstruction; still, the eponym was attributed to his idea of the interruption of the sympathetic supply. Ogilvie syndrome is associated with a 25-30% mortality in severe cases, which increases to 50% if the patient develops ischemia and perforation [19]. Early recognition and management are essential. The specific pathophysiology of acute colonic pseudo- obstruction is still unclear. It may include many etiologies, risk factors, pathological conditions, and multiple associations leading to altered autonomic regulation of colonic motility [8,34]. Excessive parasympathetic suppression, sympathetic stimulation, or both are thought to produce an imbalance in the colonic autonomic innervation. All these factors produce temporary suppression of intestinal motility, inducing acute dilatation of the colon. Nimodipine has rarely been associated with the development of Ogilvie syndrome in aneurysmal SAH. In this report, we presented two cases in which its use could have been associated with the development of acute colonic pseudo-obstruction. Abbreviations SAH = subarachnoid hemorrhage CT = computed tomographic GCS = Glasgow coma scale DSA = digital subtraction angiogram KUB = kidney, ureter, and bladder SAH = subarachnoid hemorrhage Discussion This report was the first to associate the oral use of nimodipine with the development of Ogilvie syndrome. They continued treatment with nimodipine for the recommended time, despite the colonic dilatation, as they thought the risk of removing the medication was more detrimental than its continuation. The pseudo- obstruction was ultimately resolved after endoscopic colon decompression and rectal tube placement. In one of our patients, we also implemented similar measures to improve the pseudo-obstruction. Our other patient required a rectal tube after an unsuccessful colonoscopy. Patients with Ogilvie syndrome are initially managed with supportive therapy for decompression of the gastrointestinal tract, including gastric and rectal tubes. Among the conservative treatments, Page 5/9 discontinuation of the oral intake, placement of a nasogastric tube for proximal gut decompression, correction of fluid and electrolyte abnormalities, treatment of any underlying concomitant illnesses, and the cessation of medications such as narcotics and anticholinergics that adversely affect colonic motility had been effective [16]. Those who do not respond within 1-2 days may require neostigmine use. The use of intravenous neostigmine, an acetylcholinesterase inhibitor, is the best-documented pharmacological treatment for acute colonic pseudo-obstruction with initial response rates of 60-90% [8,16,20,28]. Although neostigmine has a short elimination half-life, most patients in the study by Ponec et al. achieved a sustained response after the initial dose [28]. We had to use neostigmine in one of our patients; however, its use only partially improved the distention, reappearing when the patient was started again on a diet. Haj et al. reported similar outcomes using conservative management or interventional management to manage Ogilvie syndrome [14]. Patients who are refractory to treatment should receive endoscopic decompression [19]. Colonoscopic decompression is successful in approximately 80% of patients. Exploratory laparotomy is limited to those patients in whom complications occur [9]. Conclusion Even though the cause and mechanism of acute colonic pseudo-obstruction are not yet clearly understood, calcium channel blockers may predispose patients to develop the condition. Our two cases and prior reported cases provide additional evidence about the possible causal relation between nimodipine use and Ogilvie syndrome in patients treated for aneurysmal SAH. The continued use of nimodipine after the development of the acute colonic pseudo-obstruction had not been established, and each physician should decide based on the risk of withdrawing the medication. Our data suggest that further investigation of the impact of oral nimodipine on the gastrointestinal tract of aneurysmal SAH patients is justified. Availability of data and material: Not applicable in this study type (review) Availability of data and material: Not applicable in this study type (review) Availability of data and material: Not applicable in this study type (review) Code availability: Not applicable in this study type (review) Ethics approval: There are no patient identifiers in this publication. The University Institutional Review Board verified the manuscript and allowed its submission and publication. Consent to participate: Informed verbal consent was obtained from both individual participants included in the study. Consent for publication: The participants have consented to the submission of the review manuscript to the journal. No photography is included in the study. Consent for publication: The participants have consented to the submission of the review manuscript to the journal. No photography is included in the study. Author’s contributions: Page 7/9 Authors contributions: Concept and design: Orlando De Jesus, José Sánchez Jiménez,MD Data acquisition: Orlando De Jesus, José Sánchez Jiménez,MD, Juan C. Vicenty Drafting the manuscript: Orlando De Jesus, José Sánchez Jiménez,MD, Juan C. Vicenty Revising the manuscript: Orlando De Jesus, José Sánchez Jiménez,MD, Juan C. Vicenty Approval of the final manuscript: Orlando De Jesus, José Sánchez Jiménez,MD, Juan C. Vicenty References 1. Addison NV (1983) Pseudo-obstruction of the large bowel. J R Soc Med 76:252-255 2. Allen GS, Ahn HS, Preziosi TJ, Battye R, Boone SC, Boone SC, et al (1983) Cerebral arterial spasm--a controlled trial of nimodipine in patients with subarachnoid hemorrhage. N Engl J Med 308:619- 24. https://doi.org/10.1056/NEJM198303173081103. 3. Allen GS (1985) Role of calcium antagonists in cerebral arterial spasm. Am J Cardiol 55:149 B-153 B. https://doi.org/10.1016/0002-9149(85)90624-1 4. Auer LM (1984) Acute operation and preventive nimodipine improve outcome in patients with ruptured cerebral aneurysms. Neurosurgery 15:57-66. https://doi.org/10.1227/00006123- 198407000-00011 5. Bachulis BL, Smith PE (1978) Pseudoobstruction of the colon. Am J Surg 136:66-71 6. Bullock PR, Thomas WE (1984) Acute pseudo-obstruction of the colon. Ann R Coll Surg Engl 66:327- 330 Concept and design: Orlando De Jesus, José Sánchez Jiménez,MD Data acquisition: Orlando De Jesus, José Sánchez Jiménez,MD, Juan C. Vicenty Drafting the manuscript: Orlando De Jesus, José Sánchez Jiménez,MD, Juan C. Vicenty Revising the manuscript: Orlando De Jesus, José Sánchez Jiménez,MD, Juan C. Vicenty Concept and design: Orlando De Jesus, José Sánchez Jiménez,MD Approval of the final manuscript: Orlando De Jesus, José Sánchez Jiménez,MD, Juan C. Vicenty Declarations Funding: No funding was received to assist with the preparation of this manuscript. Page 6/9 Conflict of interest: The authors have no conflicts of interest to declare relevant to this article’s content. All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript. References 1. 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Kazner E, Sprung C, Adelt D, Ammerer HP, Karnick R, Baumann H, et al (1985) Clinical experience with nimodipine in the prophylaxis of neurological deficits after subarachnoid hemorrhage. Neurochirurgia (Stuttg) 28:110-113. https://doi.org/10.1055/s-2008-1054114 18. Kazner E, Sprung C, Adelt D, Ammerer HP, Karnick R, Baumann H, et al (1985) Clinical experience with nimodipine in the prophylaxis of neurological deficits after subarachnoid hemorrhage. Neurochirurgia (Stuttg) 28:110-113. https://doi.org/10.1055/s-2008-1054114 19. Keller J, Layer P (2015) Akute Kolonpseudoobstruktion: Ogilvie-Syndrom [Acute colonic pseudo- obstruction: Ogilvie syndrome]. Med Klin Intensivmed Notfmed 110:506-509. German. https://doi.org/10.1007/s00063-015-0081-4 19. Keller J, Layer P (2015) Akute Kolonpseudoobstruktion: Ogilvie-Syndrom [Acute colonic pseudo- obstruction: Ogilvie syndrome]. Med Klin Intensivmed Notfmed 110:506-509. German. https://doi.org/10.1007/s00063-015-0081-4 20. 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Nimodipine: pharmacological and clinical properties. Proceeding of the 1st International Nimotop Symposium, Munich. Stuttgart, New York: Schattauer, pp 431-441 24. Neifert SN, Chapman EK, Martini ML, Shuman WH, Schupper AJ, Oermann EK, Mocco J, Macdonald RL (2021) Aneurysmal subarachnoid hemorrhage: the last decade. Transl Stroke Res 12:428- 446. https://doi.org/10.1007/s12975-020-00867-0 25. References Ogilvie H (1948) Large-intestine colic due to sympathetic deprivation; a new clinical syndrome. Br Med J 2:671-673. https://doi.org/10.1136/bmj.2.4579.671 26. Philippon J, Grob R, Dagreou F, Guggiari M, Rivierez M, Viars P (1986) Prevention of vasospasm in subarachnoid haemorrhage. A controlled study with nimodipine. Acta Neurochir (Wien) 82:110- 114. https://doi.org/10.1007/BF01456369 27. Pickard JD, Murray GD, Illingworth R, Shaw MD, Teasdale GM, Foy PM, et al (1989) Effect of oral nimodipine on cerebral infarction and outcome after subarachnoid haemorrhage: British aneurysm nimodipine trial. BMJ 298:636-42. 10.1136/bmj.298.6674.636 27. Pickard JD, Murray GD, Illingworth R, Shaw MD, Teasdale GM, Foy PM, et al (1989) Effect of oral nimodipine on cerebral infarction and outcome after subarachnoid haemorrhage: British aneurysm nimodipine trial. BMJ 298:636-42. 10.1136/bmj.298.6674.636 28. Ponec RJ, Saunders MD, Kimmey MB (1999) Neostigmine for the treatment of acute colonic pseudo- obstruction. N Engl J Med 341:137-41. https://doi.org/10.1056/NEJM199907153410301 28. Ponec RJ, Saunders MD, Kimmey MB (1999) Neostigmine for the treatment of acute colonic pseudo- obstruction. N Engl J Med 341:137-41. https://doi.org/10.1056/NEJM199907153410301 29. Seiler RW, Grolimund P, Zurbruegg HR (1987) Evaluation of the calcium-antagonist nimodipine for the prevention of vasospasm after aneurysmal subarachnoid haemorrhage. A prospective transcranial Doppler ultrasound study. Acta Neurochir (Wien) 85:7-16 28. https://doi.org/10.1007/BF01402363 30. Sheftell FD, Rapoport AM, Weeks RE, Arrowsmith FRN, Kontaxis CRN, Baskin SM (1985) Nimodipine in the prevention of mixed headache disorders: a preliminary report. In: Betz E, Deck K, Hoffmeister F, eds. Nimodipine: pharmacological and clinical properties. Proceedings of the 1st International Nimotop Symposium, Munich. Stuttgart, New York: Schattauer, pp 445-450. 31. Tettenborn D, Dycka J, Volberg E, Düdden P (1985) Blood pressure and heart rate during treatment with nimodipine in patients with subarachnoid hemorrhage. Neurochirurgia (Stuttg) 28(Suppl 1):84- 86. https://doi.org/10.1055/s-2008-1054109 32. Torrealba G, Sharp A, Soto B. (1987) Nimodipine-treated subarachnoid hemorrhage associated with acute pseudo-obstruction of the colon. Surg Neurol 28:150-152. https://doi.org/10.1016/0090- 3019(87)90090-5 33. Traube M, McCallum RW (1984) Calcium-channel blockers and the gastrointestinal tract. American College of Gastroenterology's Committee on FDA related matters. Am J Gastroenterol 79:892-896 34. Wells CI, O'Grady G, Bissett IP (2017) Acute colonic pseudo-obstruction: A systematic review of aetiology and mechanisms. World J Gastroenterol 23:5634-5644. https://doi.org/10.3748/wjg.v23.i30.5634 Page 9/9 Page 9/9
https://openalex.org/W2108377687	https://www.scielo.br/j/sa/a/N9DLLrhzrnzkwbbxM6LWx7g/?lang=pt&format=pdf	Portuguese	null	Espectrofotometria de proteínas totais em plasma de sangue bovino por análise em fluxo	Scientia Agrícola	2,002	cc-by	5,178	ESPECTROFOTOMETRIA DE PROTEÍNAS TOTAIS EM PLASMA DE SANGUE BOVINO POR ANÁLISE EM FLUXO Gilmara Caseri de Luca1,3; Boaventura Freire dos Reis2* 1Pós-Graduanda do Instituto de Química de São Carlos/USP - CEP: 13560-970 - São Carlos, SP. 2Lab. de Química Analítica - USP/CENA, C.P. 96 - CEP: 13400-970 - Piracicaba, SP. 3Bolsista FAPESP. Autor correspondente <reis@cena usp br> 1Pós-Graduanda do Instituto de Química de São Carlos/USP - CEP: 13560-970 - São Carlos, SP. 2Lab. de Química Analítica - USP/CENA, C.P. 96 - CEP: 13400-970 - Piracicaba, SP. 3Bolsista FAPESP. Autor correspondente <reis@cena usp br> Autor correspondente <reis@cena.usp.br> RESUMO: A concentração de proteína total em plasma é um parâmetro utilizado no controle da saúde e nutrição animal, sendo que a faixa de concentração considerada normal para animais em bom desenvolvimento situa-se entre 60 e 85 g L -1. Os métodos analíticos propostos para esta determinação geralmente apresentam como limitação a excessiva manipulação das amostras. Este trabalho descreve o desenvolvimento de um procedimento de análises em fluxo para a determinação de proteína total em plasma de sangue bovino, empregando o método do Biureto. O sistema em fluxo, constituído por um injetor comutador automático e uma válvula solenóide de três vias controlados por um microcomputador, foi projetado visando permitir diluição em linha das amostras. Um fator de diluição era estimado procedendo-se a diluição em linha de uma solução com concentração conhecida de albumina e este fator era empregado para o cálculo final das concentrações das amostras após diluição em linha. As soluções das amostras eram inseridas através da válvula solenóide, a qual permitia precisas diluições, diminuindo operações manuais. A faixa analítica estabelecida foi entre 2,5 e 20,0 g L -1, e considerando a diluição gerada, amostras de plasma bovino contendo entre 12,5 e 100,0 g L -1 de proteína total puderam ser analisadas. O procedimento apresentou desvio padrão relativo de 2,8 %, e a freqüência analítica alcançada foi de 76 determinações por hora. Os resultados foram comparados com o método tradicional de análises (Biureto) e não foram observadas diferenças estatisticamente significativas a 95% Palavras-chave: determinação de proteína, diluição em linha, sistema de análise em fluxo, plasma animal SPECTROPHOTOMETRY OF TOTAL PROTEIN IN BOVINE BLOOD PLASMA BY FLOW ANALYSIS ABSTRACT: Total protein concentration in plasma samples is normally used as a parameter to control animal health and nutritional conditions. Normal concentration levels are in the range of 60 to 85 g L -1 total protein for animals of good development. The methods proposed for its determination generally present as a disadvantage an excessive handling during operation. In this work an automated flow procedure for total protein determination in bovine serum samples, employing the Biuret method, was developed. The system, including an automatic injector commutator and a three way solenoid valve computer controlled, was designed in order to permit on line sample dilution. Since protein standard solutions were introduced with and without dilution. A dilution factor was estimated and used to calculate protein concentration obtained after on-line dilution of the samples. Solutions samples were inserted through a three way solenoid valve that allows precise dilution minimizing hand operations. The analytical range for total protein determination was 2.5 to 20.0 g L -1, and considering the dilution employed, bovine plasma samples with 12.5 to 100.0 g L -1 of total protein were analysed. The procedure presented a 2.8% rsd and analytical frequency of 76 determinations per hour. Results were in good agreement with the traditional method (Biuret) and no significant difference was verified at 95%. Key words: protein, on line dilution, flow system, blood plasma 251 251 MATERIAL E MÉTODOS como limitação o tempo longo exigido por análise e possíveis erros nos resultados ocasionados pela presença de nitrogênio não protéico na amostra. Outros procedimentos instrumentais de análises têm sido também empregados utilizando cromatografia (Hayakawa et al., 1997), fluorimetria (Yokoyama et al., 1990), polarografia (Perez & Frutos, 1995) e espectrofotometria (Bradford, 1976; Goren & Li, 1986; Guo & Shen, 1999) como técnicas de detecção e quantificação, visando suprir aplicações nas áreas clinica e nutricional, entre outras. Os métodos espectrofotométricos são os mais utilizados, e revisões recentes desses métodos apontaram as vantagens e desvantagens das principais reações empregadas (Zaia et al., 1998; Sapan et al., 1999). Parâmetros importantes a serem considerados para a escolha da metodologia são a concentração da espécie de interesse, que pode variar muito dependendo da natureza da amostra (plasma ou soro de sangue, urina, fluido espinal, leite, etc.), e possíveis interferências. No caso específico de soro ou plasma sangüíneo, o método do Biureto, cujo procedimento é manual (FAO, 1993), tem sido o mais indicado para a determinação de proteínas totais (Zaia et al., 1998). Um dos fatores negativos para a utilização de procedimentos manuais é a manipulação excessiva das amostras, aumentando o risco de contaminações. Além disso, as etapas analíticas envolvidas nestes procedimentos são lentas, trabalhosas e dispendiosas, potencializando a necessidade de se recorrer a procedimentos automatizados para diminuir o consumo de reagentes e o tempo para obtenção dos resultados. Todos os reagentes utilizados foram de grau analítico e as soluções foram preparadas com água destilada e deionizada. Soluções padrão de proteína contendo 0,0; 2,5; 5,0; 10,0; 15,0 e 20,0 g L -1 foram preparadas dissolvendo-se 0,00; 0,125; 250,0; 500,0; 750,0 e 1.000 mg de albumina de soro bovino (fração V, Merck) em 50,0 mL de cloreto de sódio 0,14 mol L -1. Estas soluções eram preparadas a cada três dias, e armazenadas em frascos de vidro. Quando não em uso, eram mantidas sob refrigeração. Reagente Biureto contendo 6,0 g L -1 de sulfato de cobre (CuSO4.5H2O; Merck), 5,0 g L -1 de iodeto de potássio (KI; Merck), 18,0 g L -1 tartarato de sódio e potássio KNaC4H4O6.4H2O) Merck), em 0,2 mol L -1 de hidróxido de sódio (NaOH). Esta solução era preparada semanalmente, armazenada em frasco de polietileno e protegida da luz. Amostras de plasma bovino, foram cedidas pelo Laboratório de Ciências Animais do Centro de Energia Nuclear na Agricultura (CENA/USP). MATERIAL E MÉTODOS As amostras de sangue animal foram coletadas diretamente dos animais por punção venosa na cauda ou veia jugular empregando agulha de calibre 18 para evitar hemólise. As amostras foram recolhidas em tubos de ensaio do tipo Vacutainer , os quais são comercializados esterilizados, à vácuo e com anticoagulante (heparina sódica). Após coleta, o tubo foi invertido cuidadosamente para que ocorresse a mistura e centrifugados durante 12 min a 2500 rpm. Uma vez separado o plasma das células, o mesmo foi removido com o auxílio de pipetador automático e transferido para frascos de polietileno esterilizados, para posterior análise. Esse procedimento de coleta e preparo da amostra é recomendado por Nogueira et al. (1998). Para facilitar a operação, minimizar contaminações, manipulação das amostras e aumentar a freqüência analítica, sistemas de análise em fluxo - FIA (Ruzicka & Hansen, 1988) podem ser empregados. Os sistemas FIA são ótimos gerenciadores de soluções, apresentam facilidade operacional, podendo ser adaptados ao desenvolvimento analítico. Em tais sistemas, reações rápidas ou lentas podem ser implementadas como adição de reagentes, diluição ou pré-concentração das amostras, separação de espécies potencialmente interferentes, entre outros procedimentos, que normalmente são realizados manualmente. Com a proposta de sistemas empregando multicomutação (Reis et al., 1994) os sistemas de análise em fluxo tornaram-se muito mais versáteis e robustos. p g ( ) O sistema em fluxo era constituído por uma bomba peristáltica Ismatec IPC-8, com tubos de Tygon de diferentes diâmetros internos; um espectrofotômetro Femto, modelo 482, equipado com cubeta de fluxo (100 µL, 10 mm passo óptico); um registrador potenciométrico ECB, modelo RB201; um injetor comutador automático; uma válvula solenóide de três vias (161T031) controlada por um microcomputador equipado com uma interface de controle PCL 711s da American Advantech. As bobinas de mistura e reação foram confeccionadas com tubos de polietileno de 0,8 mm de diâmetro interno. p O sistema em fluxo utilizado para a determinação de proteína total em plasma de sangue bovino é mostrado na Figura 1. O sistema de inserção das soluções é constituído por duas partes: uma válvula solenóide de três vias (V1) e um injetor comutador. Ambos podem funcionar independentemente ou simultaneamente. Na posição representada na Figura. 1, a válvula está desligada e um fluxo de água é aspirado através da bobina de mistura B1 e da alça de amostragem L em direção ao descarte. INTRODUÇÃO ser observados casos de desidratação bem como doenças crônicas ou intermediárias (FAO, 1993; Lindsey, 1996). A determinação de proteínas totais em plasma de sangue bovino é utilizada como um parâmetro no controle da saúde e nutrição animal (FAO, 1993). Os valores de referência em soro sangüíneo de bovinos situam-se entre 60 a 85 g L -1, sendo que níveis mais baixos são verificados em casos de deficiência de proteína na dieta; insuficiência hepática; aproveitamento inadequado da proteína ingerida; hemorragias; e perda da proteína intestinal ou renal. Quando a concentração de proteína total no plasma sangüíneo aumenta, podem Ao longo de décadas, inúmeros pesquisadores têm se dedicado ao desenvolvimento de metodologias e procedimentos visando a determinação de proteína total em diversos tipos de amostras como por exemplo sangue, urina e leite. O método de Kjedahl (AOAC, 1995), é um método clássico de análises no qual se determina a concentração de nitrogênio total após decomposição da amostra com ácido sulfúrico. É empregado como método de referência, mas apresenta Scientia Agricola, v.59, n.2, p.251-256, abr./jun. 2001 Luca & Reis Luca & Reis 252 MATERIAL E MÉTODOS A solução Explorando tais potencialidades, neste trabalho foi desenvolvido um procedimento em fluxo automatizado para a determinação de proteínas totais em plasma de sangue bovino empregando a reação do Biureto (Lindsey, 1996). O sistema proposto é composto por um injetor comutador e uma válvula solenóide de três vias cujo funcionamento é controlado por um microcomputador. Scientia Agricola, v.59, n.2, p.251-256, abr./jun. 2001 Espectrofotometria de proteínas totais Espectrofotometria de proteínas totais 253 RESULTADOS E DISCUSSÃO Em sistemas FIA, o volume da solução de amostra é definido pela dimensão da alça de amostragem. Desta forma, fixou-se o comprimento da bobina de reação (B2) em 300 cm e variou-se o comprimento de L em 20, 50 e 100 cm (0,8 mm d.i.), Durante o dimensionamento do sistema e definição dos principais parâmetros envolvidos na determinação de proteína total, a válvula solenóide foi excluída do sistema (Figura 1) e os estudos foram conduzidos apenas empregando-se o injetor comutador. O programa para controle de acionamento da válvula solenóide e do injetor comutador automático foi escrito em linguagem QuickBASIC 4.5. O fluxograma do programa é mostrado na Figura 2. Os estudos para definir o volume de amostra, forma de introdução do reagente e tempo de reação foram conduzidos empregando o sistema sem a válvula solenóide, e desta forma, a solução da amostra era introduzida diretamente através da alça de amostragem (L). Após estas otimizações, a válvula solenóide foi adaptada ao injetor comutador (Figura 1), visando promover diluição em linha das amostras. Estudaram-se, então, possíveis diluições em linha das amostras, introduzindo-se no sistema uma solução de trabalho com concentração conhecida de albumina. Para estimar os fatores de diluição, variou-se o tempo da válvula solenóide nas condições ligada-desligada, e o número de repetições desta seqüência. Em sistemas FIA, o volume da solução de amostra é definido pela dimensão da alça de amostragem. Desta forma, fixou-se o comprimento da bobina de reação (B2) em 300 cm e variou-se o comprimento de L em 20, 50 e 100 cm (0,8 mm d.i.), Início Troca variáveis? Troca variáveis Troca amostra? Troca amostra Amostragem S N S N Repete procedimento? Fim S N Figura 2 - Fluxograma do programa de controle. O fluxograma refere-se ao funcionamento do sistema mostrado na Figura 1, empregado na determinação de proteínas totais. Troca variáveis? Definidas as condições de trabalho, a exatidão do procedimento foi avaliada comparando-se o procedimento proposto com o método oficial para a determinação de proteína total em fluídos biológicos. Troca amostra? Scientia Agricola, v.59, n.2, p.251-256, abr./jun. 2001 x B2 Det L1 Dv • • Rc H2O D • • S H2O Ca R1 V1 B1 Figura 1 - Diagrama do sistema em fluxo empregado para a determinação de proteína total em plasma sangüíneo. RESULTADOS E DISCUSSÃO V1 = válvula solenóide de três vias; B1 = bobina de mistura; B2 = bobina de reação; Ca = H2O (4,0 mL min-1); R1 = reagente biureto (4,0 mL min-1); L1 = alça de amostragem; S = amostra (2,0 mL min-1); Det = espectrofotômetro (λ = 546 nm); Dv = vaso de descontaminação; x = ponto de confluência; D = descarte. Scientia Agricola, v.59, n.2, p.251-256, abr./jun. 2001 x B2 Det L1 Dv • • Rc H2O D • • S H2O Ca R1 V1 B1 Figura 1 - Diagrama do sistema em fluxo empregado para a determinação de proteína total em plasma sangüíneo. V1 = válvula solenóide de três vias; B1 = bobina de mistura; B2 = bobina de reação; Ca = H2O (4,0 mL min-1); R1 = reagente biureto (4,0 mL min-1); L1 = alça de amostragem; S = amostra (2,0 mL min-1); Det = espectrofotômetro (λ = 546 nm); Dv = vaso de descontaminação; x = ponto de confluência; D = descarte. Troca amostra? Troca amostra Amostragem S N Repete procedimento? Fim S N Figura 2 - Fluxograma do programa de controle. O fluxograma refere-se ao funcionamento do sistema mostrado na Figura 1, empregado na determinação de proteínas totais. x B2 Det L1 Dv • • Rc H2O D • • S H2O Ca R1 V1 B1 Figura 1 - Diagrama do sistema em fluxo empregado para a determinação de proteína total em plasma sangüíneo. V1 = válvula solenóide de três vias; B1 = bobina de mistura; B2 = bobina de reação; Ca = H2O (4,0 mL min-1); R1 = reagente biureto (4,0 mL min-1); L1 = alça de amostragem; S = amostra (2,0 mL min-1); Det = espectrofotômetro (λ = 546 nm); Dv = vaso de descontaminação; x = ponto de confluência; D = descarte. Figura 1 - Diagrama do sistema em fluxo empregado para a determinação de proteína total em plasma sangüíneo. V1 = válvula solenóide de três vias; B1 = bobina de mistura; B2 = bobina de reação; Ca = H2O (4,0 mL min-1); R1 = reagente biureto (4,0 mL min-1); L1 = alça de amostragem; S = amostra (2,0 mL min-1); Det = espectrofotômetro (λ = 546 nm); Dv = vaso de descontaminação; x = ponto de confluência; D = descarte. Figura 2 - Fluxograma do programa de controle. Scientia Agricola, v.59, n.2, p.251-256, abr./jun. 2001 RESULTADOS E DISCUSSÃO transportadora é bombeada através do percurso analítico e o reagente R1 é reciclado. Acionando-se a válvula V1, a solução da amostra é aspirada através de B1, preenche L e seu excesso é descartado. Após um período de tempo selecionado para preencher L, o injetor é comutado para a posição alternativa, a alça de amostragem é inserida no percurso analítico, e a válvula V1 é desligada. O reagente R1 é adicionado à zona da amostra através do ponto de confluência x, e a mistura amostra e reagente flui através da bobina de reação B2 em direção ao detector. Após a obtenção do máximo do sinal analítico, a parte central do injetor volta à posição de origem e um novo ciclo se inicia acionando-se a válvula V1 para preenchimento de L com a solução da amostra. transportadora é bombeada através do percurso analítico e o reagente R1 é reciclado. Acionando-se a válvula V1, a solução da amostra é aspirada através de B1, preenche L e seu excesso é descartado. Após um período de tempo selecionado para preencher L, o injetor é comutado para a posição alternativa, a alça de amostragem é inserida no percurso analítico, e a válvula V1 é desligada. O reagente R1 é adicionado à zona da amostra através do ponto de confluência x, e a mistura amostra e reagente flui através da bobina de reação B2 em direção ao detector. Após a obtenção do máximo do sinal analítico, a parte central do injetor volta à posição de origem e um novo ciclo se inicia acionando-se a válvula V1 para preenchimento de L com a solução da amostra. Os estudos para automatização do método direcionado à determinação de proteínas totais em plasma de sangue bovino foram conduzidos buscando estabelecer uma faixa analítica de concentração adequada a esta determinação. Embora os níveis de referência indiquem concentrações na faixa entre 60,0 e 85,0 g L -1, a faixa analítica estabelecida inicialmente foi entre 2,5 e 20,0 g L -1 de albumina, pressupondo diluições das amostras. Esta estratégia foi adotada considerando que o reagente empregado no preparo das soluções padrão tem um alto custo e as soluções são pouco estáveis e apresentam certa dificuldade no preparo. p p Durante o dimensionamento do sistema e definição dos principais parâmetros envolvidos na determinação de proteína total, a válvula solenóide foi excluída do sistema (Figura 1) e os estudos foram conduzidos apenas empregando-se o injetor comutador. RESULTADOS E DISCUSSÃO O fluxograma refere-se ao funcionamento do sistema mostrado na Figura 1, empregado na determinação de proteínas totais. Scientia Agricola, v.59, n.2, p.251-256, abr./jun. 2001 & Reis Figura 3 - Efeito da concentração do reagente. -n- : 3,0 g L-1 CuSO4.5H2O; -l- : 4,5 g L-1 CuSO4.5H2O; -s -: 6,0 g L-1 CuSO4.5H2O. Resultados obtidos empregando-se o sistema da Figura 1. 0 2 4 6 8 10 12 0,00 0,16 0,32 0,48 0,64 Absorbância 0 2 4 6 8 10 12 0,00 0,16 0,32 0,48 0,64 Concentração de albumina (g L-1) 254 Luca & Reis 0 2 4 6 8 10 12 0,00 0,16 0,32 0,48 0,64 Absorbância 0 2 4 6 8 10 12 0,00 0,16 0,32 0,48 0,64 correspondendo a volumes de 100, 250 e 500 µL e, em função da resposta do sinal analítico obtido, fixou-se o volume da solução da amostra em 100 µL (20 cm). Variou-se, então a dimensão da bobina de reação B2, correspondente ao tempo disponível para o desenvolvimento da reação, em 100, 200 e 300 cm. Foi observado um aumento de 30% nos sinais quando variou-se a dimensão de B2 de 100 para 200 cm. Acima de 200 cm, apesar do tempo de reação ser maior, observou-se uma diminuição de 5% do sinal monitorado. Esta diminuição pode ser atribuída à dispersão do produto formado na solução transportadora durante seu transporte até o detector. A bobina de reação foi fixada em 200 cm (0,8 mm d.i.). ( ) A relação entre as vazões da solução transportadora e da solução do reagente R1, era inicialmente 1:1 (2,4 mL min -1 cada uma). Com estas vazões, quando a zona da amostra atingia o ponto de confluência x, sua concentração, bem como a do reagente R1, eram reduzidas à metade. Visando promover uma menor diluição da zona da amostra no percurso analítico, a vazão do reagente foi alterada de 2,4 mL min -1 para 1,2 mL min -1. Esta redução na vazão da solução do reagente R1 diminuiu a diluição da zona da amostra, mas aumentou a diluição do reagente, acarretando perda em linearidade. Fixou-se a vazão do reagente em 1,2 mL min -1 e variou-se a concentração do reagente em 3,0; 4,5 e 6,0 g L -1 de sulfato de cobre. A concentração de tartarato de sódio e potássio, empregado para estabilizar os íons cobre em solução, também foi aumentada proporcionalmente. RESULTADOS E DISCUSSÃO A solução contendo 6,0 g L -1 CuSO4.5H2O, 18,0 g L -1 KNaC4H4O6.4H2O, e 5,0 g L -1 KI em 0,2 mol L -1 NaOH, foi a que apresentou melhores resultados em termos de linearidade (r=0,998) e sensibilidade (Figura 3), sendo fixada para a continuidade do procedimento. Concentração de albumina (g L-1) Figura 3 - Efeito da concentração do reagente. -n- : 3,0 g L-1 CuSO4.5H2O; -l- : 4,5 g L-1 CuSO4.5H2O; -s -: 6,0 g L-1 CuSO4.5H2O. Resultados obtidos empregando-se o sistema da Figura 1. Figura 3 - Efeito da concentração do reagente. -n- : 3,0 g L-1 CuSO4.5H2O; -l- : 4,5 g L-1 CuSO4.5H2O; -s -: 6,0 g L-1 CuSO4.5H2O. Resultados obtidos empregando-se o sistema da Figura 1. desta válvula permitia que as soluções de referência fossem inseridas sem diluição, acionando-se a válvula V1 por um período de tempo suficientemente longo para que a alça de amostragem L fosse preenchida. No caso das amostras cuja concentração do analito era maior, diluições podiam ser realizadas variando-se o tempo de acionamento de V1, intercalando um volume da solução da amostra com o do diluente. Empregando-se esta configuração (Figura 1), o volume da solução da amostra inserido no percurso analítico era sempre o mesmo, sendo portanto o fator dispersão inalterado. Desta forma, estudou-se possíveis diluições da amostra, variando-se os intervalos de tempo de V1 nas posições liga-desliga, para inserção das soluções da amostra e do diluente. Quando a V1 era acionada, uma alíquota da solução da amostra era inserida em B1, e quando V1 era desligada, inseria-se uma alíquota do diluente. O número de ciclos selecionado para repetir esta seqüência, era estabelecido em função do tempo necessário para completo preenchimento de L com a solução a ser analisada. Uma vez definidas as condições para o desenvolvimento da reação, alterou-se a forma de inserção do reagente: de bombeamento contínuo para inserção intermitente. O sistema empregando fluxos intermitentes foi proposto por Zagatto et al. (1980), que demonstraram a funcionalidade desta proposta na determinação de nitrato em águas. Esta estratégia foi empregada visando diminuir o consumo de reagente, que era desperdiçado durante a etapa de limpeza. Na posição do injetor na qual R1 era reciclado, um fluxo de água com maior vazão era introduzido através do ponto de confluência x (Figura 1), o que favorecia também uma limpeza mais rápida do sistema. Scientia Agricola, v.59, n.2, p.251-256, abr./jun. 2001 RESULTADOS E DISCUSSÃO O intervalo de tempo de V1 acionada foi variado em 0,2; 0,3; 0,4; 0,5; 0,6; 0,7 e 0,8 s e observou-se que quando períodos menores que 0,5 s eram empregados, o desvio padrão relativo estimado era mais expressivo (~7%). Fixou-se o tempo de inserção da alíquota da amostra em 0,5 s e variou-se o tempo de inserção da solução diluente em 0,5; 1,0; 1,5; 2,0; 2,5; 3,0 s. Pode- se observar através da Tabela 1 uma boa correlação entre os fatores obtidos e os valores teóricos quando o tempo de inserção do diluente foi fixado em 2,0 s, sendo o fator de diluição estabelecido de 5 vezes. O fator diluição (F), era estimado inserindo-se a solução de referência de maior concentração (20,0 g L -1) com (Cd) e sem (Ci) o procedimento de diluição. As concentrações eram calculadas através da curva analítica, e a razão entre Ci e Cd gerava o fator de diluição real (F = Ci/Cd). Este fator era utilizado para cálculo das concentrações Conforme mencionado anteriormente, o desenvolvimento deste procedimento para a determinação de proteína total em plasma sangüíneo previa diluição das amostras, para que a concentração das mesmas estivesse dentro da faixa de resposta linear do método. Incluiu-se então no sistema, uma válvula solenóide de três vias, a qual tinha a função de permitir a diluição em linha das amostras (Figura 1). A inclusão Scientia Agricola, v.59, n.2, p.251-256, abr./jun. 2001 255 Espectrofotometria de proteínas totais Figura 4 - Registro de rotina para a determinação de proteína totalem plasma de sangue animal. Da esquerda para a direita os sinais identificados pelas letras a; b; c; d; e e f correspondem respectivamente às soluções contendo 0,0; 2,5; 5,0; 10,0; 15,0 e 20,0 g L-1 de albumina. D1 e D2 solução contendo 20,0 g L-1 após diluição de 4 e 5 vezes. Demais sinais: amostras de plasma animal. A seta indica o sentido do papel. a b c d e f D1 D2 0,10 A 5 min. a b c d e f D1 D2 0,10 A 5 min. Tabela 1 - Fatores de diluição obtidos empregando-se uma solução contendo 20,0 g L-1 de albumina. Tabela 1 - Fatores de diluição obtidos empregando-se uma solução contendo 20,0 g L-1 de albumina. referem-se aos intervalos de tempo para inserção da solução da amostra e da solução do diluente. RESULTADOS E DISCUSSÃO De 1 a 8 os resultados correspondem a amostras de plasma de sangue eqüino e de 9 a 15 a amostras de plasma de sangue bovino. Os resultados são médias de 3 determinações. Com o sistema proposto, 84 µL de amostra e 67 µL do reagente (0,4 µg CuSO4.5H2O; 1,4 µg KNaC4H4O6.4H2O); e 0,33 µg KI) foram gastos por determinação, para amostras com concentrações entre 25 e 100 g L -1 de proteína total. finais das amostras, multiplicando-se as concentrações encontradas pelo fator estabelecido. Para verificar se a mistura amostra-diluente era eficiente, variou-se o dimensionamento de B1 em 10, 20 e 40 cm, aumentando-se proporcionalmente o número de ciclos da seqüência de acionamento de V1 para completo preenchimento de L. Não foi observada melhoria em repetibilidade com o aumento na dimensão de B1, sendo esta fixada em 10 cm (0,8 mm d.i.). CONCLUSÃO O procedimento desenvolvido apresentou maior rapidez na emissão de resultados, menor consumo de reagentes e menor manipulação de amostras quando comparado ao método oficial de análises. A montagem do módulo de análises requer poucos conhecimentos de informática e eletrônica, conhecimentos estes que não representam limitações para sua viabilização. Uma vez montado, o sistema é facilmente operado, sendo recomendado para análises de rotina. 1 Um conjunto de amostras de plasma de sangue bovino foi analisado e os resultados foram comparados com aqueles obtidos empregando o método tradicional de análises (Tabela 2). A análise estatística dos resultados (teste t-pareado) demonstrou que não há diferença estatisticamente significativa em nível de 95% de confiança entre os procedimentos utilizados. O desvio padrão relativo de 2,8% (n = 10) foi estimado para uma amostra típica de plasma de sangue bovino (76 g L -1 de proteína). RESULTADOS E DISCUSSÃO V1 Liga - desliga (s) Fator de diluição teórico Fator de diluição encontrado Erro (%) 0,5 - 0,5 2 2,3 15 0,5 - 1,0 3 3,1 3 0,5 - 1,5 4 4,2 5 0,5 - 2,0 5 5,3 6 0,5 - 2,5 6 7,4 23 0,5 - 3,0 7 9,4 34 0,5 - 4,5 10 17,3 73 referem-se aos intervalos de tempo para inserção da solução da amostra e da solução do diluente. Tabela 2 - Comparação de resultados obtidos na determinação de proteína total. Tabela 2 - Comparação de resultados obtidos na determinação de proteína total. determinação de proteína total. De 1 a 8 os resultados correspondem a amostras de plasma de sangue eqüino e de 9 a 15 a amostras de plasma de sangue bovino. Os resultados são médias de 3 determinações. Amostra* Sistema Proposto Método Tradicional (Biureto) ---------- g L-1 ----------- 1 92 ± 3 88 ± 2 2 85 ± 2 89 ± 3 3 81 ± 2 82 ± 2 4 72 ± 2 76 ± 1 5 81 ± 2 77 ± 1 6 91 ± 3 89 ± 2 7 89 ± 3 90 ± 3 8 86 ± 2 83 ± 1 9 86 ± 2 86 ± 1 10 76 ± 2 82 ± 3 11 77 ± 2 87 ± 2 12 78 ± 3 83 ± 3 13 70 ± 2 84 ± 2 14 85 ± 2 88 ± 2 15 86 ± 2 87 ± 2 Figura 4 - Registro de rotina para a determinação de proteína totalem plasma de sangue animal. Da esquerda para a direita os sinais identificados pelas letras a; b; c; d; e e f correspondem respectivamente às soluções contendo 0,0; 2,5; 5,0; 10,0; 15,0 e 20,0 g L-1 de albumina. D1 e D2 solução contendo 20,0 g L-1 após diluição de 4 e 5 vezes. Demais sinais: amostras de plasma animal. A seta indica o sentido do papel. proposto, a aquisição de dados foi realizada através de um registrador potenciométrico e parte de um registro de rotina é apresentado na Figura 4. 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Manual de Laboratórios: solo, água, nutrição vegetal, nutrição animal e alimentos. 1. Coleta, Acondicionamento e preparo de amostras. São Carlos: EMBRAPA, 1998. 72p. Recebido em 17.01.01
https://openalex.org/W2968242843	https://europepmc.org/articles/pmc6141628?pdf=render	English	null	Developmental Plasticity and Robustness of a Nematode Mouth-Form Polyphenism	Frontiers in genetics	2,018	cc-by	7,511	Developmental Plasticity and Robustness of a Nematode Mouth-Form Polyphenism Bogdan Sieriebriennikov and Ralf J. Sommer* When complemented with evolutionary and ecological analyses, these studies suggest that plasticity represents a mechanism facilitating adaptive change, increasing diversity and fostering the evolution of novelty. Here, we summarize genetic, molecular and evolutionary studies on developmental plasticity of feeding structures in nematodes, focusing on the model organism Pristionchus paciﬁcus and its relatives. Like its famous cousin Caenorhabditis elegans, P. paciﬁcus reproduces as a self-fertilizing hermaphrodite and can be cultured in the laboratory on E. coli indeﬁnitely with a four-day generation time. However, in contrast to C. elegans, Pristionchus worms show more complex feeding structures in adaptation to their life history. Pristionchus nematodes live in the soil and are reliably found in association with scarab beetles, but only reproduce after the insects’ death. Insect carcasses usually exist only for a short time period and their turnover is partially unpredictable. Strikingly, Pristionchus worms can have two alternative mouth-forms; animals are either stenostomatous (St) with a single tooth resulting in strict bacterial feeding, or alternatively, they are eurystomatous (Eu) with two teeth allowing facultative predation. Laboratory-based studies revealed a regulatory network that controls the irreversible decision of individual worms to adopt the St or Eu form. These studies revealed that a developmental switch controls the mouth-form decision, conﬁrming long-standing theory about the role of switch genes in developmental plasticity. Here, we describe the current understanding of P. paciﬁcus mouth-form regulation. In contrast to plasticity, robustness describes the property of organisms to produce unchanged phenotypes despite environmental perturbations. While largely opposite in principle, the relationship between developmental plasticity and robustness has only rarely been tested in particular study systems. Based on a study of the Hsp90 chaperones in nematodes, we suggest that robustness and plasticity are indeed complementary concepts. Genetic switch networks regulating plasticity require robustness to produce reproducible responses to the multitude of environmental inputs and the phenotypic output requires robustness because the range of possible phenotypic outcomes is constrained. Thus, plasticity and robustness are actually not mutually exclusive, but rather complementary concepts. Developmental Plasticity and Robustness of a Nematode Mouth-Form Polyphenism Bogdan Sieriebriennikov and Ralf J. Sommer* Max Planck Institute for Developmental Biology, Department of Integrative Evolutionary Biology, Tübingen, Germany In the last decade, case studies in plants and animals provided increasing insight into the molecular mechanisms of developmental plasticity. When complemented with evolutionary and ecological analyses, these studies suggest that plasticity represents a mechanism facilitating adaptive change, increasing diversity and fostering the evolution of novelty. Here, we summarize genetic, molecular and evolutionary studies on developmental plasticity of feeding structures in nematodes, focusing on the model organism Pristionchus paciﬁcus and its relatives. Like its famous cousin Caenorhabditis elegans, P. paciﬁcus reproduces as a self-fertilizing hermaphrodite and can be cultured in the laboratory on E. coli indeﬁnitely with a four-day generation time. However, in contrast to C. elegans, Pristionchus worms show more complex feeding structures in adaptation to their life history. Pristionchus nematodes live in the soil and are reliably found in association with scarab beetles, but only reproduce after the insects’ death. Insect carcasses usually exist only for a short time period and their turnover is partially unpredictable. Strikingly, Pristionchus worms can have two alternative mouth-forms; animals are either stenostomatous (St) with a single tooth resulting in strict bacterial feeding, or alternatively, they are eurystomatous (Eu) with two teeth allowing facultative predation. Laboratory-based studies revealed a regulatory network that controls the irreversible decision of individual worms to adopt the St or Eu form. These studies revealed that a developmental switch controls the mouth-form decision, conﬁrming long-standing theory about the role of switch genes in developmental plasticity. Here, we describe the current understanding of P. paciﬁcus mouth-form regulation. In contrast to plasticity, robustness describes the property of organisms to produce unchanged phenotypes despite environmental perturbations. While largely opposite in principle, the relationship between developmental plasticity and robustness has only rarely been tested in particular study systems. Based on a study of the Hsp90 chaperones in nematodes, we suggest that robustness and plasticity are indeed complementary concepts. Genetic switch networks regulating plasticity require robustness to produce reproducible responses to the multitude of environmental inputs and the phenotypic output requires robustness because the range of possible phenotypic outcomes is constrained. Thus, plasticity and robustness are actually not mutually exclusive, but rather complementary concepts. In the last decade, case studies in plants and animals provided increasing insight into the molecular mechanisms of developmental plasticity. REVIEW published: 11 September 2018 doi: 10.3389/fgene.2018.00382 published: 11 September 2018 doi: 10.3389/fgene.2018.00382 Keywords: Pristionchus paciﬁcus, developmental plasticity, robustness, switch genes, Hsp chaperones, Caenorhabditis elegans Edited by: Jean-Michel Gibert, Centre National de la Recherche Scientiﬁque (CNRS), France Reviewed by: Adrienne H. K. Roeder, Cornell University, United States Morris Maduro, University of California, Riverside, United States *Correspondence: Ralf J. Sommer ralf.sommer@tuebingen.mpg.de Specialty section: This article was submitted to Epigenomics and Epigenetics, a section of the journal Frontiers in Genetics Received: 20 July 2018 Accepted: 27 August 2018 Published: 11 September 2018 Citation: Sieriebriennikov B and Sommer RJ (2018) Developmental Plasticity and Robustness of a Nematode Mouth-Form Polyphenism. Front. Genet. 9:382. doi: 10.3389/fgene.2018.00382 Specialty section: This article was submitted to Epigenomics and Epigenetics, a section of the journal Frontiers in Genetics Received: 20 July 2018 Accepted: 27 August 2018 Published: 11 September 2018 INTRODUCTION It is unknown in how many instances the origination of novel traits has followed this pattern, because careful phylogenetic studies of novel traits using the comparative method are scarce. Nonetheless, plasticity may increase evolutionary change through the simultaneous employment of multiple developmental pathways. Since every alternative pathway is only expressed in a fraction of the population or in a limited number of generations, selective constraints are relaxed and mutations can accumulate more quickly, thereby accelerating evolution (West-Eberhard, 2005; Susoy et al., 2015). Together, this makes phenotypic plasticity an important concept in both developmental and evolutionary biology. Another fundamental feature of development is robustness, which is deﬁned as the ability to generate identical phenotypes in the face of environmental perturbations and genetic variation (Wagner, 2005). Apart from the obvious role in maintaining the function of the organism under challenging conditions, robustness is argued to accelerate evolution by enabling accumulation of cryptic variation, which can be subsequently released and become material for selection (Rutherford and Lindquist, 1998; de Visser et al., 2003). Since the deﬁnition of plasticity entails sensitivity to the environment, whereas the deﬁnition of robustness entails insensitivity to it, the two phenomena are often contrasted. And yet, both plasticity and robustness have been suggested to accelerate evolution by releasing selective constraints – plasticity through conditional expression and robustness through concealing mutations from the forces of selection. This enigmatic relationship prompts the question if the two phenomena may, in fact, be complementary rather than opposing. Citation: September 2018 \| Volume 9 \| Article 382 1 Frontiers in Genetics \| www.frontiersin.org Plasticity and Robustness in Pristionchus Sieriebriennikov and Sommer INTRODUCTION existence of a genetic control of plasticity and, therefore, the involvement of developmental switch gene networks and the execution gene network as separate developmental modules has long been debated (Bradshaw, 1965; Schlichting and Pigliucci, 1993; West-Eberhard, 2003). Developmental switches are genes that can change the developmental trajectory (Mather and de Winton, 1941; Ptashne et al., 1980). They do so by activating one or the other set of genes required for an alternative developmental pathway – sets, which we refer to as execution gene networks. For example, feminizing transcription factors Sex-lethal and tra-1 act as developmental switches in Drosophila and Caenorhabditis elegans, respectively, because the level of their activity determines whether the animal will develop as a male or as a female/hermaphrodite. The targets of these switch genes are gene execution networks that generate traits typical of one or the other sex (Hodgkin, 1987; Bell et al., 1988). In this example, the developmental choice is mandatory and determined chromosomally, but studies on the genetic regulation of plasticity also conﬁrmed the long-standing prediction about the involvement of switch genes in plastic development (for recent comprehensive reviews, see Fielenbach and Antebi, 2008; Projecto-Garcia et al., 2017; Sommer et al., 2017) (Figure 1A). Therefore, we suggest that the question of robustness of plastic traits can be addressed at two levels and that indeed robustness and plasticity are complementary concepts. First, genetic switch networks regulating plasticity require robustness to produce reproducible responses to the multitude of environmental inputs. Second, the phenotypic output requires robustness because the range of possible phenotypic outcomes is constrained. In the following we concentrate on a case study in a nematode, which explores the interplay between plasticity and robustness. Phenotypic plasticity is a feature of development whereby identical genotypes generate diﬀerent phenotypes upon perception of environmental input (West-Eberhard, 2003). Examples of plasticity are ubiquitous and in extreme cases the alternative phenotypes produced are discrete, such as the various caste systems in social insects (Abouheif and Wray, 2002). However, the evolutionary signiﬁcance of phenotypic plasticity is still widely debated. One view is that plasticity hampers evolution by enabling adaptation without genetic assimilation (Price et al., 2003). Conversely, the so called “ﬂexible stem hypothesis” suggests a possibility that a phase of plasticity may be an obligatory step in the evolution of novel traits, whereby their expression remains conditional in the beginning before it becomes fully integrated into the development and ﬁxed (Gibert, 2017). Frontiers in Genetics \| www.frontiersin.org September 2018 \| Volume 9 \| Article 382 GENETICS OF PLASTICITY: CONTINUOUS VS. DISCRETE PHENOTYPES In theory, the relationship between plasticity and robustness can be interrogated in any organism, however, some experimental systems possess features which greatly facilitate such studies. These features are, ﬁrst, availability of isogenic lines, which simplify the genetics of the study system, and second, discreteness of alternative phenotypes. Speciﬁcally, studying genetically uniform individuals oﬀers the possibility to unambiguously separate plasticity from polymorphisms generated by diﬀerent genetic variants. As for the ability to generate discrete, as opposed to continuous, alternative phenotypes, such an ability potentially allows a sharper contrast between a constrained phenotypic distribution and conditions when developmental buﬀering is impaired and atypical phenotypes are produced. Much of the discussion on the signiﬁcance of plasticity and robustness for evolutionary change is based on theoretical arguments. In contrast, few experimental case studies address the molecular, genetic, and developmental mechanisms underlying these phenomena. Even more importantly, only a few experimental studies have investigated the potential crosstalk between plasticity and robustness simultaneously in the same study system. This is surprising as arguably knowledge about the mechanisms of plastic development can help address the interplay between plasticity and robustness in a more nuanced way than simply contrasting the two phenomena. The More importantly, the hypothesis that genetic switch networks and phenotype execution networks are separate developmental modules whose robustness is provided by diﬀerent mechanisms can only be explicitly tested in organisms in which impairment of the binary switches can be disentangled from expansion or displacement of the phenotypic distribution. Although a series of developmental switches is thought to underlie both September 2018 \| Volume 9 \| Article 382 2 Plasticity and Robustness in Pristionchus Sieriebriennikov and Sommer continuous and discontinuous distributions of plastic phenotypes (West-Eberhard, 2003), phenotypic changes resulting from the manipulation of the switch and of the structural genes can be interpreted in diﬀerent ways depending on the distribution of phenotypes. In the case of continuous traits, inactivation of genes channeling and integrating environmental inputs (the switch network) can either constrain the phenotypic distribution through a decrease in sensitivity to an inducing signal, or make it more variable as a consequence of improper integration of various environmental signals (Figure 1B). At the same time, tampering with the gene network executing the phenotype will also increase the variance of the phenotypic distribution (Figure 1B). GENETICS OF PLASTICITY: CONTINUOUS VS. DISCRETE PHENOTYPES Thus, developmental perturbations at the same level can potentially lead to diﬀerent phenotypic outcomes, while manipulating diﬀerent gene networks can lead to similar change. Together, this obscures the potential interplay between plasticity and robustness when continuously plastic traits are studied. FIGURE 1 \| (A) Conceptual mechanism of the genetic regulation of plast development. Environmental inputs are processed and integrated by a network of genes, referred to as switch genes. The switch network takes decision to activate one of the alternative developmental programs and passes the signal down to a genetic network that executes the selected phenotype. (B,C) Hypothetical scenarios of change in the reaction norm continuously (B) or discontinuously (C) plastic trait in the conditions when switch network or the phenotype execution network is impaired. The orig distribution of phenotypes is shown in black in the foreground or gray in t background, altered distribution resulting from impairment of the switch network is shown in red and altered distribution resulting from incorrect ti f th h t i h i bl FIGURE 1 \| (A) Conceptual mechanism of the genetic regulation of plasti development. Environmental inputs are processed and integrated by a network of genes, referred to as switch genes. The switch network takes decision to activate one of the alternative developmental programs and passes the signal down to a genetic network that executes the selected phenotype. (B,C) Hypothetical scenarios of change in the reaction norm o continuously (B) or discontinuously (C) plastic trait in the conditions when switch network or the phenotype execution network is impaired. The origi distribution of phenotypes is shown in black in the foreground or gray in th background, altered distribution resulting from impairment of the switch network is shown in red and altered distribution resulting from incorrect ti f th h t i h i bl In contrast, manipulation of the switch and the execution networks will change the phenotypic distribution of discrete traits in a diﬀerent manner. Interfering with the switch network will only aﬀect the distribution of individual phenotypes between the discrete clusters, whereby the most extreme case would be the absence of individuals from some clusters (corresponding, e.g., to a loss of a morph). However, the distribution of phenotypes within the clusters is expected to be constant (Figure 1C). GENETICS OF PLASTICITY: CONTINUOUS VS. DISCRETE PHENOTYPES In contrast, loss of robustness of the gene network executing the phenotype is expected to aﬀect the phenotypic distribution within the clusters (Figure 1C). Thus, only using organisms exhibiting discrete plasticity as a study model allows falsiﬁcation of the hypothesis that plastic traits require robustness of both the switch and the execution gene network. THE STUDY SYSTEM: Pristionchus paciﬁcus MOUTH-FORM PLASTICITY On the right, overlay of the DIC image and an image of ﬂuorescein-stained cuticle at the base of t ich includes teeth. Arrows point at the tips of teeth. (D) Current model of the regulation of mouth-form plasticity in P. paciﬁcus. The genes shown a h network, i.e., mutations in these genes only change the frequencies of alternative phenotypes in the population. (E,F) Phenotypic effects caused Hsp90 heat shock proteins, which are known to provide robustness to phenotype execution networks. In these conditions, both morphs are still e morphologies are abnormal. (E) PCA ordination of sets of landmarks representing control individuals and individuals exposed to heat stress and cicol, a pharmacological inhibitor of Hsp90. (F) Morphological disparity within different groups shown in the PCA ordination in panel E. Error bars ot signiﬁcant (P-value > 0.05); ∗∗P-value < 0.01; ∗∗∗P-value < 0.001. Panels E and F reproduced from Sieriebriennikov et al. (2017). FIGURE 2 \| (A) The eurystomatous morph of P. paciﬁcus devouring a larva of C. elegans. (B,C) The mouth of the eurystomatous (B) and of the stenostomatous (C) morph. On the left, differential interference contrast (DIC) image. On the right, overlay of the DIC image and an image of ﬂuorescein-stained cuticle at the base of the buccal cavity, which includes teeth. Arrows point at the tips of teeth. (D) Current model of the regulation of mouth-form plasticity in P. paciﬁcus. The genes shown are part of the switch network, i.e., mutations in these genes only change the frequencies of alternative phenotypes in the population. (E,F) Phenotypic effects caused by impairment of Hsp90 heat shock proteins, which are known to provide robustness to phenotype execution networks. In these conditions, both morphs are still produced but the morphologies are abnormal. (E) PCA ordination of sets of landmarks representing control individuals and individuals exposed to heat stress and treatment by radicicol, a pharmacological inhibitor of Hsp90. (F) Morphological disparity within different groups shown in the PCA ordination in panel E. Error bars show SD. n.s., not signiﬁcant (P-value > 0.05); ∗∗P-value < 0.01; ∗∗∗P-value < 0.001. Panels E and F reproduced from Sieriebriennikov et al. (2017). FIGURE 2 \| (A) The eurystomatous morph of P. paciﬁcus devouring a larva of C. elegans. (B,C) The mouth of the eurystomatous (B) and of the stenostomatous (C) morph. On the left, differential interference contrast (DIC) image. THE STUDY SYSTEM: Pristionchus paciﬁcus MOUTH-FORM PLASTICITY The nematode Pristionchus paciﬁcus is a dimorphic species that belongs to the same order as the classical model C. elegans and shares its amenability to genetic manipulation, as well as the hermaphroditic mode of reproduction, which enables creation of isogenic lines (Sommer and McGaughran, 2013). Depending on the culture conditions, genetically identical individuals of P. paciﬁcus can develop into two morphs – eurystomatous (Eu) (literally “wide-mouthed”) and stenostomatous (St) (“narrow- mouthed”) morphs. Eu animals possess a wide buccal cavity with two hooked teeth, which worms can use to kill other nematodes (Figures 2A,B). In contrast, the buccal cavity in St animals is narrow, the dorsal tooth is ﬂint-shaped and the right ventrosublateral tooth is reduced to a cuticular ridge with a minute denticle (Figure 2C), which precludes killing, leaving such animals as obligatory microbial grazers (Bento et al., 2010; Wilecki et al., 2015). The decision on mouth-form development is taken during larval development and is irreversible in the adult stage (Serobyan et al., 2013). The developmental decision is inﬂuenced by the presence of pheromones, diet composition and the state of the culture medium (solid vs. liquid) (Bose et al., 2012; Sanghvi et al., 2016; Werner et al., 2017) (Figure 2D). FIGURE 1 \| (A) Conceptual mechanism of the genetic regulation of plastic development. Environmental inputs are processed and integrated by a network of genes, referred to as switch genes. The switch network takes a decision to activate one of the alternative developmental programs and passes the signal down to a genetic network that executes the selected phenotype. (B,C) Hypothetical scenarios of change in the reaction norm of a continuously (B) or discontinuously (C) plastic trait in the conditions when the switch network or the phenotype execution network is impaired. The original distribution of phenotypes is shown in black in the foreground or gray in the background, altered distribution resulting from impairment of the switch network is shown in red and altered distribution resulting from incorrect execution of the phenotype is shown in blue. September 2018 \| Volume 9 \| Article 382 Frontiers in Genetics \| www.frontiersin.org 3 Plasticity and Robustness in Pristionchus Sieriebriennikov and Sommer The eurystomatous morph of P. paciﬁcus devouring a larva of C. elegans. (B,C) The mouth of the eurystomatous (B) and of the stenostomatous ( ft, differential interference contrast (DIC) image. THE STUDY SYSTEM: Pristionchus paciﬁcus MOUTH-FORM PLASTICITY On the right, overlay of the DIC image and an image of ﬂuorescein-stained cuticle at the base of the buccal cavity, which includes teeth. Arrows point at the tips of teeth. (D) Current model of the regulation of mouth-form plasticity in P. paciﬁcus. The genes shown are part of the switch network, i.e., mutations in these genes only change the frequencies of alternative phenotypes in the population. (E,F) Phenotypic effects caused by impairment of Hsp90 heat shock proteins, which are known to provide robustness to phenotype execution networks. In these conditions, both morphs are still produced but the morphologies are abnormal. (E) PCA ordination of sets of landmarks representing control individuals and individuals exposed to heat stress and treatment by radicicol, a pharmacological inhibitor of Hsp90. (F) Morphological disparity within different groups shown in the PCA ordination in panel E. Error bars show SD. n.s., not signiﬁcant (P-value > 0.05); ∗∗P-value < 0.01; ∗∗∗P-value < 0.001. Panels E and F reproduced from Sieriebriennikov et al. (2017). September 2018 \| Volume 9 \| Article 382 Frontiers in Genetics \| www.frontiersin.org Plasticity and Robustness in Pristionchus Sieriebriennikov and Sommer Importantly, changing environmental conditions only alters the ratio between the phenotypes in populations, whereas intermorphs are extremely scarce. Together, these conditions make P. paciﬁcus mouth-form plasticity an ideal study system to investigate the genetics, molecular biology and epigenetics of developmental plasticity and, building on the availability of such mechanistic insight, the potential relationship between plasticity and robustness. during all larval instars after hatching (Serobyan et al., 2013). Finally, development is inherently noisy and the precision of developmental decision-making processes, such as morphogen gradients, is generally limited (Gregor et al., 2007). Therefore, the ability to generate reproducible responses to multiple and potentially contradicting environmental inputs, while staying insensitive to noise, is crucial for developmental switches. Such an ability is believed to be an intrinsic property of the architecture of gene regulatory networks (Masel and Siegal, 2009), with known examples in both vertebrates and invertebrates. For instance, the ecdysone receptor EcR in Drosophila positively regulates its own transcription and small ﬂuctuations in ecdysone level or spontaneous transcriptional bursts could lead to a premature self-amplifying response (Herranz and Cohen, 2010). This is prevented by a negative feedback loop between EcR and microRNA miR-14. THE STUDY SYSTEM: Pristionchus paciﬁcus MOUTH-FORM PLASTICITY When the level of ecdysone is low, miR- 14 represses the expression of the EcR gene, which ensures that EcR does not self-activate and remains poised for response to the elevated level of the hormone (Varghese and Cohen, 2007). An additional example is the circuit regulating neuronal subtype speciﬁcation in mice in response to Sonic Hedgehog signaling, dissection of which revealed a network that consists of the transcription factors Olig2, Nkx2.2, and Pax6 connected with feedback and feedforward loops (Balaskas et al., 2012). Experiments with knockout lines and in silico modeling showed that negative feedback loops in the network provide robustness to small signal ﬂuctuations, such that the network can respond to the morphogen by generating a highly reproducible pattern despite developmental noise (Gregor et al., 2007). Recent studies on the genetics of mouth-form plasticity in P. paciﬁcus began to elucidate how the developmental decision is controlled. Forward and reverse genetic experiments implicated a locus on the X chromosome in switching between phenotypes (Figure 2D). This locus contains three functionally relevant genes, which are expressed in sensory and interneurons and which aﬀect the phenotype ratio in the opposite manner. Speciﬁcally, the gene eud-1 encodes an extracellular sulfatase and promotes the Eu morph, whereas nag-1 and its paralog nag-2 encode α-N-acetylgalactosaminidases, which additively promote the St morph (Ragsdale et al., 2013; Sieriebriennikov et al., 2018). Furthermore, the chromatin remodelers MBD-2, a methyl binding protein, and LSY-12, a histone acetyltransferase regulate eud-1 levels (Serobyan et al., 2016; Serobyan and Sommer, 2017). The downstream transcription factor in the switch network is NHR-40, and the cytosolic sulfotransferase SULT-1 presumably acts downstream of nhr-40 (Kieninger et al., 2016; Namdeo et al., 2018). Similar to manipulating environmental conditions, the manipulation of genes in this switch network only aﬀects the ratio between the alternative morphs produced, but the morphologies of individual morphs remain intact. For example, eud-1 mutant animals are all-St, whereas nag-1 nag-2 double mutants are all-Eu. It is important to note that the current information on the genetic network is likely to be incomplete for several potential reasons. For example, genes that are part of the execution network might have essential functions earlier in development and as such, would go unnoticed in genetic screens as their phenotype would be lethal. Nonetheless, this genetic network for P. THE STUDY SYSTEM: Pristionchus paciﬁcus MOUTH-FORM PLASTICITY paciﬁcus mouth-form plasticity provides a genetic and molecular platform for studying environmental inﬂuences, the evolution of plasticity and its relationship with robustness. In P. paciﬁcus, a phenomenon dependent on the robustness of the switch network during the regulation of mouth-form plasticity is the stochasticity of the phenotypic output. Although the phenotypic implementation of plasticity in this species is binary under normal circumstances, and as such intermediate morphs are extremely rare, there is apparent stochasticity as to which morph is ﬁnally adopted by an individual. The proportion of Eu morphs on agar plates under standard laboratory conditions ﬂuctuates between 70 and 98%, even though all animals are genetically identical and grow in the same environment (Ragsdale et al., 2013; Werner et al., 2017). This pattern is consistent with the theoretical expectation of the phenotypic distribution produced by erratic action of the switch network (Figure 1C). September 2018 \| Volume 9 \| Article 382 Frontiers in Genetics \| www.frontiersin.org ROBUSTNESS OF DEVELOPMENTAL SWITCHES It is possible that such stochasticity is adaptive and represents a bet-hedging strategy – as the developmental decision is irreversible, it may be advantageous to always have some individuals of the underrepresented morph in the population in case the environmental conditions change more rapidly than a new generation can grow and develop suitable phenotypes (Losick and Desplan, 2008; Susoy and Sommer, 2016). In general, bet-hedging strategies are well-known in microbes that often face unpredictable environments (Veening et al., 2008). While the exact mechanism of how such stochasticity is generated is unknown, the gene regulatory network regulating plasticity may be susceptible to noise or it may even have a special mechanism to convert noise into phenotypic response. For example, cells forming sensory organ precursors in Drosophila are randomly We suggested that plastic traits require the robustness of the switch network and of the network producing the phenotype (Figure 1A). The switch network integrates all the relevant environmental signals and takes the developmental decision, during which it faces several challenges. First, multiple contradictory environmental inputs can be perceived simultaneously. For example, the pheromone dasc#1 induces the Eu morph in P. paciﬁcus, whereas consumption of the yeast Cryptococcus albidus represses it (Bose et al., 2012; Sanghvi et al., 2016). Additionally, the developmental decision is often not taken instantaneously. Instead, the environmental signals accumulate over a prolonged time period, such as in P. paciﬁcus, in which crowding has inﬂuence on the mouth-form ratio September 2018 \| Volume 9 \| Article 382 Frontiers in Genetics \| www.frontiersin.org 5 Plasticity and Robustness in Pristionchus Sieriebriennikov and Sommer networks, followed by large-scale mutant screens, demonstrated that functional knockdowns of 5% of all genes in S. cerevisiae decrease phenotypic robustness (Bergman and Siegal, 2003; Levy and Siegal, 2008; Bauer et al., 2015). Studies of developmental stability in recombinant inbred lines in Arabidopsis thaliana also unraveled multiple quantitative trait loci (QTL) associated with robustness against genetic and environmental variation (Hall et al., 2007; Fu et al., 2009). Nevertheless, heat shock proteins remain the best studied capacitors of morphological variation to date. Importantly, the emergence of aberrant phenotypes after developmental buﬀering by Hsp90 is alleviated was observed in laboratory populations of Drosophila and Arabidopsis, which are nearly isogenic (Rutherford and Lindquist, 1998; Queitsch et al., 2002). ROBUSTNESS OF DEVELOPMENTAL SWITCHES This indicates that not only cryptic genetic variation, but also environmental micro-heterogeneity and developmental noise are likely sources of stochastic variation buﬀered by heat shock proteins. selected from a ﬁeld of equipotent cells due to noisy expression of the transcription factor Senseless, followed by lateral inhibition of the neighboring cells (Jafar-Nejad et al., 2003). In such cases, sensitivity to developmental noise may be advantageous for the organism. The alternative view is that the response of the gene network is robust to noise and the apparent stochasticity in the phenotypic outcome results from environmental micro- heterogeneity. Indeed, there are known examples of seemingly stochastic plastic outputs when the environmental conditions approach the so called “neutral point,” i.e., a set of conditions near the threshold zone of responsiveness (Nijhout, 1975; Emlen, 1996; West-Eberhard, 2003). In such a case, reactiveness to the environment is still robust, but only a fraction of organisms happens to experience the amount of combined inducing signal above the threshold value. However, given our current knowledge it is diﬃcult to disentangle between the two scenarios. Therefore, additional studies are needed to demonstrate if mechanisms converting developmental noise to stochastic phenotypic output exist in P. paciﬁcus. Nevertheless, the seemingly stochastic action of the switch network does not lead to the production of intermediate morphologies, corroborating our expectation that studying discontinuous plasticity allows to disentangle the robustness of the switch network and the robustness of the phenotype. The proposed independence of the phenotypic buﬀering from the action of the switch suggests that the inhibition of the Hsp90 machinery in P. paciﬁcus should lead to a change in the distribution of mouth morphologies, whereby the Eu and St morphs could nevertheless still be distinguishable even if distorted (Figure 1C). To visualize the extent of morphological diﬀerences between individuals, morphologies can be quantiﬁed using geometric morphometric analysis. As expected, in P. paciﬁcus and other dimorphic species of the same nematode family, the Eu and St morphs form separate clusters in the morphospace with no overlap (Susoy et al., 2015). In accordance with the prediction, manipulation of Hsp90 activity through life-long exposure to elevated temperature, pharmacological inhibition or knockout of the Hsp90-encoding gene daf-21 increased the morphological variation of the Eu and St morphs in P. paciﬁcus (Sieriebriennikov et al., 2017) (Figures 2E,F). ROBUSTNESS OF DEVELOPMENTAL SWITCHES Speciﬁcally, rearing animals at the highest sublethal temperature displaced the distribution of the mouth morphologies in the morphospace, an eﬀect that was observable in both morphs. In contrast, applying the pharmacological inhibitor of Hsp90 function induced expansion of morphology distributions without any evident shift. Finally, daf-21/Hsp90 knockout generated using CRISPR/Cas9 resulted in a combined eﬀect, whereby the distribution of morphologies was displaced in the morphospace and morphological disparity was increased (Figures 2E,F). These observations demonstrate that the mechanism that provides developmental buﬀering against genetic and environmental perturbations acts to canalize the development of the discrete morphs in P. paciﬁcus. Importantly, although some treated animals exhibited intermediate morphologies, most individuals could still be classiﬁed into Eu and St. Additionally, introduction of the daf-21/Hsp90 mutation in the Eu-constitutive nhr-40 mutant line did not lead to the appearance of St animals, but only increased the morphological variation of the Eu morphs (Sieriebriennikov et al., 2017). This ﬁnding strongly supports the hypothesis that the two types of robustness of plastic traits described here – robustness of environmental responsiveness and robustness of phenotypic output – are provided by at least partially non-overlapping mechanisms. Frontiers in Genetics \| www.frontiersin.org REFERENCES Bradshaw, A. D. (1965). “Evolutionary signiﬁcance of phenotypic plasticity in plants,” in Advances in Genetics, eds E. W. Caspari and J. M. Thoday (Cambridge, MA: Academic Press), 115–155. Abouheif, E., and Wray, G. A. (2002). Evolution of the gene network underlying wing polyphenism in ants. 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A naive idea that chaperones maintain normal functioning of cells because they can refold proteins destabilized by weakly deleterious mutations or by environmental inﬂuences prompted a wave of experiments in various organisms, which showed that cryptic variation is indeed uncovered once the Hsp90 function is compromised (Rutherford and Lindquist, 1998; Queitsch et al., 2002; Rohner et al., 2013). Interestingly, subsequent studies provided evidence that the role of Hsp90 proteins may be more complex than simply exhibiting chaperone activity. Namely, they were implicated in the regulation of piRNA production, which in turn may silence deleterious gene variants (Gangaraju et al., 2011). Further research in yeast, animals and plants demonstrated that complementary mechanisms also exist. For example, the prion form [PSI+] of the translation release factor Sup35 in Saccharomyces yeasts allows stop codon readthrough and thus releases the cryptic genetic variation accumulated in the 3′ untranslated regions of genes (Masel and Griswold, 2009). In C. elegans, the remarkable reproducibility of the division pattern of seam cells (epidermal stem cells) is provided by the action of a basic helix-loop-helix (bHLH) transcription factor LIN- 22 (Katsanos et al., 2017). In Arabidposis, robust timing and positioning of organs on the stem is generated by a common action of two hormone-based ﬁelds (Besnard et al., 2014). In addition to studies on single genes, simulations of complex gene September 2018 \| Volume 9 \| Article 382 Frontiers in Genetics \| www.frontiersin.org 6 Plasticity and Robustness in Pristionchus Sieriebriennikov and Sommer ACKNOWLEDGMENTS We would like to thank Dr. Michael Werner and Ms. Sara Wighard for their comments on the manuscript. We would like to thank Dr. Michael Werner and Ms. Sara Wighard for their comments on the manuscript. FUNDING The authors were ﬁnancially supported by the Max Planck Society. The authors were ﬁnancially supported by the Max Planck Society. BS and RS conceived and wrote the manuscript. BS and RS conceived and wrote the manuscript. CONCLUSION of morphologies of both morphs. Yet, both morphs were still produced even though their ratio was somewhat shifted. Together, these observations corroborate our hypothesis that robustness of the switch and robustness of the execution network are provided by at least partially non-overlapping mechanisms. In summary, the P. paciﬁcus mouth-form polyphenism allows two major conclusions with regard to the relationship of plasticity and robustness. First, we propose that robustness and plasticity are complementary rather than opposing phenomena. Second, we argue that knowledge about the mechanisms of plastic development enables formulation of testable hypotheses about the interplay between plasticity and robustness. Speciﬁcally, separation between the developmental switch gene network and the gene network executing the selected phenotype (Figure 1A) strongly suggests that plastic traits require robustness at two levels. Firstly, the switch network must generate a robust response to the multitude of environmental inputs despite developmental noise and other stochastic perturbations. Secondly, the execution network must generate a robust phenotypic outcome within a constrained range of possible phenotypes. While our knowledge of plastic development of the feeding structures in P. paciﬁcus is far from being complete, the approaches discussed here pave the way to reconcile plasticity and robustness. Both phenomena are suggested to promote evolution and more mechanistic studies are necessary to elucidate the genetic and physical basis of their interaction. Therefore, we would like to encourage similar studies in other models, which will verify our conceptualizations and provide new insight into addressing the relationship between plasticity and robustness, and their role in evolution. To demonstrate how these questions can be addressed, we use an example of a self-fertilizing nematode that exhibits a discrete plasticity of feeding structures. In P. paciﬁcus, manipulation of culture conditions or introduction of mutations in the switch network only inﬂuences the ratio between the morphs and not the alternative morphologies themselves, supporting the long-standing prediction that the switch genetic network is developmentally independent from the network involved in building the morphologies. 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https://openalex.org/W3195257556	https://escholarship.org/content/qt2pb3b5w5/qt2pb3b5w5.pdf?t=rm98js	English	null	The Inclusion of Women in Global Oncology Drug Trials Over the Past 20 Years	JAMA oncology	2,021	cc-by	1,909	UCSF UC San Francisco Previously Published Works Title The Inclusion of Women in Global Oncology Drug Trials Over the Past 20 Years Permalink https://escholarship.org/uc/item/2pb3b5w5 Journal JAMA Oncology, 7(10) ISSN 2374-2437 Authors Jenei, Kristina Meyers, Daniel E Prasad, Vinay Publication Date 2021-10-01 DOI 10.1001/jamaoncol.2021.3686 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UCSF UC San Francisco Previously Published Works Title The Inclusion of Women in Global Oncology Drug Trials Over the Past 20 Years Permalink https://escholarship.org/uc/item/2pb3b5w5 Journal JAMA Oncology, 7(10) ISSN 2374-2437 Authors Jenei, Kristina Meyers, Daniel E Prasad, Vinay Publication Date 2021-10-01 DOI 10.1001/jamaoncol.2021.3686 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UCSF UC San Francisco Previously Published Wo Title The Inclusion of Women in Global Oncology Drug Trials Ove Permalink https://escholarship.org/uc/item/2pb3b5w5 Journal JAMA Oncology, 7(10) ISSN 2374-2437 Authors Jenei, Kristina Meyers, Daniel E Prasad, Vinay Publication Date 2021-10-01 DOI 10.1001/jamaoncol.2021.3686 Copyright Information This work is made available under the terms of a Creative C availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UCSF UC San Francisco Previously Published Works Title The Inclusion of Women in Global Oncology Drug Trials Over the Past 20 Ye Permalink https://escholarship.org/uc/item/2pb3b5w5 Journal JAMA Oncology, 7(10) ISSN 2374-2437 Authors Jenei, Kristina Meyers, Daniel E Prasad, Vinay Publication Date 2021-10-01 DOI 10.1001/jamaoncol.2021.3686 Copyright Information This work is made available under the terms of a Creative Commons Attribu availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed Letters identified 6 common solid tumor types for women (lung, colon, thyroid, melanoma, kidney, and pancreas). Powered by the California Digital Library University of California eScholarship.org © 2021 American Medical Association. All rights reserved. The Inclusion of Women in Global Oncology Drug Trials Over the Past 20 Years Methods\|Weconductedasystematicsearchofcancerdrugtrials registered on ClinicalTrials.gov between 2000 and 2020 for 6 common cancers for women (excluding breast and uterus). The investigation of publicly available trial protocols was ex- empt from institutional review board approval. We included completed drug trials with results for adults (≥18 years). Be- havioral, device, procedure, and radiotherapy trials were ex- cluded (eFigure in the Supplement). Pearson χ2 test of inde- pendencewasusedtoevaluatetheassociationbetweenfemale and male enrollment and phase (1, 2, 3), tumor type, and fund- ing source. P values used 2-tailed tests, with P < .05 being sig- nificant. We explored changes in sex-specific enrollment pat- terns over 2 decades by stratifying trials as 2000 to 2010 and 2011 to 2020. Cancer incidence rates were calculated by di- viding the number of new male and female cases by the total numberofnewcasespertumortype(2020).6Dataanalysiswas performedusingRstatisticalsoftware,version1.1.463(RFoun- dation). Thirty years have passed since the enactment of the National Institutes of Health (NIH) Revitalization Act, which encour- aged NIH-funded investigators to include adequate numbers of women in clinical studies.1 Since then, there have been important steps taken to en- sure better representation of women and racial and ethnic minority groups in biomedical trials.2 However, lack of representation remains problematic in oncology. Previous research suggests that women repre- sent approximately 30% to 40% of participants in trials lead- ing to drug approvals in the US.3,4 Supplemental content Supplemental content Downloaded From: https://jamanetwork.com/ by a UCSF LIBRARY User on 12/02/2022 Accepted for Publication: June 21, 2021. Accepted for Publication: June 21, 2021. Accepted for Publication: June 21, 2021. Published Online: August 26, 2021. doi:10.1001/jamaoncol.2021.3686 Corresponding Author: Kristina Jenei, BSN, MSc, School of Population and Public Health, The University of British Columbia, 2206 East Mall, Vancouver, BC V6T 1Z3, Canada (kjenei@mail.ubc.ca). Corresponding Author: Kristina Jenei, BSN, MSc, School of Population and Public Health, The University of British Columbia, 2206 East Mall, Vancouver, BC V6T 1Z3, Canada (kjenei@mail.ubc.ca). Author Contributions: Ms Jenei had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Author Contributions: Ms Jenei had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Comparing 2000 to 2010 with 2011 to 2020, we observed a marginal increase in female participation, from 40% to 42%, respectively (P < .001). We evaluated the association be- tween industry and NIH-sponsored studies in the US (n = 358) and found that NIH-funded trials enrolled a higher propor- tion of women (48%) compared with industry trials (41%) (P < .001). Concept and design: All authors. Concept and design: All authors. Concept and design: All authors. Acquisition, analysis, or interpretation of data: Jenei, Meyers. Drafting of the manuscript: Jenei. Critical revision of the manuscript for important intellectual content: All authors. Statistical analysis: Jenei. Administrative, technical, or material support: Meyers. Supervision: Prasad. Acquisition, analysis, or interpretation of data: Jenei, Meyers. Critical revision of the manuscript for important intellectual content: All authors. Statistical analysis: Jenei. Administrative, technical, or material support: Meyers. Conflict of Interest Disclosures: Dr Prasad reported receiving research funding from Arnold Ventures during the conduct of the study; and royalties from Johns Hopkins Press, Medscape, and MedPage; consulting fees from UnitedHealthcare; speaking fees from New Century Health and Evicore; and personal fees from Patreon Plenary Session (podcast has Patreon backers) outside the submitted work. No other disclosures were reported. Discussion\|Itisessentialthatwomenbeenrolledinclinicaltrials in numbers that, at least, mirror the distribution of the dis- ease in the population so that potential biological differences can be understood. Our analysis, although limited to 6 tumor types, suggests that sex differences persist in clinical trials. In 2020, women represented 77% of newly diagnosed thyroid cancer cases worldwide6 yet comprised only 51% of partici- pants in trials investigating thyroid drugs (Figure). Accepted for Publication: June 21, 2021. Similarly, women represented 48% of global colon cancer cases6 yet ac- countedforonly33%oftrialparticipantsforcoloncancerthera- peutics (Figure). 1. NIH Revitalization Act of 1993 Public Law 103-43. In: Mastroianni AC, Faden R, Federman D, eds. Women and Health Research: Ethical and Legal Issues of Including Women in Clinical Studies: Volume I. National Academies Press; 1994:appendix B. Accessed May 5, 2021. https://www.ncbi.nlm.nih.gov/books/ NBK236531/ 2. Mazure CM, Jones DP. Twenty years and still counting: including women as participants and studying sex and gender in biomedical research. BMC Womens Health. 2015;15(1):94. doi:10.1186/s12905-015-0251-9 Higher enrollment of women in NIH-funded studies (48%) compared with industry studies (41%) warrants further explo- ration, but the stagnant rates of women in trials suggest that regulatory initiatives2 over the past 2 decades may be insuf- ficient. Our analysis, although limited to sponsor- and manu- facturer-disclosed information, demonstrates that persistent inequities remain in the recruitment of female participants in trials investigating new therapeutics for certain tumor types in oncology. 3. Murthy VH, Krumholz HM, Gross CP. Participation in cancer clinical trials: race-, sex-, and age-based disparities. JAMA. 2004;291(22):2720-2726. doi:10.1001/jama.291.22.2720 4. Duma N, Vera Aguilera J, Paludo J, et al. Representation of minorities and women in oncology clinical trials: review of the past 14 years. J Oncol Pract. 2018;14(1):e1-e10. doi:10.1200/JOP.2017.025288 4. Duma N, Vera Aguilera J, Paludo J, et al. Representation of minorities and women in oncology clinical trials: review of the past 14 years. J Oncol Pract. 2018;14(1):e1-e10. doi:10.1200/JOP.2017.025288 5. National Institutes of Health Office of Research on Women’s Health. Strategic plan. moving into the future with new dimensions and strategies: a vision for 2020 for women’s health research. Accessed July 22, 2021. https://orwh.od.nih. gov/sites/orwh/files/docs/ORWH_StrategicPlan2020_Vol1.pdf 6. Global Cancer Observatory. Accessed June 16, 2021. https://gco.iarc.fr/ Kristina Jenei, BSN, MSc Daniel E. Meyers, MD, MSc Vinay Prasad, MD, MPH Supplemental content In 2010, the NIH Office of Research on Women’s Health5 set forth a vision to advance the understanding of sex- specific disease differences by 2020. As clinical trials become international in scope, we sought to evaluate the global move- ment toward this vision in oncology. In this cohort study, we reviewed enrollment patterns of completed cancer drug trials over the past 20 years to compare sex-specific trial participa- tion to current cancer incidence rates. Using data from the International Agency for Research on Cancer (IARC),6 we Results \| We identified 505 oncology clinical trials that met the eligibility criteria between 2000 and 2020. Of the total 182 416 participants, 73 103 (40%) were women, while 109 313 (60%) Table. Comparison Between Sex-Specific Enrollment and Clinical Trial Characteristics Characteristic Sex, No. (%) P valuea Female Male Total enrolled 73 103 (40) 109 313 (60) <.001 Trial phase 1 3034 (48) 3322 (52) .001 2 18 838 (43) 24 508 (57) <.001 3 40 139 (38) 66 611 (62) <.001 Year 2000-2010 23 350 (40) 34 745 (60) <.001 2011-2020 49 753 (42) 68 022 (58) Tumor type Lung 40 829 (41) 57 979 (59) <.001 Colon 7600 (33) 15 266 (67) <.001 Thyroid 904 (51) 875 (49) .50 Melanoma 11 317 (42) 15 529 (58) <.001 Kidney 6586 (33) 13 127 (67) <.001 Pancreas 5867 (47) 6537 (53) <.001 Sites US 49 911 (40) 75 755(60) <.001 Canada 29 603 (39) 45 372 (61) <.001 China 23 456 (41) 33 645 (56) <.001 United Kingdom 28 472 (39) 44 478 (61) <.001 Australia 28 505 (39) 44 332 (61) <.001 Funding (US) Industry 41 391 (41) 60 473 (59) <.001 NIH 6828 (48) 7285 (52) Abbreviation: NIH, National Institutes of Health. a P values from Pearson χ2 test of independence. jamaoncology.com (Reprinted) JAMA Oncology October 2021 Volume 7, Number 10 15 Table. Comparison Between Sex-Specific Enrollment and Clinical Trial Characteristics Downloaded From: https://jamanetwork.com/ by a UCSF LIBRARY User on 12/02/2022 Downloaded From: https://jamanetwork.com/ by a UCSF LIBRARY User on 12/02/2022 Letters Figure. Supplemental content Composition of Trial Enrollment and Incidence by Sex per Tumor Type 100 80 60 40 20 0 Incidence, %a Lung Colon Thyroid Melanoma Kidney Pancreas 60 100 80 40 20 0 Clinical trial participants by sex, % Female diagnoses Female enrollment Male diagnoses Male enrollment a New cases by sex per International Agency for Research on Cancer 2020 data.6 100 80 60 40 20 0 Incidence, %a Lung Colon Thyroid Melanoma Kidney Pancreas 60 100 80 40 20 0 Clinical trial participants by sex, % Female diagnoses Female enrollment Male diagnoses Male enrollment a New cases by sex per International Agency for Research on Cancer 2020 data.6 a New cases by sex per International Agency for Research on Cancer 2020 data.6 were men. We observed significant differences between the enrollment of sexes across all trial phases and tumor types ex- cept thyroid (Table). Drug trials for colon and kidney cancer enrolled the least number of women, with 33% participation rates (Figure). were men. We observed significant differences between the enrollment of sexes across all trial phases and tumor types ex- cept thyroid (Table). Drug trials for colon and kidney cancer enrolled the least number of women, with 33% participation rates (Figure). Use of an Analytics and Electronic Health Record– Based Approach for Targeted COVID-19 Vaccine Outreach to Marginalized Populations Author Affiliations: School of Population and Public Health, The University of British Columbia, Vancouver, British Columbia, Canada (Jenei); Department of Medicine, University of Calgary, Calgary, Alberta, Canada (Meyers); Department of Epidemiology and Biostatistics, University of California, San Francisco (Prasad). Equity in vaccine outreach and delivery has been prioritized given the disproportionate harms of the COVID-19 pandemic on communities of color and those with lower socioeco- JAMA Oncology October 2021 Volume 7, Number 10 1570 jamaoncology.com © 2021 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ by a UCSF LIBRARY User on 12/02/2022
https://openalex.org/W4211223413	https://europepmc.org/articles/pmc6604998?pdf=render	English	null	Foodborne pathogens	AIMS microbiology	2,017	cc-by	19,968	* Correspondence: Email: tbintsis@gmail.com; bintsis@kastoria.teikoz.gr; Tel: +306948721720; Fax: +302463024995. Abstract: Foodborne pathogens are causing a great number of diseases with significant effects on human health and economy. The characteristics of the most common pathogenic bacteria (Bacillus cereus, Campylobacter jejuni, Clostridium botulinum, Clostridium perfringens, Cronobacter sakazakii, Esherichia coli, Listeria monocytogenes, Salmonella spp., Shigella spp., Staphylococccus aureus, Vibrio spp. and Yersinia enterocolitica), viruses (Hepatitis A and Noroviruses) and parasites (Cyclospora cayetanensis, Toxoplasma gondii and Trichinella spiralis), together with some important outbreaks, are reviewed. Food safety management systems based on to classical hazard-based approach has been proved to be inefficient, and risk-based food safety approach is now suggested from leading researchers and organizations. In this context, a food safety management system should be designed in a way to estimate the risks to human health from food consumption and to identify, select and implement mitigation strategies in order to control and reduce these risks. In addition, the application of suitable food safety education programs for all involved people in the production and consumption of foods is suggested. Keywords: foodborne pathogens; pathogenic bacteria; pathogenic viruses; pathogenic parasites; foodborne outbreaks AIMS Microbiology, 3(3): 529-563. DOI: 10.3934/microbiol.2017.3.529 Received: 13 March 2017 Accepted: 19 June 2017 Published: 29 June 2017 http://www.aimspress.com/journal/microbiology Thomas Bintsis * Department of International Trade, TEI of West Macedonia, Kastoria, Greece Department of International Trade, TEI of West Macedonia, Kastoria, Greece AIMS Microbiology, 3(3): 529-563. DOI: 10.3934/microbiol.2017.3.529 Received: 13 March 2017 Accepted: 19 June 2017 Published: 29 June 2017 1. Introduction The association between the consumption of food and human diseases was recognized very early and it was Hippocrates (460 B.C.) who reported that there is a strong connection between food consumed and human illness [1]. Foodborne pathogens (e.g. viruses, bacteria, parasites) are biological agents that can cause a foodborne illness event. A foodborne disease outbreak is defined as 530 the occurrence of two or more cases of similar illness resulting from the ingestion of a common food [2]. rrence of two or more cases of similar illness resulting from the ingestion of a common Foodborne illness occurs when a pathogen is ingested with food and establishes itself (and usually multiplies) in the human host, or when a toxigenic pathogens establishes itself in a food product and produces a toxin, which is then ingested by the human host. Thus, foodborne illness is generally classified into: (a) foodborne infection and (b) foodborne intoxication. In foodborne infections, since an incubation period is usually involved, the time from ingestion until symptoms occur is much longer than that of foodborne intoxications. More than 200 different food-borne diseases have been identified [3]. The most severe cases tend to occur in the very old, in the very young, in those who have compromised immune system function, and in healthy people exposed to a very high dose of an organism [2]. The symptoms, onset of symptoms and the most common responsible microorganisms for the major foodborne illnesses are shown on Table 1. In the European Union (EU) for the year 2015, 26 member states reported a total of 4,362 food-borne outbreaks, including waterborne outbreaks. Overall, these outbreaks caused 45,874 cases of illness (209 more than 2014), 3,892 hospitalisations (2,546 less than 2014) and 17 deaths (10 less than 2014) [4]. The overall reporting rate of food-borne outbreaks in the EU was 0.95 per 100,000 population, which represents a slight decrease compared with data provided for 2014 [4]. Most of the outbreaks reported in 2015 were caused by bacterial agents (33.7% of all outbreaks), in particular Salmonella spp. (21.8% of all outbreaks) and Campylobacter spp. (8.9% of all outbreaks), even though the reporting of outbreaks involving these agents has been declining over the recent years. 1. Introduction Bacterial toxins ranked second among the causative agents in food- and waterborne outbreaks and were reported in 19.5% of the total outbreaks while viruses, which were the agents most frequently reported in 2014, accounted for 9.2% of total outbreaks in 2015 [4]. Parasites and other causative agents, in particular histamine, were reported in less than 3% of the outbreaks. Furthermore, for a third of the reported outbreaks (34%) the causative agent remained unknown [4]. The implicated food vehicles were mostly of animal origin, in particular eggs and egg products and pig meat (both accounting for 10% of all strong-evidence outbreaks), broiler meat (9%) and cheese (8%) followed by fish and fish products (7%), milk and dairy products (5%), bovine meat (4%) and crustaceans (3%) [4]. In 2015, Salmonella spp. in eggs was associated with the highest number of reported foodborne outbreaks and was among the top-5 food-pathogen combinations in terms of the overall number of cases of illness and hospitalisations in outbreaks. However, the number of reported outbreaks caused by Salmonella spp. and associated with the consumption of “eggs and egg products” has been decreasing in the last 5 years [4]. Household was by far the most frequent place of exposure. In strong-evidence foodborne outbreaks, Salmonella spp. was the most common agent reported in private households, whereas, “bacterial toxins other than Clostridium botulinum toxins”, viruses and other causative agents were more frequently reported in public settings such as canteens, workplace catering, restaurants and pubs [4]. The characteristics of the most important foodborne pathogens, the illnesses they cause, together with some of the most important outbreaks they have been implicated are studied in this review. Volume 3, Issue 3, 529-563. AIMS Microbiology AIMS Microbiology 531 Table 1. Symptoms, onset of symptoms and responsible microorganisms or toxin for the major foodborne illnesses. Approximate onset time to symptoms Predominant symptoms Associated organism or toxin 1–7 h, mean 2–4 h Nausea, vomiting, retching, diarrhea, abdominal pain, prostration Staphylococcus aureus and its enterotoxins 8–16 h (2–4 h if emesis predominant) Vomiting or diarrhea, depending on whether diarrheic or emetic toxin present; abdominal cramps; nausea Bacillus cereus (emetic toxin) 12–48 h Nausea, vomiting, watery non-bloody diarrhea, dehydration Norovirus 2–36 h (mean 6–12 h) Abdominal cramps, diarrhea, putrefactive diarrhea (Cl. perfringens), sometimes nausea and vomiting Clostridium perfringens 6–96 h (usually 1–3 days) Fever, abdominal cramps, diarrhea, vomiting, headache Salmonella spp., Shigella spp., E. 2.1. Bacillus cereus Bacillus cereus are members of the family Bacillaceae; they are Gram-positive, motile rods, and they have the ability to form spores [7]. Most Bacillus spp. are found throughout the environment, including soils, fresh and marine water environments. Spores produced by B. cereus possess appendages and/or pili and are more hydrophobic than any other Bacillus spores. These properties enable the spores to adhere to many different types of surfaces and to resist removal during cleaning and sanitation [8]. Vegetative cells of B. cereus grow at temperatures ranging from 4–15 to 35–55 °C but prefer 30–40 °C, depending on the strain [9]. The organism grows at pH 4.9–9.3, but the inhibitory effect of pH is reduced in foods as evidenced by limited growth on meat at pH 4.35 [5,7] The minimum aw, for growth has been established at 0.93, but it has been suggested to use 0.912 as the minimum required for growth, because fried rice tends to have aw values ranging from 0.912 to 0.961 and readily supports B. cereus growth [8,10]. B. cereus produces two types of toxins, the emetic (vomiting) and the diarrhoeal one, causing two types of illness. The emetic syndrome is caused by emetic toxin produced by the bacteria during the growth phase in the food. The diarrhoeal syndrome is caused by diarrhoeal toxins produced during growth of the bacteria in the small intestine. The rapid onset of the emetic type is characterized by nausea and vomiting while the late onset of the diarrheal type is characterized by diarrhea and abdominal pain. Both syndromes (i.e., diarrheal and emetic) are a result of B. cereus endospores surviving the cooking process, after which germination and subsequent proliferation of vegetative cells occurs at some point during storage. Foods that are frequently implicated in B. cereus diarrheic food poisoning include meat products, soups, vegetables, puddings, sauces, milk and milk products [8]. Symptoms are characterized by abdominal pain, nausea, and diarrhea after an incubation period of approximately 8–16 h (Table 1). Diarrheal syndrome symptoms generally persist no longer than 12–24 h. After a 1–5 h incubation period, emetic syndrome symptoms include primarily nausea and vomiting and persist for 6–24 h (Table 1). Foods implicated in B. cereus emetic food poisoning include fried and cooked rice, pasta, noodles, and pastry [8]. 2. Foodborne Bacteria Bacteria are the most common cause of foodborne diseases and exist in a variety of shapes, types and properties. Some pathogenic bacteria are capable of spore formation and thus, highly heat-resistant (e.g. Clostridium botulinum, C. perfringens, Bacillus subtilus, Bacillus cereus) [7]. Some are capable of producing heat-resistant toxins (e.g. Staphylococcus aureus, Clostridium botulinum). Most pathogens are mesophilic with optimal growth temperature range from 20 °C to 45 °C. However, certain foodborne pathogens (i.e. psychrotrophs), such as Listeria monocytogenes, and Yersinia enterocolitica are capable of growth under refrigerated conditions or temperatures less than 10 °C [7]. 1. Introduction coli 6 h to 5 days Abdominal cramps, diarrhea, vomiting, fever, malaise, nausea, headache, dehydration Vibrio cholearae (O1 and non-O1), Vibrio parahaemolyticus 1–10 (median 3–4) days Diarrhea (often bloody), abdominal pain, nausea, vomiting, malaise, fever (uncommon with E. coli O157:H7) Enterohaemorrhagic E. coli, Campylobacter spp. 3–5 days Fever, vomiting, watery non-inflammatory diarrhea Rotavirus, Astrovirus, enteric Adenovirus 3–7 days Fever, diarrhea, abdominal pain Yersinia enterocolitica 1 to several weeks Abdominal pain, diarrhea, constipation, headache, drowsiness, ulcers, variable—often asymptomatic Entamoeda histolytica 3–6 months Nervousness, insomnia, hunger pains, anorexia, weight loss, abdominal pain, sometimes gastroenteritis Taenia saginata, Taenia solium 2 h to 6 days, usually 12–36 h Vertigo, double or blurred vision, loss or light reflex, difficulty in swallowing, dry mouth, weakness, respiratory paralysis Clostridium botulinum and its neurotoxins 4–28 days Gastroenteritis, fever, oedema around eyes, perspiration, muscular pain, chills, prostration, laboured breathing Trichinella spiralis 7–28 days Malaise, headache, fever, fever, cough, nausea, vomiting, constipation, abdominal pain, chills, rose spots, bloody stools Salmonella Tympi 10–13 days Fever, headache, myalgia, rash Toxoplasma gondii Varying periods Fever, chills, headache, arthalgia, prostration, malaise, swollen lymph nodes and other specific symptoms of disease in question Listeria monocytogenes, Campylobacter jejuni After: [5,6]. Associated organism or toxin Volume 3, Issue 3, 529-563. AIMS Microbiology 532 2.1. Bacillus cereus The diarrheal syndrome type of food poisoning results from the action of a thermolabile enterotoxic complex, whereas the emetic syndrome type involves the action of a thermostable toxin. Due to the formation of adhesive endospores, B. cereus is commonly present in food production environments and then spreading to all kinds of foods. They produce a range of virulence factors that may cause unpleasant disease in humans when present in food or the gastrointestinal tract and it is one of the major foodborne pathogenic bacteria, although in most cases disease is mild and of short duration [11]. Volume 3, Issue 3, 529-563. AIMS Microbiology AIMS Microbiology 533 Genetic and genomic analyses have revealed that B. cereus is very similar to Bacillus anthracis and that some strains have plasmids resembling the toxin plasmids of Bacillus anthracis. 310 genomes have been completed up to now according to the data retrieved from NCBI, 2017. The median total length of the genome is 5.6626 Mb [12]. B. cereus related food poisoning is not a notifiable disease in most countries; therefore, incidence data is limited. It is recognized that there may be significant under reporting of B. cereus illness due to the generally mild, short duration and self-limiting symptoms, however, fatal incidences have been reported [11]. It is estimated that B. cereus caused 0.7% of foodborne illness among the 31 major pathogens in the US [13,14]. In a study for B. cereus outbreaks [15] were most often attributed to rice dishes (50%); fried rice was the most common type of rice dish (68%). Rice dishes were most commonly cooked and served immediately (42%) or were part of large, solid masses of food (33%) [15]. Twenty-four percent of B. cereus outbreaks were associated with meat or poultry dishes. Meat or poultry dishes were cooked and served immediately (50%), roasted (33%), or part of liquid or semisolid mixtures (17%) [15]. In an outbreak at a birthday party in Bari, Italy, the characteristics were consistent with available reports on foodborne outbreaks caused by B. cereus [16]. The short incubation period and the predominance of vomiting suggested an emetic toxin. The distribution of cases by time of onset suggested a common source of contamination by a bacteria or a toxin. B. cereus was isolated from basmati rice and fecal specimens. Poor food handling and storage was most probably the cause of the outbreak [16]. 2.1. Bacillus cereus At a college sport day in Thailand, 470 individuals were ill with vomiting, nausea, and abdominal pain; approximately half of the individuals reported diarrhea [17]. The ingestion of cream-filled eclairs was significantly associated with illness and the mean incubation period was 3.2 hours, which suggested a preformed toxin in the food; initial laboratory investigation indicated presence of B. cereus [17]. B. cereus was reported as a major causative agent of foodborne illness in the Netherlands in 2006 (causing 5.4% of the foodborne outbreaks) and in Norway in 2000 (causing 32% of foodborne outbreaks) [17]. Pasta salad and spaghetti leftovers were the cause of two outbreaks, where the clinical data and the rapid onset of symptoms, together with the microbiological and molecular study, pointed to B. cereus as the causative agent [18,19]. 2.2. Campylobacter jejuni Campylobacter spp. are members of the family Campylobacteriaceae and Campylobacter jejuni is one of the most common causes of diarrheal illness. C. jejuni is responsible for approximately 850,000 illnesses, 8,500 hospitalizations, and 76 deaths in the US each year [13]. The World Health Organization (WHO) estimates that ~1% of the population of Western Europe will be infected with campylobacters each year [20]. Extensively found throughout nature, C. jejuni can colonize the intestines of both mammals and birds, and transmission to humans occurs via contaminated food products. This organism can invade the epithelial layer by first attaching to epithelial cells, then penetrating through them. Diarrhea results from damage to the epithelial cells. Systemic infections can also occur causing more severe illnesses [12]. 932 genomes have been completed up to now according to the data retrieved from NCBI. The median total length of the genome is 1.686 Mb [12]. Campylobacter spp. are small (0.2–0.9 μm wide and 0.2–5.0 μm long), spiral formed, Gram-negative bacteria with 18 species, six sub-species and two biovars [20]. Campylobacter Volume 3, Issue 3, 529-563. AIMS Microbiology AIMS Microbiology 534 genomes are relatively unstable; several mechanisms that may lead to this genetic instability have been proposed, including bacteriophage activity, DNA recombination and transformation [5]. They are very different from other pathogens associated with foodborne disease in that they are essentially microaerophilic, growing best in an atmosphere containing approximately 10% CO2 and approximately 5% O2. The species pathogenic for man also have a rather narrow temperature range for growth with a maximum temperature of ~46 °C and a minimum of 30 °C. These are classified as thermophilic campylobacters [20]. In 2015, Campylobacter continued to be the most commonly reported gastrointestinal bacterial pathogen in humans in the EU and has been so since 2005 [4]. The number of reported confirmed cases of human campylobacteriosis was 229,213, a 5.8% decrease compared with the rate in 2014 [4]. Campylobacter spp. are part of the normal intestinal flora of a wide variety of healthy domestic and wild animals, including cattle, sheep, goats, pigs, chickens, ducks, geese, wild birds, dogs, cats, rodents, and marine mammals, and are often found associated with bodies of water such as water troughs and streams. Most cases of campylobacteriosis are associated with eating raw or undercooked poultry meat, unpasteurized milk, contaminated water, or from cross-contamination of other foods by these items. 2.2. Campylobacter jejuni All animals used for food can be campylobacter-positive as can many companion species (domestic pets). Samples from the natural environment, such as groundwater, will also frequently contain these pathogens [21]. Ready-to-eat fresh produce contaminated with enteric pathogens presents a risk to consumers. However, its importance as a source of campylobacters is unclear. The number of documented foodborne outbreaks associated with raw fruits, vegetables and unpasteurised fruit juices has increased. Such foods can present a campylobacteriosis risk to public health as a consequence of using contaminated irrigation or washing water. When stressed, campylobacters enter a “viable but non-culturable state”, characterized by uptake of amino acids and maintenance of an intact outer membrane but inability to grow on selective media; such organisms, however, can be transmitted to animals [22,23]. In June 2012, 44 persons who attended a wedding reception in Sweden became ill [24]. The outbreak investigation identified chicken liver pâtè as the suspected source of the infection; the liver pâtè had been deliberately undercooked, lightly fried to keep the right texture and mixed with spices [24]. Several Campylobacter spp. outbreaks associated with consumption of poultry liver pâtè have been described, especially in the UK [25–29], but also in other countries such as Australia [24] and US [30]. A serious outbreak of Campylobacter spp. was associated with the consumption of raw milk [31]. C. jejuni was isolated in 50 of 88 raw milk samples in New Zealand after a gastrointestinal illness among children in two different camp sites [31]. A drink prepared with raw milk was associated with an outbreak of C. jejuni enteritis involving more than 500 participants in a jogging rally in Switzerland, with an attack rate of over 75%. An outbreak of C. jejuni enteritis followed the consumption of unpasteurized milk at an attack rate of around 50%; there were cases in all age groups, with the highest number in the 1 to 10 year old group [31]. C. coli was isolated from a 9-year-old British boy with persistent diarrhea, whose family had consumed raw goat’s milk from a local farm. C. jejuni and E. coli were found in the feces of goats from the farm, and C. jejuni was identified in samples of bulk milk [31]. 2.3. Clostridium botulinum Clostridium spp. are spore-forming bacteria, members of the family Bacillaceae and includes obligately anaerobic or aerotolerant, sporeforming rods that do not form spores in the presence of air and, at least in early stages of growth, are usually Gram-positive. In most species, vegetative cells appear as straight or curved rods, varying from short coccoid rods to long filamentous forms with rounded, tapered, or blunt ends, that occur singly, in pairs, or in various chain lengths [7]. Clostridia are found throughout the environment but are most prevalent in the soil and in the intestinal tract of animals. The characteristic shape of clostridia is attributed to the presence of endospores that develop under conditions unfavorable for vegetative growth and distend single cells terminally or sub-terminally [7]. The endospores of many species are extremely sturdy and survive extended boiling in water and exposure to air. Spores germinate under conditions favorable for vegetative growth, such as anaerobiosis and presence of organic substrates [7]. Cl. botulinum are motile by means of peritrichous flagella and produce botulinum neurotoxins, the most lethal poison known. There are seven types of botulinum neurotoxin, A through G, based on the antigenic specificity of the toxin produced by each strain [5]. Types A, B, E, and F causing botulism in humans, types C and D causing botulism in birds and mammals, and type G, which has yet to be clearly implicated in a botulism case [5,7]. Thermal processing is the most common method used to produce shelf-stable, low-acid, moist foods by inactivating Cl. botulinum spores. From the evolutionary perspective, clostridia are considered to be the most ancient bacteria. It is believed that present day Mollicutes (Eubacteria) have evolved regressively (i.e., by genome reduction) from gram-positive clostridia-like ancestors with a low GC content in DNA. Several species of clostridia (e.g., Cl. perfringens, Cl. botulinum, Cl. tetani) are known opportunistic toxin-producing pathogens in animals and humans [12]. Some species are capable of producing organic solvents (acetone, ethanol, etc.), molecular hydrogen and other useful compounds. There are also species that can fix molecular nitrogen and thus are important participants in biological turnaround of nitrogen compounds in nature. The most common and widely distributed are strains and serovars of Cl. botulinum that produce type A toxin. This toxin finds its use in various applications requiring neuroparalitic intervention, including cosmetology (Botox®). 177 genomes have been completed up to now according to the data retrieved from NCBI. 2.2. Campylobacter jejuni In October 2013, public health authorities in Australia were notified of a suspected outbreak of gastroenteritis in students and guests following a catered function at a university residential college; a total of 56 cases of gastroenteritis, including seven laboratory-confirmed cases of C. jejuni Volume 3, Issue 3, 529-563. AIMS Microbiology AIMS Microbiology 535 infection, were identified in 235 eligible respondents [32]. C. jejuni diversity in epidemiologically related human and food isolates recovered during outbreaks linked to poultry liver [32]. infection, were identified in 235 eligible respondents [32]. C. jejuni diversity in epidemiologically related human and food isolates recovered during outbreaks linked to poultry liver [32]. 2.3. Clostridium botulinum The median total length of the genome is 3.898 Mb [12]. Cl. botulinum is present in soils, freshwater, marine sediments, and the intestinal tracts of animals. Food sources commonly sampled include primarily honey, which should not be fed to infants less than 1 year of age, as well as fish, meats, vegetables, and infant foods. A variety of foods, such as canned corn, peppers, green beans, soups, beets, asparagus, mushrooms, ripe olives, spinach, tuna fish, chicken and chicken livers, liver pate, luncheon meats, ham, sausage, stuffed eggplant, lobster, and smoked and salted fish have been associated with botulinum toxin [5]. Traditionally, foodborne botulism has been associated with underprocessed and abused sausages or home canned foods; however, in recent years botulism has been acquired through the consumption of contaminated foods such as potato salad, sauteed onions, garlic sauce, cheese, yogurt, bean paste, and olives. Volume 3, Issue 3, 529-563. AIMS Microbiology AIMS Microbiology 536 Symptoms of botulinum neurotoxin ingestion appear 12–36 h after consumption of contaminated food and initially may include nausea and vomiting (Table 1). However, these symptoms are followed by the more characteristic neurological signs including visual impairment and acute flaccid paralysis that begins with the muscles of the face, head, and pharynx, descending to involve muscles of the thorax and extremities and leading to possible death from respiratory failure caused by upper airway or diaphragm paralysis [7]. The minimum toxic dose of Cl. botulinum neurotoxin has not been determined, but from a human health and food safety standpoint, there should be no tolerance either for the neurotoxin itself or for conditions allowing growth of the organism in foods [7]. Botulinum neurotoxin is synthesized during cellular growth and is subsequently released during cell lysis, where proteolytic cleavage activates the molecule [7]. There are four categories of botulism, which include the classic foodborne botulism derived from the ingestion of preformed toxin in foods, wound botulism resulting from toxin production after organism growth in an infected wound, infant botulism from toxin elaboration in the intestinal tract of infants, and botulism due to intestinal colonization in older children and adults with intestinal disorders or complications resulting in a lack of microbial competition [7]. Botulinum neurotoxin introduced in any of these categories is transported via the bloodstream to neuromuscular junctions, where the toxin irreversibly binds to receptors on peripheral nerve endings and subsequently is internalized into the nerve cell [7]. 2.3. Clostridium botulinum Recent developments in whole genome sequencing have made a substantial contribution to understanding the genomes, neurotoxins and biology of Cl. botulinum Group I (proteolytic Cl. botulinum) and Cl. botulinum Group II (non-proteolytic Cl. botulinum). Two different approaches were used to study genomics in these bacteria; comparative whole genome microarrays and direct comparison of complete genome DNA sequences [33]. The failure to effectively apply the botulinum cook (121 °C for 3 min) to canned or bottled foods has led to many outbreaks of foodborne botulism associated with Cl. botulinum Group I. For example, a large outbreak in Thailand in 2006 (209 cases) was associated with consumption of inadequately home-canned bamboo shoots [33]. Inadequate thermal processing of cans of a commercial hot dog chilli sauce in 2007 in US was associated with eight botulism cases, and initially led to the recall of 39 million cans, then an expanded recall of 111 million cans [34]. Temperature abuse of foods intended to be stored chilled has also been responsible for several severe outbreaks of foodborne botulism, including those associated with commercial chilled carrot juice [35] and commercial chicken enchiladas [33,36]. AIMS Microbiology 2.4. Clostridium perfringens Clostridium perfringens, previously known as Clostridium welchii, belongs to the family Bacillaceae and is an important cause of foodborne disease. They are nonmotile, encapsulated rod-shaped cells that produce protein toxins and form spores resistant to various environmental stresses such as radiation, desiccation, and heat [7]. Vegetative cells grow at temperatures ranging from 6 to 50 °C but prefer an optimum temperature between 43 and 47 °C. Growth requires a minimum aw, of 0.93, a sodium chloride concentration less than 5–8% depending on the strain, and a pH of 5.0–9.0, although 6.0–7.2 is preferred [7,10]. Cl. perfringens are the most prevalent Clostridium species found in human clinical specimens, excluding faeces, and has been implicated in simple wound infections to myonecrosis, clostridial Volume 3, Issue 3, 529-563. AIMS Microbiology AIMS Microbiology 537 cellulitis, intra-abdominal sepsis, gangrenous cholecystitis, postabortion infection, intravascular hemolysis, bacteremia, pneumonia, thoracic and subdural empyema, and brain abscesses [7,8]. The spores and cells of the organism are frequently associated with dust contamination on many surfaces, including foods such as meat and shellfish, as a result of its ubiquity throughout the environment [7]. Cl. perfringens are estimated to be the second most common bacterial causes of foodborne illness in the US, causing one million illnesses each year [37]. Local, state, and territorial health departments voluntarily report Cl. perfringens outbreaks to the US CDC through the Foodborne Disease Outbreak Surveillance System. From 1998 to 2010, 289 confirmed outbreaks of Cl. perfringens illness were reported with 15,208 illnesses, 83 hospitalizations, and eight deaths [37]. The number of outbreaks reported each year ranged from 16 to 31 with no apparent trend over time [37]. The annual number of outbreak-associated illnesses ranged from 359 to 2,173, and the median outbreak size was 24 illnesses [37]. Restaurants (43%) were the most common setting of food preparation; other settings included catering facility (19%), private home (16%), prison or jail (11%), and other (10%) [37]. Among the 144 (50%) outbreaks attributed to a single food commodity, beef was the most common commodity (66 outbreaks, 46%), followed by poultry (43 outbreaks, 30%), and pork (23 outbreaks, 16%) [37]. Outbreaks caused by Cl. perfringens occur regularly, are often large, and can cause substantial morbidity yet are preventable if contamination of raw meat and poultry products is prevented at the farm or slaughterhouse or, after contamination, if these products are properly handled and prepared, particularly in restaurants and catering facilities [37]. AIMS Microbiology 2.5. Cronobacter sakazakii The genus Cronobacter consists of a diverse group of Gram-negative bacilli and comprises seven species: Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter muytjensii, Cronobacter turicensis, Cronobacter dublinensis, Cronobacter universalis, and Cronobacter condimenti [39]. Among these, Cr. sakazakii, formerly Enterobacter sakazakii, is associated with infant septicemia, meningitis, and necrotizing enterocolitis. Originally isolated from powdered formula, it has also been shown to compartmentalize cerebral ventricles and cause brain abcesses in neonates. This species produces a yellow pigment when grown at 30 °C, but this fades at 37 °C [12]. 49 genomes have been completed up to now according to the data retrieved from NCBI. The median total length of the genome is 4.5475 Mb [12]. Infections in elderly and immunocompromised adults have also been reported [40], and the epidemiology of these cases suggests that other potential sources of contamination exist, such as the home environment [41] and retail foods (e.g. dried milk powder, dried meats, legumes, nuts, dried flours and spices) [42]. Although Cronobacter spp. have been detected in this wide assortment of foods, only contaminated powdered infant formula has been linked epidemiologically with infant infections and outbreaks caused by Cr. sakazakii [43]. The source of this contamination is thought to be powdered infant formula manufactured under poor Good Manufacturing Practice (GMP); however, extrinsic contamination of opened cans and human carriage may also be possible [39]. 2.4. Clostridium perfringens Foodborne illness almost always is a result of temperature abuse, and in many instances, the food vehicle has been improperly cooked meat or meat product that has been left to cook and/or cool too slowly or has undergone insufficient reheating, allowing surviving spores to germinate leading to vegetative cell proliferation. After ingestion and an incubation period of 7–30 h, symptoms typically include cramping and abdominal pain, although nausea and vomiting may also ensue, persisting for 24–48 h [7]. Five toxin-producing types of Cl. perfringens have been identified (A through E), and all produce an alpha-toxin (phospholipase) that plays a role in myonecrosis [7]. Type B strains produce beta- and epsilon-toxins, type D strains also produce epsilon-toxin, and type E strains produce an iota-toxin [7]. Almost all reported cases of foodborne gastroenteritis in the US that involve Cl. perfringens are a result of type A infection after the ingestion of highly contaminated foods with greater than 106–107 viable vegetative cells, which undergo sporulation in the small intestine and produce enterotoxin [7]. The enterotoxin produced during sporulation is released with the spores during cell lysis. After release, the enterotoxin binds to epithelial cells, causing cytotoxic cell membrane damage and subsequent alteration of permeability, leading to diarrhea and abdominal cramping [7]. An outbreak of Cl. perfringens occurred in a care home and fifteen residents reported illness. The likely cause was consumption of mince and vegetable pie and/or gravy [38]. There were a number of issues with food served, in particular the mince products had been cooked, cooled, reheated and served again over a period of several days; fecal sampling revealed the presence of Cl. perfringens enterotoxin gene and four samples were indistinguishable by fluorescent amplified fragment length polymorphism, indicating a likely common source [38]. The operator of the home was charged with three offences under the General Food Regulations 2004 and the Food Hygiene (England) Regulations 2006 and was convicted on all counts [38]. Epidemiological evidence can be used to help prosecute businesses with food safety offences in such circumstances [38]. Volume 3, Issue 3, 529-563. AIMS Microbiology 538 2.6. Escherichia coli Escherichia coli is a Gram-negative, non-spore forming rod. It may or may not be mobile; some rods are flagellated and some are not [43]. The organism is a facultative anaerobe and ferments simple sugars such as glucose to form lactic, acetic, and formic acids; the optimum pH for growth is 6.0 to 8.0; however, growth can occur as low as pH 4.3 and as high as 9 to 10 pH [43]. E. coli comprise a large and diverse group of bacteria. Most strains of E. coli are harmless; other strains have acquired characteristics, such as the production of toxins, which make them pathogenic to humans [44]. 5351 genomes have been completed up to now according to the data retrieved from NCBI. The median total length of the genome is 5.171 Mb [12]. Pathogenic variants of E. coli (pathovars or pathotypes) cause much morbidity and mortality worldwide; many of these pathotypes are a major public health concern as they have low infectious doses and are transmitted through ubiquitous mediums, including food and water [45]. Transmission of E. coli occurs when food or water that is contaminated with feces of infected humans or animals is consumed. Contamination of animal products often occurs during the slaughter and processing of animals [44]. The use of manure from cattle or other animals as fertilizer for agricultural crops can contaminate produce and irrigation water [44]. E. coli can survive for long periods in the environment and can proliferate in vegetables and other foods. Pathogenic E. coli have been categorized into six groups according to the pathogenic mechanism: (1) Enteropathogenic E. coli (EPEC); (2) Enterohemorrhagic E. coli (EHEC, also known as Shiga toxin—producing E. coli [STEC] and formerly referred to as verotoxin-producing E. coli [VTEC]); (3) Enterotoxigenic E. coli (ETEC); (4) Enteroaggregative E. coli (EAggEC); (5) Enteroinvasive E. coli (EIEC); and (6) Attaching and Effacing E. coli (A/EEC) [44,45]. Volume 3, Issue 3, 529-563. AIMS Microbiology AIMS Microbiology 539 STEC infection can cause episodes of mild to severe diarrhea, and 5–10% of infections develop into Hemolytic Uremic Syndrome (HUS)—a severe complication marked by profuse bleeding that can lead to kidney failure and death. STEC strain O157:H7 is estimated to cause 63,000 illnesses, 2,100 hospitalizations, and 20 deaths each year [13]. The principal reservoir for this zoonotic pathogen is the intestinal tract of cattle, but other animals may also serve as reservoirs. 2.6. Escherichia coli O157:H7 emerged as a significant public health threat in 1982 during two outbreaks of disease that investigators associated with the consumption of undercooked ground meat [13]. A wide variety of foods, including fresh produce, have since served as a vehicle for E. coli O157:H7 outbreaks. Food producers must report the presence of E. coli O157:H7 to health authorities [13]. In 1982, an investigation by the CDC of two outbreaks of severe bloody diarrhea, associated with the same fast-food restaurant chain, led to the identification of a strain of E. coli, one that expressed O-antigen 157 and H-antigen 7, that had not previously been recognized as a pathogen [46]. Subsequently, this strain was shown to belong to a category of E. coli that produce toxins which are similar to Shiga toxin of Shigella dysenteriae and distinct from previously described E. coli heat-stable and heat-labile toxins. As data were accumulating on the role of E. coli O157:H7 as a pathogen, parallel work in Canada was uncovering high rates of infection with this and other Shiga toxin-producing E. coli in patients with the HUS [44]. Subsequent research has indicated that E. coli O157:H7 is the cause of 85–95% of cases of hemolytic uremic syndrome in North America, and that non-O157 Shiga toxin-producing E. coli are responsible for another 5–15% [47]. In 2015, 5,901 confirmed cases of STEC infections were reported in the EU [4]. The EU notification rate was 1.27 cases per 100,000 population, which was slightly lower than the notification rate in 2014. The EU notification rate following the large outbreak in 2011 was higher in 2012–2015 than before the outbreak but stabilised in the last 2 years in 2014–2015 [4]. In 2011, a rare strain of E. coli O104:H4 caused the second largest and the deadliest outbreak of E. coli-associated disease ever recorded. Between May 21 and July 22, 2011, more than 4,000 people became ill in 16 countries, and 50 individuals died [48]. By the time the outbreak ended in early July, 2011, there were reports of more than 4,000 illnesses, 800 cases of HUS, and 50 deaths in Germany and 15 other countries [49]. The outbreak was unusual because of the high proportion of adult patients (~25%) with HUS and the frequent development of neurological symptoms in these patients [50]. 2.6. Escherichia coli Traceback studies of disease clusters in five German provinces that were affected early in the outbreak pointed to sprouts produced by an organic grower in Lower Saxony [58]. A smaller, second wave of illnesses around the French city of Bordeaux also resulted from the consumption of sprouts, and patient isolates from both outbreaks were identical [59]. It was later discovered that sprout seeds associated with both outbreaks had a common origin in a 16.5 tons shipment of fenugreek seeds from Egypt [59]. Upon the shipment’s arrival in Germany in 2009, various distributors in Germany and other European countries subdivided, packaged, repackaged, and widely distributed these seeds as part of thousands of packets of “seed mixes” [59]. Despite extensive recall efforts, the complex chain of packaging and distribution may mean that contaminated seeds could remain on store shelves until their expiration date in 2014 [59]. The pathogen was not isolated from any remaining batches of the suspect seeds, and questions remain as to the source and reservoir of the contaminating pathogen [60]. Interestingly, for the outbreak in Germany, investigators initially identified fresh produce—including leafy greens, tomatoes, and cucumbers as likely sources [57]. Traceback studies of disease clusters in five German provinces that were affected early in the outbreak pointed to sprouts produced by an organic grower in Lower Saxony [58]. A smaller, second wave of illnesses around the French city of Bordeaux also resulted from the consumption of sprouts, and patient isolates from both outbreaks were identical [59]. It was later discovered that sprout seeds associated with both outbreaks had a common origin in a 16.5 tons shipment of fenugreek seeds from Egypt [59]. g p g gyp [ ] Upon the shipment’s arrival in Germany in 2009, various distributors in Germany and other European countries subdivided, packaged, repackaged, and widely distributed these seeds as part of thousands of packets of “seed mixes” [59]. Despite extensive recall efforts, the complex chain of packaging and distribution may mean that contaminated seeds could remain on store shelves until their expiration date in 2014 [59]. The pathogen was not isolated from any remaining batches of the suspect seeds, and questions remain as to the source and reservoir of the contaminating pathogen [60]. Flour and flour-based mixes have been suspected or implicated as the source of other foodborne Salmonella and STEC O157 outbreaks [61,62]. 2.6. Escherichia coli Evidence obtained at one restaurant showed that dessert pizzas were made with the same dough mix used in traditional pizzas, but used thicker dough and might have been undercooked at some locations [62]. On May 31, 2016, General Mills recalled several sizes and varieties of flours due to possible E. coli contamination; in June 2016, laboratory testing by FDA isolated STEC O121 in open samples of General Mills flour collected from the homes of ill people in Arizona, Colorado, and Oklahoma [62]. This outbreak is a reminder that is it not safe to taste or eat raw dough or batter; flour or other ingredients used to make raw dough or batter can be contaminated with STEC and other germs that can make people sick [62]. 2.6. Escherichia coli Research suggests that these clinical characteristics were due to the unique combination of traits carried by the pathogen, which included features typical of enteroaggregative E. coli and the capacity to produce Shiga toxin [50]. This strain also has a distinct set of additional virulence and antibiotic-resistance factors [50]. In addition, eight deaths due to STEC infection were reported in the EU which resulted in an EU case fatality of 0.2% among the 3,352 confirmed cases for which this information was provided. As in previous years, the most commonly reported STEC serogroup in 2015 was O157 (41.7%), although its relative proportion compared to other serogroups declined. This is possibly an effect of increased awareness and of more laboratories testing for other serogroups. Serogroup O157 was followed by serogroups O26, O103, O91, O145, O146 and O128. The proportion of non-typable STEC strains continued to increase in 2015 [4]. Since a 1993 outbreak associated with hamburgers purchased from a fast food chain resulted in more than 500 laboratory-confirmed infections with E. coli O157:H7 and at least 4 deaths [51], several interventions have been introduced to reduce the contamination of beef during processing and in the retail and restaurant industries [52]. Volume 3, Issue 3, 529-563. AIMS Microbiology AIMS Microbiology 540 In 2006, investigators linked at least 183 illnesses and one death to the consumption of fresh spinach contaminated with E. coli O157:H7 [53]. Among the ill persons, 95 (52%) were hospitalized and 29 (16%) had HUS [53]. In response to the growing outbreak—which included cases across 26 states and Canada—FDA advised consumers to stop eating all uncooked, fresh spinach, or products containing uncooked spinach [53]. Epidemiological studies traced the contamination to a single shift at a Natural Selections Foods processing plant in San Juan Batista, California, which had produced 42,000 bags of pre-washed and ready-to-eat baby spinach [54]. Based on isolates from contaminated produce from sick consumers, investigators matched the outbreak strain to environmental samples from a single field of organic spinach in central California [55]. Environmental sampling revealed the presence of the outbreak strain in river water and the feces of cattle and wild pigs less than 1 mile away from the spinach field [55,56]. Interestingly, for the outbreak in Germany, investigators initially identified fresh produce—including leafy greens, tomatoes, and cucumbers as likely sources [57]. AIMS Microbiology 2.7. Listeria monocytogenes Listeria monocytogenes is one of the leading causes of death from food-borne pathogens especially in pregnant women, newborns, the elderly, and immuno-compromised individuals [63]. Infections in pregnant women can be devastating to the fetus, resulting in miscarriages, stillbirths, and birth defects [63]. It is found in environments such as decaying vegetable matter, sewage, water, and soil, and it can survive extremes of both temperatures (1–45 °C) and salt concentration marking it as an extremely dangerous food-born pathogen, especially on food that is not reheated and is carried asymptomatically by numerous animal species. The bacterium has been found in a variety of raw foods, such as uncooked meats and vegetables, as well as in foods that become contaminated AIMS Microbiology AIMS Microbiology Volume 3, Issue 3, 529-563. 541 after cooking or processing. It can spread from the site of infection in the intestines to the central nervous system and the fetal-placental unit. It can cause meningitis (inflammation of the membrane surrounding spinal cord and brain), gastroenteritis (inflammation of mucous membranes of stomach and intestine), and septicemia (systemic spread of bacteria and toxins in the blood) can result from infection [63]. It has 13 serotypes, including 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e, and 7; among them, serotypes 1/2a, 1/2b, and 4b have been associated with the vast majority of foodborne infections [5]. 1243 genomes have been completed up to now according to the data retrieved from NCBI. The median total length of the genome is 2.974 Mb [12]. Listeriosis is a serious infection usually caused by eating food contaminated with L. monocytogenes. Although it is a relatively rare disease with a high mortality rate (20–30 %) that makes it one of the deadliest food-borne threats [64]. Unlike many other foodborne pathogens, Listeria multiplies in cold environments such as refrigerators [65]. It can quickly spread in damp buildings, dripping off pipes or ceilings onto food. Once Listeria bacteria get into a food-processing factory, they can live there for years, sometimes contaminating food products [64,65]. In the EU for the year 2015, 28 member states reported 2,206 confirmed human cases of listeriosis [4]. The EU notification rate was 0.46 cases per 100,000 population, which was similar to 2014 [4]. AIMS Microbiology 2.7. Listeria monocytogenes There was a statistically significant increasing trend of listeriosis over 2008–2015; nineteen member states reported 270 deaths due to listeriosis in 2015, which was the highest annual number of deaths reported since 2008 [4]. The EU case fatality was 17.7% among the 1,524 confirmed cases with known outcome [4]. Listeriosis infections were most commonly reported in the elderly population in the age group over 64 years old and particularly in the age group over 84 years [4]. It is estimated that L. monocytogenes causes on average 1,591 episodes of domestically acquired food-borne illnesses, 1,455 hospitalizations, and 255 deaths annually in the US [13]. Over the last 10 to 15 years, increasing evidence suggests that persistence of L. monocytogenes in food processing plants for years or even decades is an important factor in the transmission of this foodborne pathogen and the root cause of a number of human listeriosis outbreaks. L. monocytogenes persistence in other food-associated environments (e.g., farms and retail establishments) may also contribute to food contamination and transmission of the pathogen to humans [13]. Although the available data clearly indicate that L. monocytogenes persistence at various stages of the food chain contributes to contamination of finished products, continued efforts to quantitatively integrate data on L. monocytogenes persistence (e.g., meta-analysis or quantitative microbial risk assessment) will be needed to advance our understanding of persistence of this pathogen and its economic and public health impacts [66]. Whole apples have not been previously implicated in outbreaks of foodborne bacterial illness. A nationwide listeriosis outbreak associated with caramel apples was investigated [67]. Outbreak-associated cases were compared with non-outbreak-associated cases and environmental investigations were performed; 35 outbreak-associated cases were identified in 12 states; 34 (97%) were hospitalized and seven (20%) died [67]. This outbreak highlights the importance of minimizing produce contamination with L. monocytogenes; investigators should perform single-interviewer open-ended interviews when a food is not readily identified [67]. L. monocytogenes is killed by pasteurization and cooking; however, in some Ready-To-Eat (RTE) foods contamination may occur after factory cooking but before packaging. RTE foods pose higher risk for listeriosis as they are ingested without any further processing, such as cooking, that would kill L. monocytogenes. Many of these foods use refrigeration, among other methods, to restrict AIMS Microbiology AIMS Microbiology Volume 3, Issue 3, 529-563. 542 bacterial growth during their shelf-life. 2.7. Listeria monocytogenes While these standard practices work well for most bacteria, they are not adequate for Listeria control as the organism is capable of growth at refrigeration temperature and is often tolerant to freezing temperature, high salt and low pH [68]. RTE products, such as delicatessen (deli) meats and soft cheeses have repeatedly been identified by foodborne disease control programs as sources of outbreaks and products that put humans at risk for listeriosis. Although, most listeriosis cases tend to be sporadic in occurrence, outbreaks do occur frequently. Due to the global phenomenon of outbreaks associated with Listeria in deli meats and cheese, it requires an urgent attention from national and international authorities through rigorous procedures for its identification, surveillance procedures that can bring more awareness to the general public [68]. One of the largest and deadliest multi-state outbreaks of listeriosis in the US occurred in late summer of 2011. The incident marked the first time that Listeria spp. contamination had been linked to whole cantaloupe and one of the few times it had been linked to fresh produce [69]. 146 Individuals had become ill after being infected with the outbreak strain of listeria; 29 deaths and 1 miscarriage had also been attributed to the infection [69]. In response to the CDC outbreak investigation, the cantaloupe producer, announced a voluntary recall of the 300,000 cases of cantaloupes harvested and produced between July and September [69]. The recall included 1.5 to 4.5 million melons that were distributed at supermarkets and chain stores in at least 28 states [69]. FDA inspectors cited unsanitary conditions—such as old, corroded, and difficult-to-clean equipment and standing pools of water—and the absence of processing steps to cool the melons before cold storage as likely contributors to contamination [69]. AIMS Microbiology 2.8. Salmonella spp. This group of Enterobactericiae have pathogenic characteristics and are one of the most common causes of enteric infections (food poisoning) worldwide. They were named after the scientist Dr. Daniel Salmon who isolated the first organism, Salmonella choleraesuis, from the intestine of a pig [7]. The genus Salmonella is divided into two species that can cause illness in humans: S. enterica and S. bongori [5]. Salmonella is further subdivided into serotypes, based on the Kaufmann-White typing scheme first published in 1934, which differentiates Salmonella strains by their surface and flagellar antigenic properties. Salmonella spp. are commonly referred to by their serotype names. For example, Salmonella enterica subsp. enterica is further divided into numerous serotypes, including S. Enteritidis and S. Typhimurium [5]. Certain serovars of Salmonella enterica are responsible for more serious diseases such as Typhoid fever. The presence of several pathogenicity islands (PAIs) that encode various virulence factors allows Salmonella spp. to colonize and infect host organisms. There are two important PAIs, Salmonella pathogenicity island 1 and 2 (SPI-1 and SPI-2) that encode two different type III secretion systems for the delivery of effector molecules into the host cell that result in internalization of the bacteria which then leads to systemic spread. 5323 Salmonella enterica genomes have been completed up to now according to the data retrieved from NCBI. The median total length of the genome is 4.783 Mb [12]. Salmonella spp. are the leading bacterial causes of food-borne illness in the US [13]. The CDC estimates that more than 1 million people in the US contract Salmonella each year, with an average of 19,000 hospitalizations and 380 deaths [13]. Salmonella spp. live in the intestines of most livestock and many wild animals. Salmonella spp. infection usually occurs when a person eats food contaminated with the feces of animals or humans carrying the bacteria. Salmonella outbreaks are AIMS Microbiology Volume 3, Issue 3, 529-563. 543 commonly associated with eggs, meat, and poultry, but these bacteria can also contaminate other foods such as fruits and vegetables. More recently, the CDC has reported a total of 258 persons infected with the outbreak strain of Salmonella Bareilly (247 persons) or Salmonella Nchanga (11 persons) from 24 states and the District of Columbia [70]. Thirty-two ill persons have been hospitalized, and no deaths have been reported. 2.8. Salmonella spp. Collaborative investigation efforts of state, local, and federal public health agencies indicate that a frozen raw yellow fin tuna product, known as Nakaochi Scrape, from Moon Marine USA Corporation is the likely source of this outbreak [70]. In EU for the year 2015, a total of 94,625 confirmed salmonellosis cases (126 fatal) were reported by 28 member states, resulting in an EU notification rate of 21.2 cases per 100,000 population. This represented a 1.9% increase in the EU notification rate compared with 2014. There was a statistically significant decreasing trend of salmonellosis in the 8-year period between 2008 and 2015 [4]. As in previous years, the two most commonly reported Salmonella serovars in 2014 were S. Enteritidis and S. Typhimurium, representing 45.7% and 15.8%, respectively, of all reported serovars in confirmed human cases. Cases of Salmonella Infantis, the fourth most common serovar continued to decrease in 2015. Cases of Salmonella Stanley still remained, as in the last 2 years, at a higher level than before the large outbreak reported in 2011–2012 [4]. In 1994, 138,000 gallons of ice cream were contaminated by Salmonella. This “single batch” of ice cream was consumed by individuals in 15 states, where it sickened an estimated 225,000 individuals [71]. Salmonella spp. contamination of peanuts and peanut products led to one of the largest product recalls in US history. More than 714 people in 46 states were sickened in this outbreak and 9 individuals died [72]. Investigators traced the contamination to a single facility that produced peanuts, peanut butter, and peanut paste; more than 200 companies used these foodstuffs as ingredients in a variety of other products, such as brownie products, cake and pie products, candy products, cereal products, cookie products, cracker products, prepackaged meals, snack mix products, ice cream, pet food, and topping products [72]. The recall extended to more than 3,900 products [72,73]. In 2008, 1,450 individuals in 43 states and the District of Columbia became ill from salmonellosis and two patients died after consuming jalapeño and serrano peppers imported from Mexico; investigations traced the contaminated peppers to one farm in Mexico, but the source of contamination is unknown [73]. 2.9. Shigella spp. The genus Shigella is a member of the family Enterobacteriaceae and possesses four serogroups that have been traditionally treated as species: serogroup A as Shigella dysenteriae, serogroup B as Shigella flexneri,serogroup C as Shigella boydii, and, serogroup D as Shigella sonnei. Whereas serogroups A, B, and C consist of 38 serotypes, serogroup D possesses only one [7]. Shigella are non-motile, non-spore-forming, facultative anaerobic Gram-negative rods. They can grow at temperatures ranging from 6 to 48 °C, but prefer 37 °C, and S. sonnei appears to be able to tolerate lower temperatures better than the other serogroups. Optimum growth occurs between pH 6.0 and 8.0, although growth has been reported between pH 4.8 and 9.3 [10]. Shigella spp. are closely related to E. coli in their DNA homology and share some biochemical characteristics as well as reactivity to some of the same antibodies, but despite these similarities, their differentiation should be considered clinically significant based, at least in part, on differences in symptoms expressed by infected individuals [7]. Shigella spp. are found most frequently in environments of compromised sanitation and poor hygiene, and although the primary route of transmission is by person-to-person contact, shigellosis can occur after the ingestion of focally contaminated water or food [7]. Shigella spp. have not been associated with one specific type of food; foods associated with outbreaks of shigellosis have included milk, salads, chicken, shellfish, and other fresh produce served at a wide range of establishments including restaurants, homes, schools, sorority houses, commercial airlines, cruise ships and military mess halls [10]. Approximately 20% of all shigellosis cases in the US are related to international travel (i.e. travelers diarrhea), with S. sonnei being the most prevalent and S. flexneri being the second most common in developed countries [82]. However, in developing countries, S. flexneri and S. dysenteriae type 1 are the most common serogroups, with S. dysenteviae type 1 having been involved in a lengthy epidemic in southern Africa and major epidemics in other parts of Africa, in Asia and in Central America [12]. These epidemics have resulted in high morbidity and mortality rates, especially in malnourished children, immuno-compromised individuals, and the elderly [12]. All Shigella serogroups can cause gastrointestinal infections after an incubation period of 12–50 h, after which time individuals experience watery diarrhea in conjunction with fever, fatigue, malaise, and abdominal cramps (Table 1). Although dysentery can be caused by all four Shigella serogroups, S. 2.8. Salmonella spp. Pathogens may be passively internalized during produce processing, and this occurred in 1999, when mangoes imported to the US from Brazil were treated to kill possible Mediterranean fruit fly by dipping them in hot water, after which they were chilled in a cold-water bath [74]. The cold water was not treated, it was not potable and it was contaminated with Salmonella Newport, which infected 78 people in 13 states [74,75]. During 1973–2011, of the 1,965 outbreaks of salmonella where a food vehicle was implicated, 96 were attributed to beef, accounting for 3,684 illnesses [76]. In EU between 2014 and 2015, a total of 162 cases, mostly from France, followed by Belgium, the Netherlands, Spain, Denmark and Sweden were reported, including 86 (53%) women [77]. Using whole genome sequencing (WGS), the cause was identified as Salmonella enterica serotype Chester; S. Chester was more likely to have eaten in a restaurant and visited the coast of Morocco [77]. Outbreaks associated with S. Chester have been reported: in Australia, associated with sea turtle meat in 1998 and with tap water in 2005; in the US, associated with frozen meals (cheesy chicken and rice) in 2010 and in Canada, associated with headcheese in 2010 [78–81]. Volume 3, Issue 3, 529-563. AIMS Microbiology AIMS Microbiology 544 AIMS Microbiology 2.10.Staphylococcus aureus Staphylococcus aureus are nonmotile, gram-positive cocci that appear singly or in pairs, tetrads, short chains, or characteristic “grapelike” clusters. Staphylococci are facultative anaerobes that, with the exception of Staphylococcus saccharolyticus and Staph. aureus subsp. anaerobius, grow more rapidly under aerobic conditions [7]. Staphylococcus spp. are widespread throughout nature and can be found on the skin and skin glands of mammals and birds, in addition to the mouth, blood, mammary glands, and intestinal, genitourinary, and upper respiratory tracts of infected hosts [7]. Outside the body, Staph. aureus can survive for long periods of time in a dry state, and have been isolated from air, dust, sewage, and water, making it one of the most resistant non-spore-forming pathogens [5]. In addition to environmental sources of infection, some reported Staph. aureus containing foods include ground beef, pork sausage, ground turkey, salmon steaks, oysters, shrimp, cream pies, milk, and delicatessen salads [7]. Staph. aureus grow, depending on the strain, at temperatures ranging from 7 to 47.8 °C and produce enterotoxins between 10 and 46 °C but prefer an optimum temperature between 40 and 45 °C. The bacterium grows between pH 4.5 and 9.3, with an optimum between 7.0 and 7.5, and is very tolerant to high levels of salt (>10% sodium chloride); enterotoxin production requires a minimum aw of 0.86, whereas growth has been demonstrated at an αw of 0.83 [5,7,10]. Staph. aureus typically causes infections involving the skin, such as boils, cellulitis, impetigo, and postoperative wound infections, but can also be associated with more serious infections like bacteremia, pneumonia, osteomyelitis, cerebritis, meningitis, and abscesses of muscle, urogenital tract, central nervous system, and various abdominal organs [7]. Toxic shock syndrome, a condition resembling septic shock and resulting from the production of toxic shock syndrome toxin 1, has been attributed to Staph. aureus infection [7]. Humans are the major reservoir for Staph. aureus, and contamination of food can occur through direct contact, indirectly by skin fragments, or through respiratory tract droplets, with most staphylococcal food poisoning cases being traced to food contamination during preparation because of inadequate refrigeration, inadequate cooking or heating, or poor personal hygiene. After ingestion of the enterotoxin and an incubation period of less than 6 and up to 10 h, symptoms may include vomiting, nausea, abdominal cramps, headache, dizziness, chills, perspiration, general weakness, muscular cramping and/or prostration, and diarrhea that may or may not contain blood [7]. 2.9. Shigella spp. dysenteriae type 1 is the most frequent cause of epidemic dysentery and is associated with a particularly severe form of the illness that may be accompanied by other complications including HUS [82]. Twenty-one (32%) of 65 football players and staff developed shigellosis that was associated with consumption of cold sandwiches; the sandwiches were prepared at the airline flight kitchen [83]. Confirmed or probable shigellosis was identified among 240 passengers on 219 flights to 24 states, the District of Columbia, and four countries between September 14 and October 13 [83]. Outbreaks associated with fresh produce have emerged as an important public health concern. On 10 August 1998, the Ontario Ministry of Health was notified of a family of three persons with S. sonnei infection who attended a food fair during July 31-August 3 [84]. Laboratory-based surveillance identified 32 additional persons with S. sonnei infection who had eaten at a specific kiosk at the fair or at the restaurant that had supplied the kiosk [84]. Foodhandlers at six (75%) of the eight implicated restaurants reported washing parsley before chopping it; usually parsley was chopped in the morning and left at room temperature, sometimes until the end of the day, before it was served to customers [84]. Volume 3, Issue 3, 529-563. AIMS Microbiology AIMS Microbiology 545 2.11.Vibrio spp. The genus Vibrio, belonging to the family Vibrionaceae, contains more than 35 species, of which nearly half have been described in the last 20 years and more than one-third are pathogenic to humans [7]. Organisms in this genus are non-spore-forming, primarily motile, facultatively anaerobic, Gram-negative straight or curved rods. All pathogenic Vibrio species, including Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus, require sodium for optimum growth. They are found primarily in brackish or marine environments located in tropical or temperate areas, because their incidence decreases significantly as water temperature falls below 20 °C [7]. V. cholerae are also motile by means of a single polar-sheathed flagellum; these curved rods thrive in their environmental reservoir as part of the microflora found in estuaries. In addition to its primary environmental source, V. cholerae has been isolated from areas not associated with a marine or brackish water supply, including freshwater lakes and rivers and from birds and herbivores [7]. Vibrio cholerae O1 is composed of the classic biogroup that has been isolated during previous pandemics and El Tor, which is the predominant biogroup of the current pandemic [86]. The optimum temperature for growth of V. cholerae is between 30 and 37 °C, although growth can occur between 10 and 43 °C. Vibrio cholerae grow at pH 5.0–9.6 but prefer a pH of 7.6. They grow at a aw of at least 0.97 but prefer 0.984. Optimum growth occurs in an environment with a sodium chloride concentration of 0.5%, although V. cholerae growth can occur at concentrations of 0.1–4.0% [10]. V. cholerae typically gain entrance into the human body through ingestion of a contaminated food, such as mollusks (raw oysters) or crustaceans eaten raw, undercooked, or even contaminated after cooking, or exposure of an open wound to a contaminated water source. Conditions resulting from V. cholerae O1 infection range from asymptomatic to the most severe form known as “cholera gravis” and in part depend on which biogroup is involved, because 75% of the El Tor biogroup and 60%) of the classic biogroup lead to asymptomatic infections [7]. Additionally, the El Tor biogroup results in severe disease in 2% of the infected individuals and mild or moderate disease in 23% whereas the classic biogroup produces severe disease in 11%, of individuals and mild or moderate disease in 30% [87]. 2.10.Staphylococcus aureus The CDC estimates that, in the US, staphylococcal food poisoning causes approximately 241,188 illnesses, 1,064 hospitalizations, and 6 deaths each year [5]. The presence of Staph. aureus in food may be considered a public health hazard because of its ability to produce enterotoxin and the risk of subsequent food poisoning. Although there are nine identified staphylococcal enterotoxins, designated as A, B, C1, C2, C3, D, E, F, and G, types A and D are responsible for the majority of the outbreaks [85]. Staphylococcal enterotoxins are included in a larger family of toxins, known as pyrogenic toxins, that have the unique ability to act as superantigens, thereby stimulating an extraordinarily high percentage of T cells. They are difficult to inactivate with heat, because temperatures required to inactivate them are higher than those needed to kill the organism [7]. Staphylococcal enterotoxin A is more heat sensitive than enterotoxins B or C and requires heating at 80 or 100 °C for 180 or 60 s, respectively, to cause a loss in serological reactivity [7]. Volume 3, Issue 3, 529-563. AIMS Microbiology AIMS Microbiology 546 AIMS Microbiology 2.11.Vibrio spp. After an incubation period of several hours to 5 days, depending on inoculum size and the amount of food ingested, typical symptoms include muscle cramping caused as a result of severe dehydration (fluid loss up to 500–1000 ml/h) resulting from vomiting, increased peristalsis followed by loose stools progressing to watery stools, and mucus-flecked diarrhea that is characteristic of cholera [88]. In addition to dehydration, other complications may include hypovolemic shock, hypoglycemia, and metabolic acidosis [88]. The disease caused by V. cholerae O139 Bengal is clinically identical to the symptoms exhibited by V. cholerae O1-infected individuals. Other V. cholerae serogroups, in addition to V. cholerae O1 and V. cholerae O139 Bengal, are known as non O1, non agglutinating vibrios or noncholera vibrios and are not known to cause epidemic disease. However, noncholera vibrios are known to cause self-limiting gastroenteritis and also may cause wound infections, bacteremia, and septicemia when associated with a preexisting liver condition [89]. The infectious dose of V. cholerae is approximately 1011, but with the ingestion of food, the infectious dose is reduced to about 106 depending on the buffering capacity of the food [87]. Food sources implicated as vehicles of transmission for Vibrio parahaemolyticus include crabs, prawns, scallops, seaweed, oysters, and clams [7]. V. parahaemolyticus grow at temperatures Volume 3, Issue 3, 529-563. AIMS Microbiology AIMS Microbiology 547 between 5 and 44 °C, with an optimum temperature and pH for growth between 30 and 37 °C and 7.6 and 8.6, respectively; the organism will grow in an environment at pH 4.8–11.0, in sodium chloride concentrations of 0.5–10.0%, and in environments with a minimum aw of 0.94; however, it prefers a concentration of sodium chloride in the range of 2 to 4% and a αw of 0.981 [7,10]. p g [ ] V. parahaemolyticus is the Vibrio species most frequently isolated from clinical samples obtained in the US [7]. Gastroenteritis is typically associated with consumption of raw, inadequately cooked, or cooked but recontaminated seafood. After a 4 to 96 h incubation period, symptoms of V. parahaemolyticus induced gastroenteritis include nausea, vomiting, headache, abdominal cramps, slight fever, chills, and watery diarrhea that is occasionally bloody [7]. Additional symptoms, after exposure to contaminated water, may include infected wounds, eyes, and ears [7]. Although symptoms are usually self-limiting, lasting only 2–3 days, severe cases may result in dysentery, primary septicemia, or cholera-like illness with the possibility of death [87]. 2.11.Vibrio spp. The presence of a pre-existing condition (e.g., liver disease, alcoholism, diabetes mellitus, antacid medication, peptic ulcer disease, immune disorder, etc.) greatly enhances the likelihood of developing a clinical syndrome such as gastroenteritis, wound infection, or septicemia [7]. V. parahaemolyticus possess four hemolytic components, including a thermostable direct hemolysin (TDH), a thermolabile direct hemolysin, phospholipase A, and lysophospholipase [7]. V. parahaemolyticus are invasive and can penetrate the lamina propria and enter circulation, as they have been found in the heart, spleen, pancreas, and liver [85]. During the past two decades in China, V. parahaemolyticus has been the most common cause of the bacterial foodborne outbreaks and among the leading causes of bacterial foodborne disease outbreak in many Asian countries, including Japan and India [88]. For the years 2003–2008 V. parahaemolyticus gastroenteritis outbreaks in 12 provinces were investigated from China National Foodborne Diseases Surveillance Network. 322 gastroenteritis outbreaks due to V. parahaemolyticus were reported, resulting in 9,041 illnesses and 3,948 hospitalizations [89]. A single food commodity was implicated in 187 (58%) outbreaks, of which 58 (31%) involved meat and meat products, and 52 (28%) involved aquatic products [89]. Outbreaks most frequently occurred in restaurants (39%), cafeterias (30%), and private residences (15%); to prevent and control V. parahaemolyticus gastroenteritis outbreaks, food workers and consumers should receive training on avoiding cross contamination of ready-to-eat foods with uncooked seafoods, particularly in warm weather months [89]. V. parahaemolyticus infection has been considered the leading cause of bacterial illnesses mainly associated with seafood consumption in Guangdong province in China [90]. From 2010 to 2014, 71 outbreaks due to V. parahaemolyticus were reported China National Foodborne Diseases Surveillance Network, resulting in 933 illnesses and 117 hospitalizations without death [90]. A food item was implicated if V. parahaemolyticus was isolated from food or based on epidemiologic evidence; aquatic products (27 outbreaks, 38.0%), meat and meat products (9 outbreaks, 12.7%), plant-based foods (6 outbreaks, 8.4%), mixed foods (5 outbreaks, 7.0%) were the most commonly implicated foods. Outbreaks most frequently occurred in restaurants (50.7%), private residents (21.1%), and cafeteria (12.7%) [90]. In order to prevent V. parahaemolyticus outbreaks caused by cross contamination, improper cooking and improper storage in high-temperature seasons, regulations for seafood safety from the production stage to the consumption stage should be strengthened [90]. Consumption of raw shellfish, primarily oysters, was linked to several multistate V. parahaemolyticus illness outbreaks in the US [88]. Animal-based (i.e. AIMS Microbiology 2.12.Yersinia enterocolitica The genus Yersinia belongs to the family Enterobacteriaceae and includes 10 established species, although only 3 are considered pathogenic to either humans or animals. Yersinia pestis is the causative agent of plague, Yersinia pseudotuberculosis is primarily an animal pathogen but may infect humans after the ingestion of contaminated food or water, and Yersinia enterocolitica has surfaced as a cause of foodborne gastroenteritis in humans [7,91]. Yersinia spp. are Gram-negative or gram-variable, non-spore-forming rods that grow under both aerobic and anaerobic conditions but are considered facultative anaerobes. With the exception of Y. pestis, all Yersinia spp. possess peritrichous flagella and are motile at 22–30 °C but not at 37 °C [7]. Twenty-six member states reported 7,202 confirmed cases of yersiniosis in 2015, making it the third most commonly reported zoonosis in the EU [4]. The EU notification rate was 2.20 cases per 100,000 population which was 6.8% higher than in 2014 [4]. There was a statistically significant decreasing 8-year trend in 2008–2015; Y. enterocolitica was the most common species reported to be isolated from human cases [4]. The most common serotype was O:3 followed by O:9 and O:5,27. No fatalities were reported among the 4,304 confirmed yersiniosis cases for which this information was reported in 2015 [4]. Y. enterocolitica are widely distributed throughout the environment and have been isolated from raw milk, sewage-contaminated water, soil, seafood, humans, and many warm-blooded animals such as poultry and, most importantly, pigs [7]. As a psychrotroph, Y. enterocolitica may pose a health hazard in contaminated refrigerated foods, although under refrigeration temperatures the pathogen is usually outgrown by other competing psychrotrophs [92]. Y. enterocolitica grow at temperatures between 0 and 45 °C but prefer an optimum temperature between 25 and 30 °C [7]. This psychrotroph can survive alkaline conditions as well as any other gram-negative bacterium but does not survive well in acidic environments, because growth occurs between pH 4.0 and 10.0, with pH 7.6 being optimum [7]. Additionally, Y. enterocolitica can grow in the presence of sodium chloride at concentrations as high as 5% [7,10]. Not all serotypes of Y. enterocolitica are enteropathogenic, and the specific serotypes of Y. enterocolitica involved in hunian yersiniosis are prevalent primarily in swine. Ingestion of contaminated water or food. more specifically raw or undercooked pork, is a source of foodborne infection in humans, resulting in symptoms appearing after an incubation period of a few days to a week. 2.11.Vibrio spp. meats, such as poultry, internal organs, beef, deli meat and cured meat, and aquatic products, such as crustacean, mollusks and fish) Volume 3, Issue 3, 529-563. AIMS Microbiology AIMS Microbiology 548 foods were the most common single commodity reported (61%), followed by mixed foods (19%), and other foods (17%) [88]. foods were the most common single commodity reported (61%), followed by mixed foods (19%), and other foods (17%) [88]. 3. Foodborne Viruses Viruses are particulate in nature and multiply only in other living cells. Thus, they are incapable of survival for long periods outside the host. More than 100 types of enteric viruses have been shown to cause foodborne illness; the most common foodborne virus pathogens are Hepatitis A and Noroviruses. These viruses are frequently transmitted via food; bivalve molluscs, such as clams, cockles, mussels, and oysters, are especially prone to transmit viruses. The waters in which they grow are increasingly subject to human fecal contamination, sometimes from sewage discharges and sometimes from infected shellfish harvesters. The shellfish collect viruses in the course of their filter feeding activity. Human viruses do not infect these species, but they are harbored for days or weeks in the shellfish digestive tract and are apparently more difficult to remove than bacteria during processes intended to cleanse the shellfish (e.g. depuration) [94,95]. Unlike many other seafoods, shellfish are usually eaten with their digestive tracts in place. They are often eaten raw or lightly cooked. Shellfish, unlike other foods, may also protect viruses from thermal inactivation during cooking [96]. 2.12.Yersinia enterocolitica Intestinal yersiniosis may persist for 1–2 weeks in adults and as long as 4 weeks in children and may include symptoms such as watery, sometimes bloody, stools or bloody diarrhea in conjunction with fever, vomiting, and abdominal pain [5,7]. Immunocompromised individuals and children under the age of 15 are most commonly infected, and extraintestinal infections associated with yersiniosis include septicemia, meningitis, Reiter syndrome, myocarditis, glomerulonephritis, thyroiditis, and erythema nodosum [85,91]. Y. enterocolitica toxin is heat stable, resists enzymatic degradation, remains stable during prolonged storage, and is of similar pH stability as the thermostable enterotoxin produced by ETEC [92]. In July 2011, a cluster of Y. enterocolitica infections was detected in southwestern Pennsylvania, US [92]. The outbreak was investigated for the source, in order to prevent further transmission; Volume 3, Issue 3, 529-563. AIMS Microbiology AIMS Microbiology 549 twenty-two persons were diagnosed with yersiniosis; 16 of whom reported consuming pasteurized dairy products from a local dairy [92]. Because consumption of pasteurized milk is common and outbreaks have the potential to become large, public health interventions such as consumer advisories or closure of the dairy must be implemented quickly to prevent additional cases if epidemiological or laboratory evidence implicates pasteurized milk as the outbreak source [92]. In addition, Y. enterocolitica serogroup O:8 was isolated from 24 fecal specimens of 21 patients and 3 kitchen staff in an outbreak in Japan; fresh vegetable salad was confirmed as the incrimination food of this outbreak [93]. AIMS Microbiology 3.1. Hepatitis A Hepatitis A virus particles are environmentally hardy organisms that can be transmitted by contaminated food, water, environmental surfaces (e.g., contaminated table tops, cooking utensils) and through direct or indirect person-to-person contact [5]. Hepatitis A cannot grow in the environment, however, they are considered to be extremely stable under a wide range of environmental conditions, including freezing, heat, chemicals, and desiccation [5]. Although Hepatitis A share some major characteristics with other genera of the picornavirus family, it is sufficiently different that it is classified as the only species in the genus Hepatovirus [97]. There are six Hepatitis A genotypes (I-VI), as determined by RNA sequence analysis. Genotypes I, II, and III contain strains associated with human infections, with the majority of human strains grouped within genotypes I and III. The virus is comprised of single positive-stranded RNA genome of approximately 7.5 kilobases and is a non-enveloped (i.e., no lipid-containing envelope), hydrophobic virus 22 to 30 nm in size [5]. The first recorded outbreak of shellfish-associated viral disease resulted from storing clean oysters in a fecally contaminated harbor while awaiting sale [98,99]. Over 600 cases of Hepatitis A resulted. More recently, outbreaks of viral gastroenteritis and Hepatitis A have been associated with eating usually uncooked shellfish. A clam-associated outbreak of Hepatitis A in Shanghai may have been the largest recorded outbreak of foodborne disease in history, with 292,301 cases [98]. Sporadic viral illnesses associated with shellfish have also been demonstrated [99]; it is difficult to avoid bias entirely in such studies because, at least in coastal states, a diagnosis of Hepatitis A regularly leads to asking the patient about shellfish consumption, to the exclusion of other foods. Shellfish-growing AIMS Microbiology Volume 3, Issue 3, 529-563. 550 waters are typically monitored for fecal contamination by testing for bacteria of the fecal coliform group or for Escherichia coli. The presence of these bacteria, however, has been shown to be a poor predictor of the presence of human enteric viruses [100]. Unfortunately, no more accurate index of the presence of viruses in shellfish or their growing waters has yet been identified. Because it has no other way to guarantee the safety of raw cockles, the U.K. government allows their sale only if cooked by an approved method. In 2003, a series of Hepatitis A outbreaks resulted in 1,000 cases of illness across multiple states and 3 deaths. 3.1. Hepatitis A The outbreaks were linked to green onions imported from four farms in Mexico where hepatitis A is endemic and the FDA subsequently banned imports from these farms [101]. The multinational Hepatitis A outbreaks occurring in Europe in 2013 and 2014 with over 1,400 cases linked to fresh and frozen strawberries and berry mix evidenced the usefulness of virus sequencing to link sporadic cases reported in different EU countries in outbreaks [102,103]. However, due to different sequencing practices and protocols in EU, the interpretation of the sequencing results was often challenging and untimely. Molecular data based on WGS are increasingly replacing the numerous different methodologies currently in use in human and veterinary reference laboratories for outbreak investigation and attribution modeling. These methods have the potential for early identification of foodborne organisms with epidemic character and the resulting data is beginning to be integrated into risk assessment studies. The epidemic potential of a virus genotype or even a subtype, may vary considerably, and is a function of its inherent genetic characteristics and their capacity to mutate, survive and spread through the food chain. The numbers of reported foodborne illnesses are fewer than actually occur because the CDC’s passive data collection system records only illnesses occurring as outbreaks, rather than those occurring sporadically. Hepatitis A, which is notoriously under reported in the US [104], is the only foodborne viral disease in which official reporting is mandatory for all diagnosed cases. Thus, records of the incidence of the other viral diseases are certain to be less accurate. 3.2. Noroviruses Norovirus cause the majority of acute viral gastroenteritis cases worldwide, including an estimated 5.4 million episodes of foodborne illnesses in the US annually [13]. In addition, according to the WHO, Norovirus is nowadays the leading cause of acute gastroenteritis among children less than 5 years of age who seek medical care [105]. Noroviruses are nonenveloped viruses with a diameter of 30–35 nm and a single-stranded RNA genome of approximately 7.5 kb. The viruses are very diverse and are classified into six genogroups of which only three cause infection in humans; within these genogroups, 30 genotypes have been described to date [106]. Recent improvements to diagnostic techniques have allowed researchers to describe the significant contribution of this highly infectious RNA virus to the burden of food-borne illness, particularly as the cause of numerous outbreaks of food-borne disease in community settings such as nursing homes, hospitals, the military, and cruise ships [107,108]. Fecal-oral spread is the primary mode of transmission. The virus’s abilities to withstand a wide range of temperatures (from freezing to 60 °C) and to persist on environmental surfaces and food items contribute to rapid dissemination, particularly via secondary spread (via food handlers or to family members) [108]. Food can be contaminated at the source (via contaminated water) or during preparation [108]. Prevention of infection is difficult because these viruses can persist on AIMS Microbiology AIMS Microbiology Volume 3, Issue 3, 529-563. 551 environmental surfaces and food items. Comparison of Norovirus sequences collected from around the world over the past decade have raised the possibility that pandemic strains of Norovirus are spread through foods sold internationally, or through person-to-person contact when travelers carry the virus [108,109]. Recent evidence suggests the possibility of animal reservoirs, but direct zoonotic transmission appears to be rare [110]. Cruise ships provide ideal conditions for the introduction and the rapid, global spread of Norovirus infection. Thousands of passengers from different geographic areas are transported in close quarters to multiple destinations around the world. Passengers and crew often disembark at multiple ports throughout the cruise where they can sample the local foods and culture. Cruise ships account for 10% of all reported outbreaks of Norovirus in the US [13]. With the average carrying capacity of a cruise ship now exceeding 2,500 passengers and crew, these outbreaks often affect a large number of people. 3.2. Noroviruses In 2010, outbreaks of diarrhea and vomiting among passengers and crew on the Celebrity Cruise ship “Mercury” occurred during three consecutive sailings. More than 10–22% of the passengers and 2–4% of the crew fell ill during each trip, resulting in a total of 1,058 cases of illness over the course of a month [111]. An outbreak of Norovirus gastroenteritis that affected as many as 24 players and staff members from 13 National Basketball Association teams were affected with gastroenteritis symptoms was reported [112]. Four of 5 stool specimens from ill players and staff tested positive for Norovirus genogroup II, with the majority of illness occurring during the first week of December 2010; epidemiologic and laboratory evidence strongly suggested that person to person transmission occurred within at least 1 team during this outbreak [112]. In another study, 286 fecal specimens from 88 oyster-associated gastroenteritis outbreaks were examined for the presence of 10 human enteric viruses using antigenic or genetic detection methods in order to determine the prevalence of these infections [113]. All virus-positive patients were over 18 years old. The most common enteric virus in outbreaks (96.6%) and fecal specimens (68.9%) was Norovirus, indicating a high prevalence of Norovirus infection associated with the consumption of raw or under-cooked oysters. Rapid identification of pathogens is important for the development of means for control and prevention. The results of the present study will be useful to establish an efficient approach for the identification of viral pathogens in oyster-associated gastroenteritis in adults [113]. Norovirus outbreaks occur frequently in EU and it can be difficult to establish whether apparently independent outbreaks have the same origin [114]. Six outbreaks linked to frozen raspberries, were investigated separately over a period of 3 months. In one outbreak at a hospital canteen, frozen raspberries was associated with illness by cohort investigation. Bags of raspberries suspected to be the source were positive for genogroup I and II Noroviruses, one typable virus was genotype GI.6 [114]. These molecular investigations showed that the apparently independent outbreaks were the result of one contamination event of frozen raspberries. The contaminated raspberries originated from a single producer in Serbia and were originally not considered to belong to the same batch. The outbreaks led to consultations and mutual visits between producers, investigators and authorities. Further, Danish legislation was changed to make heat-treatment of frozen raspberries compulsory in professional catering establishments [115]. AIMS Microbiology 4. Foodborne Parasites Parasites are one-celled microorganisms without a rigid cell wall, but with an organized nucleus. They are larger than bacteria. Like viruses, they do not multiple in foods, only in hosts. The transmissible form of these organisms is termed a cyst. Parasites are organisms that derive nourishment and protection from other living organisms known as hosts. They may be transmitted from animals to humans, from humans to humans, or from humans to animals. Several parasites have emerged as significant causes of foodborne and waterborne illness. These organisms live and reproduce within the tissues and organs of infected human and animal hosts, and are often excreted in faeces. The most common foodborne parasites are Cyclospora cayetanensis, Toxoplasma gondii and Trichinella spiralis. In 2015, 156 confirmed trichinellosis and 41 cases of congenital toxoplasmosis were reported in the EU. The EU notification was 0.03 cases per 100,000 population, and decreased by 57.1% compared with 2014 when the highest notification rate was reported since 2010 [4]. Lithuania reported the highest notification rate followed by Romania and Bulgaria. France reported data with 2-year delay, 216 confirmed congenital toxoplasmosis cases in 2014 [4]. The significant burden in low- and middle-income countries where cycles of parasitic infection are highly specific to food sources all over the world has been emphasized [118]. 357 million cases, 33,900 deaths and 2.94 million disability-adjusted life years (DALYs) are due to enteric protozoa of which 67.2 million cases, 5,560 deaths and 492,000 DALYs are attributable to foodborne transmission [118]. 3.2. Noroviruses Foodborne viruses cause considerable morbidity and mortality. Controlling them still means relying on good personal and food hygiene, good agricultural practice, post-harvest controls and effective management of human sewage to prevent onward transmission [115]. The role of the asymptomatic food handlers in contributing to Norovirus outbreaks is becoming increasingly clear, AIMS Microbiology Volume 3, Issue 3, 529-563. 552 with up to one-quarter of outbreaks attributable to them; handwashing with soap and water remains the best method for removing Norovirus from fingers [115]. However, hand sanitizers formulations supplemented with urea and citric acid may be more effective against viruses such as Norovirus [116]. with up to one-quarter of outbreaks attributable to them; handwashing with soap and water remains the best method for removing Norovirus from fingers [115]. However, hand sanitizers formulations supplemented with urea and citric acid may be more effective against viruses such as Norovirus [116]. Ri k t f t i i f i i th h th f d h i h ld i l d Risk assessment for transmission of emerging viruses through the food chain should include consideration of all means by which food could pose a hazard, that is not just consumption. New technologies have demonstrated the widespread nature of viral contamination in the food chain, but this does not necessarily correlate with the risk of disease. Finally, understanding people’s knowledge and behaviour is just as important as understanding virus characteristics and epidemiology when assessing risks of foodborne transmission [114]. 4.2. Toxoplasma gondii Toxoplasma gondii is a protozoan parasite member of the phylum Apicomplexa, and an obligate intracellular pathogen that is the causal agent of toxoplasmosis in humans. T. gondii uses cats as its primary reservoir and any other warm-blooded animal as an intermediate host [7]. The protozoan may be present as tachyzoites, bradyzoites, or sporozoites, which are the three stages of its life cycle. Tachyzoites and bradyzoites occur in body tissues, where the tachyzoites proliferate and destroy infected host cells and the bradyzoites multiply within tissue cysts. Sporozoites are shed, within oocysts, in cat feces where they sporulate after 1–5 days, surviving for months by utilizing their ability to resist disinfectants, freezing, and drying [7]. 17 genomes of T. gondii have been completed up to now according to the data retrieved from NCBI. The median total length of the genome is 64.1936 Mb [12]. In humans, T. gondii can be acquired in several ways, including the ingestion of contaminated food or water containing the oocyst, contaminated blood transfusion or organ transplantation, transplacental transmission, or accidental tachyzoite inoculation. T. gondii infections typically result from the ingestion of cysts in raw or undercooked meat, with fresh pork and beef appearing to be the primary sources [7]. Toxoplasmosis can result from the ingestion of as few as 100 tissue cysts or oocysts, at which time cyst walls rupture, releasing the sporozoites or bradyzoites to move through the intestinal epithelium and circulate throughout the body [7]. Sporozoites and bradyzoites transform into tachyzoites and begin to rapidly multiply intracellularly, and after host cell death, the tachyzoites invade adjacent cells and repeat the reproduction process; these tachyzoites, by means of the host immune response, are forced to transform back into bradyzoites and form cysts in the local tissue, where they can remain throughout the life of the host organism [7]. Toxoplasmosis symptoms include fever, rash, headache, muscle aches and pain, and swelling of the lymph nodes and may persist for more than a month [5]. T. gondii is one of the world’s most common parasites. Although cats are the only known host in which the parasite can complete its life cycle, this parasite can use almost all warm-blooded vertebrates, including humans, as hosts. T. 4.1. Cyclospora cayetanensis Cyclosporra cayetanensis are protozoan parasites, belonging to the family Eimeriidae, that inhabit the small intestine, where they spend the intermediary life cycle stages in the cytoplasm of enterocytes and subsequently produce oocysts containing two sporocysts encapsulating four sporozoites [7]. After subsequent shedding of the oocysts, 7–15 days are required for sporulation to occur. 2 genomes of C. cayetanensis have been completed up to now according to the data retrieved from NCBI. The median total length of the genome is 44.2991 Mb [12]. C. cayetanensis is capable of causing prolonged illness (6 weeks or longer) in both immunocompromised and immunocompetent individuals, with characteristic symptoms including nonbloody diarrhea, nausea, vomiting, anorexia, bloating, abdominal cramping, malaise, fever, and fatigue [7]. AIMS Microbiology Volume 3, Issue 3, 529-563. 553 Between 1996 and 1998 C. cayetanensis was identified as the etiologic agent in several outbreaks in the US and Canada involving raspberries, baby lettuce, and basil. Currently in the US, C. cayetanensis is estimated to cause about 15,000 cases of foodborne illness annually [3]. In 1996, 1,465 persons in 20 states, the District of Columbia, and two Canadian provinces became ill after consuming fresh raspberries that were imported from Guatemala and infected with the parasite C. cayetanensis [119]. AIMS Microbiology 4.3. Trichinella spiralis Trichinella spirulis is a parasitic roundworm belonging to the Phylum Nematoda, responsible for most human trichinosis infections. Besides humans, T. spiralis can infect most carnivorous mammals. 2 genomes of T. spiralis have been completed up to now according to the data retrieved from NCBI. The median total length of the genome is 56.7757 Mb [12]. The adult worms are 1.4–1.8 mm in size and are found embedded in the epithelium of the host’s small intestine, where females and males mate [7]. Female adults pass larvae into the blood stream and these reach muscle fiber where they encyst; larvae encysted in muscle remain viable for a long time [7]. The symptoms and pathogenicity are mainly due to the migrating and encystment process which cause pain, fever, edema, neurological disorders and even death. Adult nematodes live in the duodenal and jejunal mucosal epithelium, where they can exist for up to 8 weeks before they are expelled; during this transient period, adult female nematodes can release approximately 1,500 larvae into the bloodstream to travel around the body and subsequently enter muscle tissue, where they can survive for several years [7]. In skeletal muscle, larvae develop, mature, and undergo encapsulation in a calcified wall 6–18 months later. Both the larval and the adult stages are passed from the same host. Encysted larvae remain viable for up to 10 years and are freed by the stomach enzymes of the new host after the ingestion of the encysted flesh [7]. Symptoms, after an incubation period of 3–14 days, include nonspecific gastroenteritis, nausea, vomiting, headaches, fever, visual deficiencies, difficulty breathing, chills, night sweating, eosinophilia, myalgia, and circumorbital edema [7]. The nematode can be thermally inactivated, and therefore the USDA recommends cooking pork products to an internal temperature of 76.7 °C [7]. Currently in the US, T. spiralis is estimated to cause about 52 cases of foodborne illness annually, with a case fatality rate of 0.003 [3]. 4.2. Toxoplasma gondii gondii infections are estimated to cause approximately 87,000 illnesses, 4,400 hospitalizations, and 330 deaths each year in the US, making it the second leading cause of foodborne mortality in the US and the third leading cause of food-borne hospi- talizations [13]. The most common sources of toxoplasma are undercooked meat, animal feces, and transmission from mother to unborn child. While most people infected with toxoplasma experience no symptoms, unborn children (who contract it from their mothers) and adults with compromised immune systems risk serious side effects. An estimated 22.5% of the US population over the age of 12 has been infected with toxoplasma. For some countries, this figure is as high as 95%. Of particular concern are women of childbearing age who have not acquired immunity against this parasite since it can be transmitted via placenta to the fetus (congenital toxoplasmosis). The consequences of congenital toxoplasmosis range from mild to severe to fatal and include: mental Volume 3, Issue 3, 529-563. AIMS Microbiology 554 retardation, seizures, blindness and death [13]. T. gondii is highly amenable to experimental manipulation in the laboratory, and serves as a model system for genetic exploration of parasite biology and host-parasite interactions; this organism has been successfully used in transformation studies with genes from the closely related apicomplexan relative Plasmodium falciparum [13]. 5. Concluding Remarks The WHO Foodborne Disease Burden Epidemiology Reference Group provided in 2015 an estimate of global foodborne disease incidence, mortality, and disease burden in terms of DALYs [105]. The global burden of foodborne hazards was 33 million DALYs in 2010; 40% affecting children under 5 years of age. The US CDC estimated that each year roughly 48 million people in the US gets sick, 128,000 are hospitalized, and 3,000 die from foodborne diseases [120]. The number of confirmed cases, hospitalizations and deaths caused by the most common foodborne pathogens reported in the Foodborne Diseases Active Surveillance Network, US, 2015 is shown in Table 2. These major foodborne pathogens also represent an important economic concern; the annual economic impact in the US from health loss alone is estimated as more than $USD 77 billion [122]. A single outbreak from E. coli O104 in Germany was estimated to cost more than $USD 3.5 billion in medical costs and a further $USD 304 million was paid by the European Commission for crop losses due to not selling the Volume 3, Issue 3, 529-563. AIMS Microbiology AIMS Microbiology 555 fresh produce [123]. The economic impact of food safety outbreaks on food businesses has been analysed recently [124]. Table 2. Number of cases, hospitalizations and deaths caused by foodborne pathogens reported in the Foodborne Diseases Active Surveillance Network, US, 2015. able 2. Number of cases, hospitalizations and deaths caused by foodborne pathogens ported in the Foodborne Diseases Active Surveillance Network, US, 2015. Pathogen No of cases Hospitalizations (%) Deaths (%) Campylobacter spp. 6,309 1,065 (17) 11 (0.2) Listeria spp. 116 111 (96) 15 (12.9) Salmonella spp. 7,728 2,074 (27) 32 (0.4) Shigella spp. 2,688 619 (23) 1 (0.0) Shiga toxin-producing Escherichia coli O157 463 180 (39) 3 (0.6) Shiga toxin-producing Escherichia coli non-O157 796 126 (16) 1 (0.1) Vibrio spp. 192 47 (24) 5 (2.6) Yersinia spp. 139 37 (27) 1 (0.7) Parasites 1,676 272 (16) 8 (0.5) Total 20,107 4,531 77 After: [121]. Continued surveillance for foodborne disease outbreaks is important to reveal trends in the foods, regions and pathogens associated. In this field, genotype and subtype information from food contaminant strains is required to trace the transmission source, and to characterize and compare strains. The use of WGS as a tool for subtyping foodborne pathogen isolates has considerable potential for improving the detection of foodborne disease outbreaks, rapidly [125]. AIMS Microbiology 5. Concluding Remarks Furthermore, as suggested by subtyping data, different strains of foodborne pathogens are differently associated with human disease and such differences can be attributed, among others, to the hardy nature of certain strains enabling them to survive and proliferate in food-related environments, or to their increased virulence towards humans [126,127,128]. Hence, strain variability data can also facilitate the assessment of the relationships among various characteristics of foodborne pathogens including their virulence, distribution and epidemiology [129]. From the studies reviewed, the foods implicated in foodborne outbreaks are: fish and seafood [70,88,89,90,94,95,96,113], liver pâtè [24–30], chicken products [33,36], meat and meat products [37,51,52,68,76], ice cream [71], raw milk [31], rice dishes [15,16], pasta and pasta salad [18,19], peanuts [72], flour [61,62], cold sandwiches [83], fruit juices [35] and fresh produce [49,50,53–60,67,69,73,74,75,84,93,101,102,103,114]. Fresh produce have attracted great attention during the last 20 years, and it seems that there is some weakness of available international networks, as detection and real-time data show [130]. Risk-based food safety approach is significantly different, compared to the classical hazard-based approach, leading to a major shift in thinking about the ways that science and policy-making in food safety should interplay [131]. In this context, a food safety management system is aiming to estimate the risks to human health from food consumption and to identify, select and implement mitigation strategies in order to control and reduce these risks. According to the Codex Alimentarius, risk analysis is a process consisting of three components: risk assessment, risk management and risk AIMS Microbiology AIMS Microbiology Volume 3, Issue 3, 529-563. 556 communication [132,133]. It is now generally recognized that the new approach allows for a sharper diagnosis of food safety problems and the identification of effective mitigation strategies to properly deal with them [132]. communication [132,133]. It is now generally recognized that the new approach allows for a sharper diagnosis of food safety problems and the identification of effective mitigation strategies to properly deal with them [132]. Foodborne diseases are a global issue, and a unified and joint approach by all countries and the relevant international organizations is a prerequisite for the identification and control of all emerging foodborne problems that threaten human health and international trade [134]. Most foodborne illnesses are preventable despite being complex in their biology, analysis and epidemiology. Certainly, a combination of knowledge and skills across disciplines is required. 5. Concluding Remarks Public health agencies, regulatory agencies, the food industry and consumers need to make continuous efforts to prevent contamination of foods on the farm, in processing, in restaurants and homes. With suitable food safety education programs for all involved people, numbers of cases of foodborne illnesses could be minimized. Conflict of Interest The author declares no conflict of interests in this paper. References 1. 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W2027108152.txt	https://journals.plos.org/plosntds/article/file?id=10.1371/journal.pntd.0002716&type=printable	en		Comparison of Three Quality of Life Instruments in Lymphatic Filariasis: DLQI, WHODAS 2.0, and LFSQQ	PLoS neglected tropical diseases	2,014	cc-by	6,249	Comparison of Three Quality of Life Instruments in Lymphatic Filariasis: DLQI, WHODAS 2.0, and LFSQQ Cristina Thomas1, Saravu R. Narahari2, Kuthaje S. Bose2, Kuthaje Vivekananda2, Steven Nwe1, Dennis P. West1, Mary Kwasny3, Roopal V. Kundu1* 1 Department of Dermatology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America, 2 Department of Integrative Dermatology, Institute of Applied Dermatology, Uliyathadka, Kasaragod, Kerala, India, 3 Department of Preventative Medicine Biostatistics Collaboration Center, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America Abstract Background: The Global Program to Eliminate Lymphatic Filariasis aims to interrupt transmission of lymphatic filariasis and manage morbidity in people currently living with the disease. A component of morbidity management is improving healthrelated quality of life (HRQoL) in patients. Measurement of HRQoL in current management programs is varied because of the lack of a standard HRQoL tool for use in the lymphatic filariasis population. Methodology/Principal Findings: In this study, the psychometric properties of three health status measures were compared when used in a group of lymphatic filariasis patients and healthy controls. The World Health Organization Disability Assessment Schedule 2.0 (WHODAS 2.0), the Dermatology Life Quality Index (DLQI), and the Lymphatic Filariasis Quality of Life Questionnaire (LFSQQ) were administered to 36 stage II and stage III lymphatic filariasis subjects and 36 age and sex matched controls in Kerala, India. All three tools yielded missing value rates lower than 10%, suggesting high feasibility. Highest internal consistency was seen in the LFSQQ (a = 0.97). Discriminant validity analysis demonstrated that HRQoL was significantly lower in the LF group than in controls for the WHODAS 2.0, DLQI, and LFSQQ, but total HRQoL scores did not differ between stage II and stage III lymphedema subjects. The LFSQQ total score correlated most strongly with the WHODAS 2.0 (r = 0.91, p,0.001) and DLQI (r = 0.81, p,0.001). Conclusions/Significance: The WHODAS 2.0, DLQI, and LFSQQ demonstrate acceptable feasibility, internal consistency, discriminate validity, and construct validity. Based on our psychometric analyses, the LFSQQ performs the best and is recommended for use in the lymphatic filariasis population. Citation: Thomas C, Narahari SR, Bose KS, Vivekananda K, Nwe S, et al. (2014) Comparison of Three Quality of Life Instruments in Lymphatic Filariasis: DLQI, WHODAS 2.0, and LFSQQ. PLoS Negl Trop Dis 8(2): e2716. doi:10.1371/journal.pntd.0002716 Editor: Sabine Specht, University Clinic Bonn, Germany Received September 11, 2013; Accepted January 10, 2014; Published February 20, 2014 Copyright: ß 2014 Thomas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was funded by the Center for Global Health at Northwestern University Feinberg School of Medicine. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: I have read the journal’s policy and have the following conflicts: the Lymphatic Filariasis-Specific Quality of Life Questionnaire was developed at the Institute of Applied Dermatology. This does not alter our adherence to all PLOS policies on sharing data and materials. * E-mail: rkundu@nmff.org Health Organization Disability Assessment Schedule 2.0 (WHODAS 2.0), have been used in the LF population because of their applicability to a number of diseases and the ease of HRQoL comparison among diseases [8,12–14]. However, most generic instruments are only able to measure 50% of LF-associated problems, often overlooking feelings of fear and embarrassment [15]. Emphasis has now shifted to disease-specific tools that are better able to measure disease-specific factors as well as disease progression [15,16]. Despite the call for development of LFspecific tools, only two such tools exist, the LF QoL Tool (LFQoL) and the LF-specific QoL Questionnaire (LFSQQ) [7,10,17,18]. Of the two tools, the LFSQQ has been used to measure intervention efficacy and has been tested in a larger patient population. Because generic measures miss relevant HRQoL content and disease-specific tools have only recently been developed, LF HRQoL has been most often measured by the Dermatology Life Quality Index, a skin-specific HRQoL tool [11,19–21]. However, the DLQI has been found to assess only 24% of disability caused by LF [15]. Introduction Lymphatic filariasis (LF) is the second leading cause of disability worldwide, affecting almost 120 million people across 81 countries [1]. Although LF is not curable, the Global Program to Eliminate Lymphatic Filariasis has reduced disease transmission and is working to expand morbidity management programs globally [2,3]. Morbidity management is expected to greatly improve health-related quality of life (HRQoL) in LF patients because LF is characterized by disfiguring lymphedema and debilitating inflammatory episodes that cause significant disability and social isolation [4–6]. In fact, HRQoL and health status assessments have become primary measures of intervention efficacy in LF studies [7–11]. Despite the reliance on HRQoL in LF management, no consensus has been made on the most suitable tool for use in the LF population. Generic instruments, including a seven domains and five levels (7D5L) tool, the International Classification of Functioning, Disability and Health checklist, and the World PLOS Neglected Tropical Diseases \| www.plosntds.org 1 February 2014 \| Volume 8 \| Issue 2 \| e2716 QoL Tool Comparison in Lymphatic Filariasis Applied Dermatology in Kasaragod, Kerala, India and the Speciality Hospital in Kannur, Kerala, India. Subjects were enrolled if they had a clinical diagnosis of LF, had never been treated for LF, and were at least 18 years of age. LF staging was performed by physicians based on the International Society of Lymphology’s consensus staging system (Table 1) [22]. Thirty-six control subjects were age and sex matched to LF subjects and were recruited from Kasaragod, Kerala. Control inclusion criteria were: no history of LF diagnosis, no blood relation to cases included in the study, and at least 18 years of age. Author Summary Lymphatic filariasis affects approximately 120 million people and is the second leading cause of life-long disability worldwide. Because lymphatic filariasis is one of the World Health Organization’s six eradicable diseases, much effort has been placed into reducing transmission of the disease and managing morbidity. Novel interventions frequently use health-related quality of life as an outcome measure to monitor efficacy of the intervention. In an effort to delineate the strengths and weaknesses of health status measures and recommend use of a single tool in the lymphatic filariasis population, we compared the use of three health status tools (The World Health Organization Disability Assessment Schedule 2.0, the Dermatology Life Quality Index, and the Lymphatic Filariasis Quality of Life Questionnaire) in lymphatic filariasis subjects and healthy controls in Kerala, India. The Lymphatic Filariasis Quality of Life Questionnaire performed the best by discriminating well between subjects and controls, possessing significant content overlap with the other two tools, yielding a low missing value rate, and being internally consistent. This is the first study to compare health status measures in lymphatic filariasis subjects and provides insight into the use of the tools in quality of life analysis. Design and Instruments All subjects completed the WHODAS 2.0, DLQI, and LFSQQ in the local language of Malayalam. Because the WHODAS 2.0 was not available in Malayalam, the original English version was translated according to standard protocols [23]. Two native Malayalam speakers independently forward-translated the WHODAS 2.0 from English to Malayalam. Both translators and the local team assessed the tool’s clarity, cultural relevance, and language, and any differing opinions were discussed. Changes to the instrument were made as needed. This version was then backtranslated to English and confirmed to be equivalent with the original English version to ensure the validity of the Malayalam version. The DLQI and LFSQQ were available in Malayalam and have been validated for use [7,24]. The sequence of instrument administration was randomized to avoid an ordering bias. Demographic information was obtained via a demographic questionnaire. Table 2 lists the domains of each tool and outlines tool domains that cover similar content. The WHODAS 2.0 is a generic health and disability assessment tool that describes effects of disease on six domains: cognition, mobility, self-care, getting along, life activities, and participation in society [25]. Disability perception is measured by responses on a 5point scale from 1 (no difficulty) to 5 (extreme difficulty or cannot do). Final scores were calculated using a WHO SPSS 36 version syntax for employed subjects and a WHO SPSS 32 version syntax for unemployed subjects. The WHO SPSS 32 version syntax is identical to the WHO SPSS 36 version syntax with the omission of four questions relating to work ability. Domain and total scores range from 0 to 100 with a higher score indicating greater impairment of health status. The DLQI is a 10-item questionnaire that assesses skin-specific HRQoL through six domains: individual’s symptoms and feelings, daily activities, leisure, work and school, personal relationships, and treatment [26]. Questions are scored on a 4-point scale, resulting in a maximum score of 30, or large negative effect on HRQoL, and a minimum score of 0, or no effect on HRQoL. The LFSQQ was developed to assess quality of life in LF subjects through seven domains: mobility, self-care, usual activities, disease burden, pain/discomfort, psychological health, and social participation. Each item is scored on a 5-point scale (no problem, mild, moderate, severe, most severe). Total scores are calculated based on the number of questions answered and the Given the lack of a standard HRQoL tool for LF and minimal research comparing the various instruments, the aim of this study was to compare the relative performances of three health status measures in LF subjects and a matched control group. The tools chosen included a generic tool, the WHODAS 2.0, a skin-specific tool, the DLQI, and a disease-specific tool, the LFSQQ. Specifically, we examined four instrument psychometric properties: feasibility, reliability, discriminative validity, and construct validity. Methods Ethics Statement Ethical clearance was obtained from the institutional review boards at Northwestern University in Evanston, Illinois USA, the Institute of Applied Dermatology in Kasaragod, Kerala India, and the Speciality Hospital in Kannur, Kerala, India. Within the LF population in Kerala, written consent was unable to be obtained because subjects did not want their name associated with the diagnosis of LF due to the disease’s cultural stigma. Because of this limitation, verbal consent was obtained from all participants. As detailed in the IRB-approved protocol, the consent document was read aloud to participants and signed by the reader upon participant’s approval to signify the participant’s consent. Study Population In this cross-sectional study, a total of 36 LF subjects were consecutively enrolled from the outpatient units of the Institute of Table 1. International Society of Lymphology consensus staging system. Stage Criteria I Fluid accumulation that subsides with limb elevation. Pitting edema may be present. II Fluid accumulation that does not subside with limb elevation. Pitting edema may be present. III Fluid accumulation that does not subside with limb elevation and presence of trophic skin changes (hyperpigmentation, fat deposition, or warts) doi:10.1371/journal.pntd.0002716.t001 PLOS Neglected Tropical Diseases \| www.plosntds.org 2 February 2014 \| Volume 8 \| Issue 2 \| e2716 QoL Tool Comparison in Lymphatic Filariasis Table 2. Content comparison of WHODAS 2.0, DLQI, and LFSQQ. WHODAS 2.0 DLQI LFSQQ Cognition – Psychological Health Mobility – Mobility – Symptoms & Feelings Pain/Discomfort Disease Burden Getting Along Participation Personal Relationships Social Participation Self-Care – Self-Care Life Activities Leisure Work/School Daily Activities Usual Activities – Treatment – doi:10.1371/journal.pntd.0002716.t002 value rates (WHODAS 2.0: #0.1%, DLQI: 0.0%). Missing values for the LFSQQ domains were ,7%, except for the usual activities domain (32.9%). Less than 10% of data for total scores of all raw scores [7]. Scores range from 0 to 100 with a higher score indicating a higher HRQoL. Statistical Analyses Median scores and interquartile ranges for the WHODAS 2.0, DLQI, and LFSQQ were calculated for subject and control data. Discriminant ability, or the ability of a questionnaire to discriminate between respondent subgroups, was gauged by comparing HRQoL scores between subjects and controls using Wilcoxon Rank Sum tests. Further comparisons were made adjusting for demographic and disease-specific variables using linear regression models. A number of factors account for the overall feasibility of any tool, including cost, completion time, and ease of administration. In this study, feasibility was evaluated by examining the number of missing item responses. Internal consistency is a measure of the consistency of results yielded by items of a single construct. Cronbach’s a-coefficient was calculated to determine the internal consistency of the WHODAS 2.0 and the LFSQQ. Coefficient values $0.70 were deemed reliable [27]. Internal consistency values were not calculated for the DLQI domains because domains consisted of only one or two items. A test demonstrates construct validity if it accurately measures the construct it intends to measure. Construct validity was assessed by comparing correlations between related constructs of the three tools using Spearman’s rank correlation coefficients. Correlations were regarded as weak if the Spearman’s coefficient was less than 0.50, moderate if the coefficient was between 0.50 and 0.69, and strong if the coefficient was greater than 0.69. Table 3. Study subjects’ demographic data. Characteristic LF Subjects (n = 36) Controls (n = 36) No. of females 28 (77.8%) 28 (77.8%) Age in years (mean6SD) 51.7616.2 52.3616.1 Education* No education 13 (36.1%) 0 (0.0%) ,5 years completed 7 (19.4%) 4 (11.1%) 5–9 years completed 5 (13.9%) 5 (13.9%) 10–12 years completed 7 (19.4%) 18 (50.0%) College completed 4 (11.1%) 9 (25.0%) Unmarried 6 (16.7%) 8 (22.2%) Married/engaged 24 (66.7%) 23 (63.9%) Divorced 1 (2.8%) 0 (0.0%) Widowed 5 (13.9%) 5 (13.9%) Full-time 9 (25.0%) 18 (50.0%) Part-time 2 (5.6%) 0 (0.0%) Unemployed 1 (2.8%) 0 (0.0%) Marital Status Occupation Results Thirty-six subjects and 36 controls participated in this study. Demographic information for LF subjects and healthy controls are summarized in Table 3. For both groups, more females participated than males. LF subjects were more likely to have no education (36.1%) than controls in which no participants lacked education. All other variables were comparable between LF subjects and the control group including mean age (51.7 vs. 52.3 years), primary source of family income (16.7% vs. 25% as primary source), and poverty level (25% vs. 44.4% below poverty line). Disease-specific variables of subjects are shown in Table 4. Of the subjects, 58.3% had stage II lymphedema and 41.7% had stage III lymphedema. Housewife 23 (63.9%) 2 (5.6%) Retired 1 (2.8%) 16 (44.4%) 6 (16.7%) 9 (25.0%) Above poverty line 27 (75.0%) 20 (55.6%) Below poverty line 9 (25.0%) 16 (44.4%) Primary source of family income Poverty level Source of drinking water Tap 11 (30.6%) 7 (19.4%) Well 24 (66.7%) 28 (77.8%) Pond, lake, or body of water 1 (2.8%) 1 (2.8%) History of hypertension 10 (27.8%) 7 (19.4%) Feasibility History of diabetes 5 (13.9%) 5 (13.9%) Missing values of tools are shown in Table 5. Individual domains of the WHODAS 2.0 and DLQI showed low missing Significant difference between groups. doi:10.1371/journal.pntd.0002716.t003 PLOS Neglected Tropical Diseases \| www.plosntds.org 3 February 2014 \| Volume 8 \| Issue 2 \| e2716 QoL Tool Comparison in Lymphatic Filariasis The majority of correlations between WHODAS 2.0 and LFSQQ were moderate to strong (Table 8). Strong correlations were observed between the WHODAS 2.0 mobility and participation domains and LFSQQ domains. The total scores of both tools exhibited an especially high correlation (r = 20.912, p,0.001). Mobility and participation subscales of both domains displayed strong correlation with their corresponding counterparts (r = 20.917, p,0.001 and r = 20.829, p,0.001, respectively). In addition to the social participation domain of the LFSQQ, the WHODAS 2.0 participation domain also strongly correlated with the psychological health dimension of the LFSQQ (r = 20.855, p,0.001). Correlations between the LFSQQ and the DLQI are shown in Table 9. Total LFSQQ score highly correlated with DLQI total score (r = 20.808, p,0.001). Additionally, LFSQQ subscales related to disease burden, psychological health, and social participation strongly correlated with DLQI total score and two DLQI domains (symptoms & feelings and leisure). Table 4. LF subjects’ disease characteristics. Characteristic LF Subjects (n = 36) Disease stage at recruitment Stage II 21 (58.3%) Stage III 15 (41.7%) Disease activity Progressive 12 (33.3%) Stable 19 (52.8%) Improving 5 (13.9%) No. of limbs affected One limb Two limbs Wounds present on affected limb or limbs 31 (86.1%) 5 (13.9%) 8 (22.2%) Age in years at onset of disease (mean6SD) 33.3617.9 Duration of disease in years (mean6SD) 17.8614.8 No. of ADLA attacks annually (mean6SD) 3.664.8 Family history of disease 8 (22.2%) Discussion Comparisons of the psychometric properties of health status measures are useful in determining the appropriateness of a certain tool in a specific population. In this study, we examined the performances of three HRQoL tools (WHODAS 2.0, DLQI, LFSQQ) in the LF population and an age and gender matched control group. Although such comparisons have been performed in other chronic conditions [28–31], to our knowledge, our study is the first of its kind in the LF community. As such, it should be regarded as a preliminary step in bettering our understanding of doi:10.1371/journal.pntd.0002716.t004 instruments were missing (WHODAS 2.0 = 0.04%, DLQI = 0.0%, LFSQQ = 7.07%). Reliability Cronbach’s alpha coefficients are presented for the three tools in Table 5. Whole instrument reliability was highest for the LFSQQ (a = 0.97) as compared to the WHODAS 2.0 (a = 0.93) and DLQI (a = 0.73). Domains of the WHODAS 2.0 demonstrated higher internal consistency (mean a = 0.85; range = 0.76–0.91) than the domains of the LFSQQ (mean a = 0.80; range = 0.69–0.90). All domains of the WHODAS 2.0 and all but one domain of the LFSQQ (pain/discomfort) showed internal consistency above 0.70, the minimum value of acceptable internal consistency. Table 5. Missing values and internal consistency. Questionnaire N (%) missing Cronbach’s a WHODAS 2.0: 1 (0.04%) 0.93 Cognition (6) 0 0.85 0.91 Mobility (5) 0 Discriminant Validity Self-Care (4) 0 0.78 Descriptive statistics for each tool in LF subjects and the control group are summarized in Table 6. All three tools demonstrated lower HRQoL in LF subjects as compared to the control group. The LFSQQ domains discriminated best between the two groups as all but one of the domains (self-care) yielded p values ,0.001. For all three tools, domains relating to mobility, symptoms, and participation in society yielded notable differences between subject and control scores. Although the WHODAS 2.0, DLQI, and LFSQQ discriminated well between LF subjects and the control group, no global tool score was able to discriminate between stage II and stage III lymphedema subjects. LF stage discrimination was only noted with the DLQI symptoms subscale (p = 0.045) and the LFSQQ disease burden subscale (p = 0.040). Getting Along (5) 0 0.85 Life Activities (8) 0 0.88 Participation in Society (8) 1 (0.1%) 0.82 0 0.73 DLQI: Symptoms & Feelings (2) 0 Daily Activities (2) 0 Leisure (2) 0 Work & School (1) 0 Personal Relationships (2) 0 Treatment (1) 0 LFSQQ: 224 (7.07%) 0.97 Mobility (8) 1 (0.1%) 0.88 Self-Care (5) 25 (6.9%) 0.74 Construct Validity Usual Activities (7) 166 (32.9%) 0.80 Results of the correlation analysis between the WHODAS 2.0 and the DLQI are presented in Table 7. Correlations between the WHODAS 2.0 and DLQI were generally low or moderate, including correlations between corresponding domains such as the WHODAS 2.0 life activities domain and the DLQI daily activities domain. The strongest correlation between the WHODAS 2.0 and DLQI was noted between the two total scores (r = 0.748, p,0.001). Disease Burden (5) 11 (3.1%) 0.71 Pain/Discomfort (7) 20 (4.0%) 0.69 Psychological Health (7) 1 (0.2%) 0.89 Social Participation (5) 0 0.90 PLOS Neglected Tropical Diseases \| www.plosntds.org Number in parentheses indicates number of items. doi:10.1371/journal.pntd.0002716.t005 4 February 2014 \| Volume 8 \| Issue 2 \| e2716 QoL Tool Comparison in Lymphatic Filariasis This domain contained items assessing the effects of disease on activities such as agrarian work, gardening, and cleaning the floors. Because the tool does not allow for subjects to choose a ‘‘not applicable’’ option, subjects may have skipped items unrelated to their lifestyle rather than choose from the Likert scale ranging from ‘‘no problem’’ to ‘‘severe’’. Questionnaire length does not seem to have an appreciable effect on the number of missing values as the 10-item DLQI performed similarly to the 36–item WHODAS 2.0. Internal consistency was acceptable (a.0.70) for all domains examined, except the LFSQQ pain/discomfort subscale (a = 0.69). Internal consistency values were highest among WHODAS 2.0 domains, and values were similar to those obtained by the WHO in a global population (a range: 0.84–0.98) [25]. Discriminant ability analysis demonstrated that the LFSQQ scales distinguished best between LF subjects and the control group. The DLQI and WHODAS 2.0 also performed well, and among all tools, the largest differences in mean scores were noted in domains relating to mobility, symptoms, and participation. Although mobility and symptoms may be directly influenced by the physical manifestations of the disease, participation in society may be an indirect effect of the disease. This finding is in agreement with other studies that demonstrate that LF affects quality of life in a more nuanced manner than solely through the disease’s physical signs. Kumari et al. found that in addition to the physical burdens of LF, subjects also coped with shame and depression [4]. The psychological effects of social stigma result in delayed treatment as patients are embarrassed to reveal their condition in society [32]. These components of health status are critical to include in HRQoL measures of intervention efficacy because although a patient’s physical symptoms may regress with treatment, the psychological stress and disease stigma may persist and can influence HRQoL. Current disability and HRQoL tools do not encompass all psychosocial disabilities that affect the LF patient, and including domains outside the realm of social participation in HRQoL instruments may further highlight the difficulties faced by patients in this component of HRQoL [21]. Despite the ability of the DLQI, WHODAS 2.0, and LFSQQ to discriminate between LF subjects and the control group, no global tool score was able to differentiate between stage II and stage III lymphedema. Of the domains, only the LFSQQ disease burden domain and the DLQI symptoms domain showed significant, albeit weak, association with LF stage. This weak association is Table 6. WHODAS 2.0, DLQI, and LFSQQ score distribution. Questionnaire Patients Controls P WHODAS 2.0 Domains: Cognition 15 (3–20) 0 (0–3) ,0.001 Mobility 50 (19–69) 0 (0–13) ,0.001 Self-Care 5 (0–10) 0 (0–0) ,0.001 Getting Along 25 (0–33) 0 (0–0) 0.002 Life Activities 20 (0–40) 0 (0–5) 0.001 Participation in Society 25 (17–38) 0 (0–8) ,0.001 Symptoms & Feelings 3 (2–4) 0 (0–0) ,0.001 Daily Activities 1 (0–2) 0 (0–0) ,0.001 DLQI Domains: Leisure 1(1–3) 0 (0–0) ,0.001 Work & School 0(0–1) 0 (0–0) 0.001 Personal Relationships 0(0–1) 0 (0–0) 0.001 Treatment 0(0–1) 0 (0–0) 0.001 LFSQQ Domains: Mobility 67 (58–80) 100 (88–100) ,0.001 Self-Care 100 (82–100) 100 (100–100) 0.007 Usual Activities 92 (75–100) 100 (100–100) ,0.001 Disease Burden 63 (60–75) 100 (100–100) ,0.001 Pain/Discomfort 90 (83–96) 100 (98–100) ,0.001 Psychological Health 75 (64–86) 100 (95–100) ,0.001 Social Participation 67 (85–50) 100 (100–100) ,0.001 Median value is given and 25th–75th quartiles in parentheses. doi:10.1371/journal.pntd.0002716.t006 the specific benefits and disadvantages of these three tools in LF research. For all tools, feasibility was defined by the percentage of missing values per item. As the order of questionnaire administration was randomized, an ordering effect does not explain the LFSQQ’s relatively low observed feasibility as compared to the other tools. Instead, the relatively high number of missing values may reflect the irrelevance of certain items to subjects. For example, the usual activities domain of the LFSQQ yielded the most missing values. Table 7. Spearman correlation coefficients among the domains of WHODAS 2.0 and DLQI. DLQI Symptoms & Feelings Daily Activities Leisure Work & School Personal Relationships Treatment Total Cognition 0.493 0.443 0.465 0.264 0.221 0.110* 0.516 Mobility 0.632 0.367 0.563 0.369 0.428 0.313 0.654 Self-Care 0.445 0.332 0.345 20.061 0.298 0.259 0.405 Getting Along 0.356 0.239 0.365 20.060 0.298 0.021* 0.327 WHODAS 2.0 Life Activities 0.386 0.242 0.419 0.291 0.413 0.091* 0.434 Participation 0.661 0.501 0.672 0.266 0.400 0.351 0.708 Total 0.700 0.504 0.697 0.389 0.454 0.259 0.748 Not significantly different from zero. Strong correlations are bolded. doi:10.1371/journal.pntd.0002716.t007 PLOS Neglected Tropical Diseases \| www.plosntds.org 5 February 2014 \| Volume 8 \| Issue 2 \| e2716 QoL Tool Comparison in Lymphatic Filariasis Table 8. Spearman correlation coefficients among the domains of WHODAS 2.0 and LFSQQ. LFSQQ Mobility Self-Care Usual Activities Disease Burden Pain/Discomfort Psychological Health Social Participation Total WHODAS 2.0 Cognition 20.471 20.391 20.406 20.440 20.366 20.671 20.497 20.580 Mobility 20.917 20.513 20.518 20.697 20.734 20.620 20.720 20.860 Self-Care 20.513 20.552 20.371 20.526 20.427 20.447 20.534 20.580 Getting Along 20.375 20.434 20.271 20.340 20.283 20.452 20.501 20.458 Life Activities 20.521 20.555 20.648 20.446 20.411 20.493 20.559 20.595 Participation 20.711 20.433 20.632 20.658 20.558 20.855 20.829 20.828 Total 20.821 20.591 20.696 20.717 20.656 20.840 20.854 20.912 Strong correlations are bolded. doi:10.1371/journal.pntd.0002716.t008 Correlations between global scores of all instruments were high, suggesting that the tools cover similar measures of health status. Domains of the LFSQQ and WHODAS 2.0 were highly correlated. The mobility and participation domains of the WHODAS 2.0 were differentiated by the LFSQQ. As expected, domains of different instruments covering similar content often significantly correlated. However, frequently domains assessing different facets of HRQoL correlated strongly and at times, were more highly correlated than domains of similar names. For example, the life activities domain of the WHODAS 2.0 correlated more strongly to the symptoms & feelings domain of the DLQI than the DLQI’s daily activities domain. This finding may be related to the depth and breadth of topics covered by each domain. The life activities domain of the WHODAS 2.0 encompasses the effect of disease on housework and work/school in depth. The DLQI covers a broader range of topics, including the effects of disease on shopping, looking after the home, gardening, and clothing choices. This difference in extent of domain coverage may have implications on the comprehensiveness of any tool’s measure of HRQoL. As a cross-sectional study, there was no assessment of test-retest reliability of the instruments, a study limitation given the growing most likely because the International Society of Lymphology’s consensus staging system is based on the clinical assessment of disease signs rather than disability. Despite the importance of stage discrimination in the use of HRQoL as an outcome measure of intervention efficacy, the abilities of the DLQI, WHODAS 2.0, and LFSQQ to discriminate between LF stages is unknown. Conflicting results exist concerning the ability of the DLQI to distinguish between LF stages. Yahathugoda et al. noted a correlation between DLQI score and lymphedema stage, and a modified version of the DLQI was also found to correlate with LF stage [20,21]. In addition, McPherson et al. reported decreased DLQI score following a clinical intervention, but baseline DLQI was only weakly associated with lymphedema grade [11]. Similar to our results, a study conducted in eastern India did not find a correlation between LF grade and DLQI total score [19]. The LFSQQ and WHODAS 2.0 have not been studied extensively in regard to their use in discriminating LF stage. Given our findings, the questionnaires in their current form are not sensitive to LF stage and modifications to each of the tools to better correlate with disease stage would improve each instrument’s ability to accurately assess health status in LF subjects. Table 9. Spearman correlation coefficients among the domains of LFSQQ and DLQI. DLQI Symptoms & Feelings Daily Activities Leisure Work & School Personal Relationships Treatment Total LFSQQ Mobility 20.634 20.356 20.562 20.340 20.410 20.304 20.648 Self-Care 20.323 20.320 20.381 20.163 20.248 20.005* 20.334 20.492 Usual Activities 20.418 20.436 20.569 20.318 20.288 20.007* Disease Burden 20.757 20.514 20.699 20.381 20.400 20.285 20.799 Pain/Discomfort 20.611 20.379 20.632 20.307 20.244 20.232* 20.648 Psychological Health 20.767 20.546 20.702 20.386 20.363 20.370 20.801 Social Participation 20.730 20.496 20.692 20.354 20.364 20.319 20.765 Total 20.778 20.524 20.731 20.394 20.393 20.321 20.808 *Not significantly different from zero. Strong correlations are bolded. doi:10.1371/journal.pntd.0002716.t009 PLOS Neglected Tropical Diseases \| www.plosntds.org 6 February 2014 \| Volume 8 \| Issue 2 \| e2716 QoL Tool Comparison in Lymphatic Filariasis interest in LF treatment modalities and the increasing reliance on HRQoL tools that accurately measure change over time. In evaluating HRQoL, variations in tool applicability are expected based on differences in culture and lifestyle of targeted populations. In fact, Zeldenryk et al. noted 43 new HRQoL-related constructs in Bangladeshi focus groups that had not yet been described in the literature [33]. This study was conducted in a rural region of southern India, and thus, our results may not be generalizable to the global LF population. Finally, the study LF population was composed of only stage II and stage III lymphedema subjects. In the study region, stage I lymphedema subjects do not often present to clinic because of their minimal health changes and associated disabilities. Including stage I subjects in future studies of HRQoL tool comparison may shed light on correlations between LF stage and HRQoL. Although many tools have been used to assess HRQoL in the LF population, no consensus has been made on the gold-standard tool. Our aim in this study was to delineate the strengths and weaknesses of the WHODAS 2.0, the DLQI, and the LFSQQ. All three tools showed moderately good performance as measured by feasibility, internal consistency, construct validity and discriminant validity. Specifically, the LFSQQ demonstrated the highest overall internal consistency, construct validity, and discriminant validity. Based on our results, the LFSQQ may be the best tool of the three to accurately assess HRQoL in the LF population. However, use of the instrument in its current state is limited by its high missing value rate. We recommend the addition of a ‘‘not applicable’’ option on the LFSQQ to increase tool feasibility. HRQoL largely depends on the cultural behaviors of the population studied. Before the LFSQQ can be used globally, the tool may require further modifications to take into account culturally-relevant lifestyle activities of LF-endemic areas. We recommend validation of the tool in LF populations outside of India to ensure proper application of the instrument. Although our results suggest that the LFSQQ should be used, further research examining additional psychometric properties, respondent burden, ease of comprehension, and differences in clinically-relevant and research-relevant tools is needed to better select a HRQoL for clinical or research use. Supporting Information Table S1 Complete study dataset. (First tab) Dataset of demographics and disease characteristics. (Second tab) Dataset of DLQI domain scores. (Third tab) Dataset of LFSQQ domain scores. (Fourth tab) Dataset of WHODAS 2.0 scores. (XLSX) Text S1 Lymphatic Filariasis Quality of Life Question- naire. (PDF) Acknowledgments We gratefully acknowledge the work of Mrs. Jeong Hae Bae and Dr. Pranathi Lingam in obtaining ethical clearance from the study sites and manuscript review. We thank Dr. Andrew Finlay, Dr. Guruprasad Aggithaya, and the World Health Organization for permission to use the DLQI, LFSQQ, and WHODAS 2.0, respectively. This study was presented at the American Academy of Dermatology 2013 Summer Academy Meeting in New York, NY USA and the American Society of Tropical Medicine & Hygiene 62nd Annual Meeting in Washington, DC USA. Author Contributions Conceived and designed the experiments: CT SRN KSB DPW RVK. Performed the experiments: CT KV. Analyzed the data: CT MK. Contributed reagents/materials/analysis tools: CT SRN SN DPW RVK. Wrote the paper: CT MK RVK. References 12. Girois S, Brantus P, Mackenzie C (2007) Lymphatic filariasis disability prevention for field managers. Ghana: Noguchi Institute. 13. Harichandrakumar KT, Krishnamoorthy K, Kumari AK, Das LK (2006) Health status of lymphatic filariasis assessed from patients using seven domains five levels (7D5L) instrument. Acta Trop 99: 137–143. 14. World Health Organization (2003) Global programme to eliminate lymphatic filariasis annual report on lymphatic filariasis 2003. Geneva: World Health Organization. 15. Zeldenryk L, Gordon S, Gray M, Speare R, Melrose W (2012) Disability measurement for lymphatic filariasis: a review of generic tools used within morbidity management programs. PLoS Negl Trop Dis 6: e1768. 16. Institute of Applied Dermatology (2008) The third national seminar on evidence-based and integrated medicine for lymphatic filariasis, other chronic dermatoses and HIV/AIDS. 1–40 p. 17. Krishnamoorthy K, Kumari A, Harichandrakumar KT, Das LK (2008) Using HRQoL to assess the impact of morbidity management. Journal of Lymphoedema 3: 7–11. 18. Narahari SR, Bose KS, Aggithaya MG, Swamy GK, Ryan TJ, et al. (2013) Community level morbidity control of lymphoedema using self care and integrative treatment in two lymphatic filariasis endemic districts of South India: a non randomized interventional study. Trans R Soc Trop Med Hyg 107: 566– 577. 19. Babu BV, Nayak AN, Rath K, Kerketta AS (2006) Use of the Dermatology Life Quality Index in filarial lymphoedema patients. Trans R Soc Trop Med Hyg 100: 258–263. 20. Chandrasena TG, Premaratna R, Muthugala MA, Pathmeswaran A, de Silva NR (2007) Modified Dermatology Life Quality Index as a measure of quality of life in patients with filarial lymphoedema. Trans R Soc Trop Med Hyg 101: 245–249. 21. Yahathugoda TC, Wickramasinghe D, Weerasooriya MV, Samarawickrema WA (2005) Lymphoedema and its management in cases of lymphatic filariasis: the current situation in three suburbs of Matara, Sri Lanka, before the introduction of a morbidity-control programme. Ann Trop Med Parasitol 99: 501–510. 22. International Society of Lymphology (2009) The diagnosis and treatment of peripheral lymphedema. 2009 Concensus Document of the International Society of Lymphology. Lymphology 42: 51–60. 1. Michael E, Bundy DA, Grenfell BT (1996) Re-assessing the global prevalence and distribution of lymphatic filariasis. Parasitology 112 (Pt 4): 409–428. 2. Ottesen EA, Hooper PJ, Bradley M, Biswas G (2008) The global programme to eliminate lymphatic filariasis: health impact after 8 years. PLoS Negl Trop Dis 2: e317. 3. World Health Organization (2010) WHO global programme to eliminate lymphatic filariasis progress report for 2000–2009 and strategic plan 2010–2020. Geneva: World Health Organization. 4. Krishna Kumari A, Harichandrakumar KT, Das LK, Krishnamoorthy K (2005) Physical and psychosocial burden due to lymphatic filariasis as perceived by patients and medical experts. Trop Med Int Health 10: 567–573. 5. Person B, Addiss D, Bartholomew LK, Meijer C, Pou V, et al. (2007) A qualitative study of the psychosocial and health consequences associated with lymphedema among women in the Dominican Republic. Acta Trop 103: 90–97. 6. Suma TK, Shenoy RK, Kumaraswami V (2003) A qualitative study of the perceptions, practices and socio-psychological suffering related to chronic brugian filariasis in Kerala, southern India. Ann Trop Med Parasitol 97: 839– 845. 7. Aggithaya MG, Narahari SR, Vayalil S, Shefuvan M, Jacob NK, et al. (2013) Self care integrative treatment demonstrated in rural community setting improves health related quality of life of lymphatic filariasis patients in endemic villages. Acta Trop 126: 198–204. 8. Budge PJ, Little KM, Mues KE, Kennedy ED, Prakash A, et al. (2013) Impact of Community-Based Lymphedema Management on Perceived Disability among Patients with Lymphatic Filariasis in Orissa State, India. PLoS Negl Trop Dis 7: e2100. 9. Coreil J, Mayard G, Addiss D (2003) Support groups for women with lymphatic filariasis in Haiti. World Health Organization. 10. Kumari AK, Krishnamoorthy K, Harichandrakumar K, Das L (2007) Health Related Quality of Life, an appropriate indicator to assess the impact of morbidity management and disability prevention activities towards elimination of lymphatic filariasis. Filaria J 6: 8. 11. McPherson T (2003) Impact on the quality of life of lymphoedema patients following introduction of a hygiene and skin care regimen in a Guyanese community endemic for lymphatic filariasis: A preliminary clinical intervention study. Filaria J 2: 1. PLOS Neglected Tropical Diseases \| www.plosntds.org 7 February 2014 \| Volume 8 \| Issue 2 \| e2716 QoL Tool Comparison in Lymphatic Filariasis 23. Brislin R (1970) Back-translation for cross-cultural research. Journal of CrossCultural Psychology 1: 158–216. 24. Department of Dermatology & Wound Healing School of Medicine Cardiff University Dermatology Life Quality Index: Different Language Versions. 25. Ustun TB, Chatterji S, Kostanjsek N, Rehm J, Kennedy C, et al. (2010) Developing the World Health Organization Disability Assessment Schedule 2.0. Bull World Health Organ 88: 815–823. 26. Finlay AY, Khan GK (1994) Dermatology Life Quality Index (DLQI)–a simple practical measure for routine clinical use. Clin Exp Dermatol 19: 210–216. 27. Nunnaly J, Bernstein I (1978) Psychometric theory. New York: McGraw-Hill. 28. Ringbaek T, Martinez G, Lange P (2012) A comparison of the assessment of quality of life with CAT, CCQ, and SGRQ in COPD patients participating in pulmonary rehabilitation. COPD 9: 12–15. 29. Kemmler G, Holzner B, Kopp M, Dunser M, Margreiter R, et al. (1999) Comparison of two quality-of-life instruments for cancer patients: the functional assessment of cancer therapy-general and the European Organization for PLOS Neglected Tropical Diseases \| www.plosntds.org 30. 31. 32. 33. 8 Research and Treatment of Cancer Quality of Life Questionnaire-C30. J Clin Oncol 17: 2932–2940. Dougherty CM, Dewhurst T, Nichol WP, Spertus J (1998) Comparison of three quality of life instruments in stable angina pectoris: Seattle Angina Questionnaire, Short Form Health Survey (SF-36), and Quality of Life Index-Cardiac Version III. J Clin Epidemiol 51: 569–575. Kopp M, Schweigkofler H, Holzner B, Nachbaur D, Niederwieser D, et al. (2000) EORTC QLQ-C30 and FACT-BMT for the measurement of quality of life in bone marrow transplant recipients: a comparison. Eur J Haematol 65: 97– 103. Perera M, Whitehead M, Molyneux D, Weerasooriya M, Gunatilleke G (2007) Neglected patients with a neglected disease? A qualitative study of lymphatic filariasis. PLoS Negl Trop Dis 1: e128. Zeldenryk L, Gray M, Gordon S, Speare R, Hossain M (2013) The use of focus groups to develop a culturally relevant quality of life tool for lymphatic filariasis in Bangladesh. Qual Life Res 1–11. doi 10.1007/s11136-013-0455-0. February 2014 \| Volume 8 \| Issue 2 \| e2716
https://openalex.org/W2030702935	https://zenodo.org/records/1787439/files/article.pdf	English	null	MEDICINE, A DETERMINING FACTOR IN WAR	Journal of the American Medical Association	1,919	public-domain	11,119	President's address before the American Medical Association at the Seventieth Annual Session, Atlantic City, N. J., June, 1919. WORK OF THE LOCAL BOARDS In the work done by the medical profession in the war, one cannot pass by that accomplished in exam¬ ining the young men drafted into the army. This work of the local boards was not done by picked specialists or by men previously trained for it. The physicians of the boards were at first usually the county and city physicians appointed by the local sheriff, and the figures form an interesting study. Of the 10,000,000 (9,952,735) called and registered, 6,750,000 (6,744,289) were not examined. ' Of the one third who were examined—that is, of the 3,208,448—70.4 per cent, were found to be fully quali¬ fied, while 29.6 per cent, were found physically to be totally or partially disqualified. Of the 2,124,293 who were sent by the local boards to the camps and were then subjected to the careful and minute examination of experts, 91.9 per cent, were accepted and only 8.1 per cent, rejected. It is interesting to note that in the draft of 1917 the local boards rejected 29.1 per cent., and in 1918 they rejected 29.6 per cent. The more this work is studied, the more one. appreciates its extent, its far-reaching influence, and the high value of the work performed. RESPONSE OF MEDICAL PROFESSION TO CALL OF DUTY The medical profession of the United States needed no draft or conscription to answer the call to duty. About 20,000 of its members volunteered and thus expanded the small regular force of physicians in the Army and the Navy. At the outbreak of the war only 447 physicians as regulars, and about 1,600 of the Medical Reserve Corps comprised the entire commissioned personnel of the Medical Department in the regular army. In the navy, 329 surgeons, 161 naval reserve officers, and twenty-five contract sur¬ geons comprised the medical staff. When the armistice was signed there were 35,000 medical officers in the army and 3,000 in the navy, which represents about 26 per cent, of the entire profession of the country. Thi bili ti dd d th h i i h MEDICINE, A DETERMINING FACTOR IN WAR PRESIDENTIAL ADDRESS ALEXANDER LAMBERT, M.D. NEW york No one can be elected President of the American Medical Association and fail to appreciate the great honor conferred, or fail to realize the responsibilities which may rest on him. In the recent past these responsibilities have at times lain dormant, but at other times they have actively exerted their full force, and the influence wielded by the President of this Associa- tion has had great possibilities for good or evil on the medical profession. profession. A year ago the problems before us were the great responsibilities of war, the conduct of that war, and the means whereby it could be pushed to a successful issue. Today, our problems still loom large, for they are those of rearrangement and reconstruction. It seems timely, therefore, to consider what the medical pro- fession has done to fulfill its duty in the war, and what medical science has done to alleviate the dreadful horrors which, in the past, have followed in the wake of warring armies, hovering like terrible specters over the civil populations, and which sooner or later would take their death toll through epidemics of disease. We have also to consider the problems of the future and to base our actions on the lessons we have learned by the experiences through which we have so recently passed. MEDICINE, A DETERMINING FACTOR IN WAR PRESIDENTIAL ADDRESS ALEXANDER LAMBERT, M.D. NEW york MEDICINE, A DETERMINING FACTOR IN WAR PRESIDENTIAL ADDRESS ALEXANDER LAMBERT, M.D. NEW york Downloaded From: http://jama.jamanetwork.com/ by a University of Manitoba User on 06/04/2015 sacrifices, both by individuals and by communities, as was specially evidenced when the influenza epidemic swept over the country. But this is part of the price for war that peoples have ever paid. This mobiliza¬ tion of physicians was accomplished by the Surgeon- General's Office, aided by the Council of National Defense and the American Medical Association, both acting through the organized state and county socie¬ ties of the American Medical Association. The wis¬ dom and the necessity of these state organizations was never more completely demonstrated than in the last two years. Simultaneously, the justification for The Journal of the Association, as a medium through which information could be quickly disseminated, and its great influence among physicians, were never shown more clearly. The men in the executive positions of the American Medical Association and the Council of National Defense deserve the greatest credit and deserve the acknowledgment of work well done. This tribute is but their just due, and it is with keen pleasure that I take the responsibility of speaking for the pro¬ fession and publicly acknowledging it. SUCCESS OF PREVENTIVE MEDICINE Few realize how crucial has.been the test of pre¬ ventive medicine in the war just finished. Appalling as has been the number of battle casualties, the death rate from disease has been held down as never before. The statistics available show conclusively that the great scourges and plagues of former armies have y This mobilization, added to those physicians who were taken by the draft, could not occur without great until Cardinal Richelieu installed them, in 1639, in vil¬ lages behind the fighting lines. been held in check : that is, typhus fever, cholera, recurrent fever, typhoid, scurvy and malaria, and, not least important, smallpox. Influenza with pneumonia, occurring in an epidemic sweeping over the eastern and western hemispheres, has been the epidemic that has baffled medical science and stands out with star¬ tling distinctness as the one uncontrolled epidemic. The death toll of pneumonia has almost equaled the battle casualties of those killed and dying of wounds in the American army. In spite of the failure to control influenza and pneumonia, this is an extraordinary rec¬ ord of disease control, and never, in any previous war, has the knowledge of medical science and sanitation, and the application of this knowledge been able to accomplish so much. We saw typhoid start as an •epidemic in Belgium, in 1914, spread with its old-time fury among the troops at that point, and then we saw it conquered by sanitation and vaccination. We have seen typhus spread with terrible fury in Serbia and in Austria, we have seen typhus and recurrent fever break out in Russia, and again through the knowledge that these diseases are carried by body lice the epidemics were controlled. Cholera has started and been con¬ trolled through sanitation and vaccination, by both Russians and Italians. Tetanus has been practically eradicated among the wounded. Smallpox, appearing among the recruits coming from civil life, has been so quickly controlled that there were, from January, 1917, to April, 1919, in the American army of over 2,000,000 men, but six deaths from this disease. To appreciate fully the meaning of this result of preventive medicine and what the American medical profession has accom¬ plished, let us study the battle casualties and disease rates of former wars, and, by this contrast, appreciate the achievement. SUCCESS OF PREVENTIVE MEDICINE lages fighting It is not usually realized that some of the greatest wars of modern times were fought with practically no provision made for the care of the sick and wounded, that the decision of wars has at times depended more on the wastage of the armies by disease than on the valor of the soldiers or the genius of the generals. The Emperor Frederick Barbarossa, in the Middle Ages, saw one army in Italy annihilated by sunstroke. Ten years later, after he had succeeded in storming and conquering the city of Rome itself, a pesti¬ lence swept away another army, and he was forced to flee, a fugitive, to Germany. In the Thirty Years War, the Swedish army under Torstenson fought its way from the Baltic Sea to the very gates of Vienna, where the bubonic plague so decimated his forces that he was compelled to withdraw and lose the campaign so brilliantly won. In the middle of the eighteenth century the bubonic plague again so raged, this time among the Austrian and Russian armies, that these nations were forced to bring the war to an unexpected ending and to make an unfavorable peace with Tur¬ key. In more modern times, the disorganization and discouragement produced by disease in the French forces before Sebastopol was not a small element in hurrying the government of France to conclude peace before the ultimate aims of the campaign were accomplished. p For a background to modern hygiene, let us con¬ sider the occurrence of disease in the Thirty Years War, 1618-1648, a war fought between Protestant Swedes and northern Germans, aided, toward the end, by the French, against the imperial Catholic armies of Spain, Bavaria and Austria. This war had all the bitterness of other religious wars. The armies were often an unpaid rabble of mercenaries greedy for loot, who lived on the country, but who deliberately burned and destroyed all they could not use. They methodically wasted entire districts, thus adding famine to pesti¬ lence, for wherever these armies went they took with them and spread typhus fever. The numbers engaged, relative to modern times, were small, an entire army representing no more than one or two modern divis¬ ions, the strength of either side averaging from 20,000 to 30,000, occasionally mòre, often less. SUCCESS OF PREVENTIVE MEDICINE Bodart sums up the battle casualties of the thirty greatest engagements of this war as averaging 15 per cent, for the victors and 30 per cent, for the defeated antagonist. These losses were small indeed compared to loss by disease. The sick and wounded were uncared for except by their comrades or by the camp- followers ; or they were left in villages or cities to be aided by the civilians, and were universally the foci of infection from which typhus fever spread far and wide. Smallpox was ever present in the communities. Dysentery and scurvy added their toll of death in the armies ; typhus fever never ceased its virulent devasta¬ tion, and, after 1632, bubonic plague imposed its ter¬ rors on the armies and stricken peoples alike. Great as was the destruction of life among the soldiers, whether from battle or by disease, the loss of life was always greater among the noncombatants, not only because of famine and pestilence, but also because of the brutal and barbaric conduct of the war. Prinzing records that the population of Württemberg lost by war, famine and pestilence, in five years, 300,000 persons, or three quarters of its inhabitants. Downloaded From: http://jama.jamanetwork.com/ by a University of Manitoba User on 06/04/2015 DESTRUCTIVE EFFECTS OF DISEASE IN FORMER WARS The pursuing Russians did not escape free from the scourge, for in the three months from October to December they lost 62,000 soldiers, most of whom died from typhus. It is stated that the Russian armies in this campaign lost 200,000 killed and 150,000 wounded. The total of death by disease is not recorded. Typhus fever still raged as an epidemic among the armies in the Wars of the Spanish Succession, and in the Seven Years War of Frederick the Great. It was not till the nineteenth century, after the Napoleonic Wars, that the French hospitals showed any improve¬ ment. On the other hand, it was because of the experiences in the Napoleonic Wars that the English were able to improve the sanitary care of their wounded. This the historian Napier realizes, and he pays tribute to the success achieved : y The German campaign, a year later, was no less dis¬ astrous, and among the French and their allies there seems to have been some 60,000 killed and 196,000 wounded during 1813. Duncan thus describes the result of this campaign : But a few fragmentary battalions followed the eagles of Napoleon across the Rhine in November. The army lay scattered amid the villages on the route from Germany, the men dying by thousands and spreading a pestilence among the inhabitants. Reliable observers say that the retreat from Leipzig was no less ruinous than the retreat from Moscow, although there was no cold nor famine. The utter ruin of the army was the legitimate fruit of utter neglect of the sick and wounded. The extraordinary excellence of the medical officers may be said to have decided the day at Vittoria, for their exertions undoubtedly added a full division to the strength of Welling¬ ton's army, and without these 5,000 it is doubtful if his Lord¬ ship, with his unrivaled talent, could have carried the day. Their efforts were directed more toward cure than prevention, although Jenner had, by the end of the eighteenth century, shown the first means of control, by preventive vaccination, of one of the decimating plagues, that of smallpox. In strange contrast to his habitual indifference to the fate of his sick and wounded, Napoleon seized on the discovery of Jenner, and by 1809 had succeeded in having his entire army vaccinated. DESTRUCTIVE EFFECTS OF DISEASE IN FORMER WARS DESTRUCTIVE EFFECTS OF DISEASE IN FORMER WARS Of course, any accurate and minute comparison requires carefully kept statistics, and such records are available only for the wars of the nineteenth cen¬ tury. Surgeons and physicians have been with the armies since antiquity, but they were as part of the retinue of some king, general or noble, and were not assigned to troops. Charles the Bold of Burgundy, in the last third of the fifteenth century, was the first one that definitely assigned surgeons to the troops as well as to the officers. This seems to be the isolated instance of a single ruler, for it was not till a hundred ye'ars later that the first surgeons were sup¬ plied to the British army, in an expedition to St. Quentin. In the middle of the sixteenth century the armies of Charles V did not possess surgeons or medical corps, for it is reported as an extraordinary occurrence that, in the siege of Metz, when Charles V, beaten by disease and famine, was forced to raise the siege of this town, his young opponent, the Duc de Guise, gathered the abandoned sick and wounded of the retreating imperial army and, contrary to the cus¬ toms and traditions of the time, had them cared for by his own physician, the renowned Ambroise Paré. il h h p ys c a , However, it was not until the seventeenth century, in the time of Louis XIII of France, that surgeons were regularly appointed to the French armies, and not till 1660, when a standing army came into exist¬ ence in England, were surgeons first regularly appointed to regiments in the English army. Not till the middle of the eighteenth century, however, was there a well-organized medical service in any of the armies. Field hospitals were not thought necessary The retreat from Moscow and the Russian campaign of 1812 was probably the greatest military disaster of modern times. From recent authoritative French figures Bodart estimates that 680,000 crossed the fron¬ tier with Napoleon. Dysentery severely attacked the armies after they had crossed the Polish frontier, 80,000 men being down with it at one time. Of the 612,000 fighting strength, there returned to the fron¬ tier, according to Bodart, but 112,000 men. Lemazu- rier says that the great majority of the 30,000 French prisoners left at Vilna died. DESTRUCTIVE EFFECTS OF DISEASE IN FORMER WARS Faure claims that all of the French soldiers who fell into the hands of the Russians succumbed to typhus fever. It seems proba¬ ble, therefore, that there were 100,000 men killed in battle, and at least 350,000 perished from starvation, cold and disease. Prinzing says that the instinct of self-preservation had kept the army together in a common line of march from Moscow to Vilna and on to Niemen. He adds : In the Electorate of Saxony, bubonic plague, typhus and dysentery, in two years, carried off 934,000 of the inhabitants. Three quarters of the entire population of Germany, over whose fields the war had chiefly been waged, were blotted out of existence, for the population dropped from 16,000,000 to 4,000,000, the logical consequences of barbarous warfare and no sanitation. By the middle of the eighteenth century the armies of England and France had regularly organized med¬ ical corps. With its increased responsibilities the medical corps of England had been given an equiva¬ lent increased authority and independence, but the French medical service, from the eighteenth century even through the war of 1870, had neither indepen¬ dence nor authority, but was a subordinate part of the intendance, or quartermaster's department. In spite of the improved ideas of care of the sick and wounded in the armies of the eighteenth century, the mortality rate diminished but little. The hospitals were still only shelters for the very sick. Two, four, or even six patients were still crowded on a single bed. Over¬ run with vermin, with absolutely no ventilation, filthy beyond description, they still propagated typhus, plague and dysentery. Sir John Pringle, in discus¬ sing the causes of mortality in war, names hospitals as an important factor. And Turpin de Crisse declared that in the wars of the decade from 1731 to 1741 more men died in hospitals from lack of care than lost their lives in combat. After crossing the river, however, at this point, the few unfortunate soldiers who had survived the awful misery of the march, hungry, with clothes in rags, with torn shoes, alive with vermin, with frozen and gangrenous limbs, scattered in all directions, some going home and others to strongholds that were in the hands of the French. Thus typhus fever, with which all parts of the army were infected, was spread in a comparatively short time over a large part of Germany. Downloaded From: http://jama.jamanetwork.com/ by a University of Manitoba User on 06/04/2015 DESTRUCTIVE EFFECTS OF DISEASE IN FORMER WARS It was the most complete experiment in army hygiene, as com¬ plete as a chemical experiment in a laboratory, but which should not be repeated, even for the benefit of inquirers at home. The Crimean War, 1854-1856, shows the highest loss from battle casualties among the Russians, and from disease among the French, of all wars of which we possess accurate records. The battle death rate among the British was sixty-nine per thousand per year, among the French seventy, and among the Rus¬ sians 120. The disease death rate was 230 per thousand among the English, 341 among the French, and 263 among the Russians. The medical and human lessons do not lie in these mere figures, extraordinary as they are. They can be brought out only by a comparison between the mortality from disease in the English and French armies. The cause of the French deterioration is plainly seen from a study of the correspondence of their med¬ ical inspectors and a study of the increase of sickness in the army. Surgeon-General Longmore of the English army, reviewing the medical lessons of this war, states that "the French medical officers were com¬ pletely subordinated to the intendance, or direct administration, and had no authority beyond that of ordinary civil practitioners at the bedside. Even the control of hospitals, ambulances, and medical service in battle was directed by the intendance." This quar¬ termaster's staff, having no medical training, were quite incompetent to advise on the means necessary to preserve the health of the troops, and quite incompe¬ tent to give directions on matters of hygiene and sani¬ tation. This situation led to the development of scurvy and typhus, with a constantly increasing viru¬ lence of these diseases, until at last their diffusion took place in such overwhelming proportion that all avail¬ able resources were powerless to cope with the sit¬ uation. In the beginning of the Crimean War, the English were sent out unprepared. They had forgotten their lessons of the Peninsular War, they had discarded the knowledge so obtained, and they were absolutely unprepared for the wrar and went out with insufficient equipment, food and clothing. The first winter was terribly severe. The French, on the other hand, were much better equipped and better prepared for war, were better rationed, better clothed and had good equipment. DESTRUCTIVE EFFECTS OF DISEASE IN FORMER WARS In the two and a half years under this sur¬ geon there were 2,699 deaths from wounds, and 14,269 from disease, which gives a death rate of killed or dying of wounds of forty-two per thousand per year, and 118 dead of disease. Typhus fever was con¬ trolled, but dysentery and typhoid caused 11,000 of the 14,000 deaths. 1855, we find that there was a decrease of 80.5 per cent, in the rate of the British mortality, and an increase of 62.8 per cent, in the rate of the French. Comparisons of the deaths occurring from January to April, 1855, and those from January to April, 1856, reveal that there was a decrease in the British mor¬ tality of 97.05 per cent, and an increase in the French mortality of 57.43 per cent. mortality per The details given by Garrison of these figures are even more striking. For example, during the first winter the British lost 164 men from typhus fever, and the French ninety. During the second winter the British losses from typhus were only sixteen, those of the French 10,278. The French lost 145 men from scurvy and the British 175 during the winters of 1854 and 1855 ; during the following winter the French lost 964, but the English had but one death from this dis¬ ease. Florence Nightingale, from whose work in this war the modern system of nursing arose, describes the situation as follows : , The Medical Corps of the American army, modeled more on English than on French lines, had not, in the Mexican War, 1846-1848, advanced far in the preven¬ tion of disease nor improved the waste of life of the Napoleonic era. The mortality from disease was 110 per thousand per year, and the battle loss was fifteen per thousand. Seven times as many men died of dis¬ ease as were killed in action. . . . the most complete example in history of an army, after a great disaster arising from neglect, having been brought into the highest state of health efficiency. During the first winter the mortality rate was 60 per cent., which exceeded the rate of the great plague of London. But during the last six months the mortality was not more than among the healthy guards at home, and during the last five months it was two thirds of that among the healthy troops at home. DESTRUCTIVE EFFECTS OF DISEASE IN FORMER WARS There seems to be a general agreement that no man ever was more indifferent or cared less for the salvage of the sick and wounded of his armies than Napoleon. Duncan says that abandonment of the wounded was the rule by the French in the Napole¬ onic Wars. When not abandoned, they were huddled in buildings of every sort and left to die. Prinzing, studying the epidemic of typhus fever of 1813 and 1814, following the Russian campaign, believes that between 2,000,000 and 3,000,000 people contracted this disease, spread broadcast by the scat¬ tered armies of Napoleon, over 10 per cent, of whom died. The Spanish War, in its six years' duration, 1808- 1814, cost France over 90,000 killed. The deaths by disease are variously estimated as from 300,000 to 460,000 among the French army. We know that typhus fever was widespread and virulent. We know that yellow fever, in 1810 and 1811, raged furiously in the southern portion of Spain. It is known that in the siege of Saragossa, for example, of the 100,000 inhabitants, 54,000 died of typhus ; and of the 30,000 soldiers, 18,000 died of the same disease before the city was forced to capitulate. I iki buildings y It is of more than passing interest that during Napoleon's war against Prussia, 1806-1807, typhoid seems to have been recognized. The French phy¬ sicians at this period differentiated and accurately described its symptoms and lesions. From this time epidemics of typhoid are recognized and separated from the real typhus, which still ravaged both armies and population. y capitulate. In striking contrast to this was the work of the English in the peninsular campaign during 1808-1811, under Sir James McGrigor. This surgeon had been in the Walcheren expedition on the coast of Holland Downloaded From: http://jama.jamanetwork.com/ by a University of Manitoba User on 06/04/201 iti 1809, when the English attempted to take Antwerp from Napoleon. This ill-fated expedition, of a strength of 42,000, lost 206 men killed and dying of wounds, but lost 8,000 through disease. Impressed by his experience on that expedition, MacGrigor made a most determined endeavor to rectify the conditions in the English army in the peninsula, and especially to fight typhus fever in the hospitals. He insisted on accurate medical statistics, so that, for the first time, the relative loss from the different causes might be known. Downloaded From: http://jama.jamanetwork.com/ by a University of Manitoba User on 06/04/2015 , p There is one achievement by the Medical Depart¬ ment of the United States Army after the Civil War which stands as a lasting monument to the industry and genius of the surgeons of that time ; it is the "Medical and Surgical History of the War of the Rebellion." This was the first great medical history ever published of any war, and remains still the standard to be attained. o get The death rate in our Civil War of killed and dying of wounds is given as thirty-three per thousand, the disease death rate as sixty-five. In the Spanish War the death rate from battle is five and the death rate from disease 30.4 per thousand. In the present war, taking the statistics up to March 28, 1919, we find the rate of death from wounds received in action is 14.191 and that of death from disease is 14.797 per thousand. This includes the army on both sides of the ocean. The statistics of the American Expeditionary Forces, with an average strength of 975,716, reveal a rate of death from wounds in action of 31.256 per thousand and a death rate from disease of 11.233. Of those who died of disease, pneumonia claimed 9.146 per thousand. As a result of the scientific medical work during and after the Spanish-American War, the investigations of three American army surgeons, Jesse Lazear, James Carroll and Walter Reed, gave to the world the solution of the problem of the transmission of yellow fever by mosquitoes. With this knowledge, came simultaneously the power to control this dread disease, which for centuries had been the scourge of the West Indies, and had time and again spread in devastating epidemics to this country and even to southern Europe. Lazear and Carroll laid down their lives to gain this knowledge, and paid the ultimate sacrifice in order that thousands, through their work, might be protected and live. The sanitary control of mosquitoes, and thus of tropical malaria and yellow fever, and the wise administration of this knowledge, made possible the building of the Panama Canal. It was an American army surgeon, William C. In the Franco-Prussian War of 1870, the Prussians reached the highest standard of protection against disease that any army had yet attained. The ratio of their battle casualties was fifty-five per thou¬ sand to a rate of death from disease of twenty-five. The French, however, were just the opposite. Still hampered by the quartermaster control of medical organization, in a demoralized, defeated army, they suffered battle casualties of sixty-eight per thousand and a rate of death from disease of 141. The average strength of the German army seems to have been 725,000, and their total losses were 28,500, of whom but a little over 12,000 died through disease. 12,000 through Three infectious diseases had a plague-like spread in this war : these were smallpox, typhoid and dysen¬ tery. For the first time in a large European war, typhus fever did not break out in the armies. The incidence of typhoid fever in the Prussian army was as high as ninety-three per thousand. The incidence of dysentery was forty-nine per thousand. Though smallpox occurred in only 6.1 per thousand of the fighting strength, it occurred in an army that was supposed to be vaccinated. Among the French pris¬ oners of war, however, smallpox broke out as a plague, about 14,000 cases occurring in Germany and about 25,000 in the interned army in Belgium. The incidence was fifty-four per thousand among the pris¬ oners in Germany, which is nine times that of the German army and shows the difference between the vaccinated and unvaccinated army. Up to this time, in Germany, the population was supposed to be vac¬ cinated, but as is usual under noncompulsory health laws, many had neglected the precaution. Smallpox followed as an epidemic in Germany, causing the death of 170,000 persons after the war. This produced a most beneficial result in causing the passage, in 1874, of a compulsory vaccination law, the workings of which have practically eradicated the disease. p y To appreciate the death rate from disease in the French army one must compare its rate of 140.8 per thousand with the death rate from disease in the Ger¬ man army of 24.5, or with the death rate of sixty-five per thousand in our Civil War. No accurate French statistics have been published, the situation being so bad that all have wished to forget it. deaths that it is not given in detail, but is put into the aggregate term of "other diseases." Typhoid fever, with typhomalaria, so called, was one of the chief causes of death from disease in both the Civil War and the Spanish-American War, causing 22.4 per cent, of the deaths of the Civil War, and being the one great uncontrolled epidemic of the Spanish-American War, causing in the fighting period of the latter war 60.5 per cent, of all deaths. But in the recent war only 0.4 per cent, of the deaths are chargeable to this scourge. Pneumonia, on the other hand, causing only 13 per cent, of deaths during the four years of the Civil War and only 3 per cent, in five months of the Spanish-American War, has become the dreaded epi¬ demic of the recent war, causing in the American army 85 per cent, of all deaths from disease. In the Civil War, meningitis caused 2 per cent, of the deaths, and 2 per cent, of the deaths in the Spanish-American War, and it caused 4 per cent, of the deaths in this war. Smallpox caused 4 per cent, of the deaths in the Civil War; in the Spanish-American War, one man died of this disease; in this war, one man died from smallpox in the-United States and five in France. In 1918 and in the first months of 1919, there were 102 patients with smallpox admitted to the hospitals in the United States. These patients came into the various camps from civil life, for the disease developed among the recruits before they could be vaccinated and thus protected, but it has not developed at all among the vaccinated troops in the United States. Dysentery caused 28 per cent, of the deaths in the Civil War, and nearly 30 per cent. (29.3 per cent.) of the 5,600,000 cases of disease reported in that war. In the Spanish- American War it caused 5.6 per cent, of the deaths. But it caused only forty-one deaths out of 48,000 cases, or 0.08 per cent, of the deaths in the recent war. The transmission of yellow fever by mosquitoes does not come into consideration in the recent war, though there were small epidemics of this disease in both the former wars, there being about 1,300 cases in the Civil War and about 1,100 in the Spanish-American War. deaths that it is not given in detail, but is put into the aggregate term of "other diseases." Typhoid fever, with typhomalaria, so called, was one of the chief causes of death from disease in both the Civil War and the Spanish-American War, causing 22.4 per cent, of the deaths of the Civil War, and being the one great uncontrolled epidemic of the Spanish-American War, causing in the fighting period of the latter war 60.5 per cent, of all deaths. But in the recent war only 0.4 per cent, of the deaths are chargeable to this scourge. Pneumonia, on the other hand, causing only 13 per cent, of deaths during the four years of the Civil War and only 3 per cent, in five months of the Spanish-American War, has become the dreaded epi¬ demic of the recent war, causing in the American army 85 per cent, of all deaths from disease. In the Civil War, meningitis caused 2 per cent, of the deaths, and 2 per cent, of the deaths in the Spanish-American War, and it caused 4 per cent, of the deaths in this war. Smallpox caused 4 per cent, of the deaths in the Civil War; in the Spanish-American War, one man died of this disease; in this war, one man died from smallpox in the-United States and five in France. In 1918 and in the first months of 1919, there were 102 patients with smallpox admitted to the hospitals in the United States. These patients came into the various camps from civil life, for the disease developed among the recruits before they could be vaccinated and thus protected, but it has not developed at all among the vaccinated troops in the United States. Dysentery caused 28 per cent, of the deaths in the Civil War, and nearly 30 per cent. (29.3 per cent.) of the 5,600,000 cases of disease reported in that war. In the Spanish- American War it caused 5.6 per cent, of the deaths. But it caused only forty-one deaths out of 48,000 cases, or 0.08 per cent, of the deaths in the recent war. The transmission of yellow fever by mosquitoes does not come into consideration in the recent war, though there were small epidemics of this disease in both the former wars, there being about 1,300 cases in the Civil War and about 1,100 in the Spanish-American War. Downloaded From: http://jama.jamanetwork.com/ by a University of Manitoba User on 06/04/2015 DESTRUCTIVE EFFECTS OF DISEASE IN FORMER WARS The two armies were living and fighting together, side by side in camps, under the same condi¬ tions in the same climate. They both suffered from two epidemics of Asiatic cholera, which cost the English 4,513 deaths and the French 10,044. Com¬ paring the French and the English death rates, exclud¬ ing deaths from wounds and cholera, we find that in the first eight months of the war the English lost from disease alone 9,762, and the French 9,523. But here the story changes. Intense indignation in England at the frightfully insanitary condition and the terrible death rate of their forces produced a tremendous reac¬ tion. England rushed the military necessities of food, equipment and transport to the Crimea, and as a con¬ sequence, from May to August, 1855, the English losses dropped to 923, but the French rose to 10,545. From September to December, 1855, the English losses were 463 and the French 8,473. In the last four months the British losses by disease were 218 and the French 17,129. Comparing the mortality of the autumn of 1854 with that of the same period in The short war of seven weeks' duration between Prussia and Austria, in 1866, is interesting from a medical point of view for two reasons : first, its statistics show that no improvement had been made in the Prussian army in safeguarding the health of troops or in checking the spread of disease ; and second, because of this fact, within the year, the Prussian government had completed a reform of the medical service in their army, and turned it into as effective a machine to obtain the results for which it was organized as the military machine proved to be four years later in the Franco-Prussian War. This Austrian War is also noteworthy as being the first one in which the organized aid of the Red Cross societies, under the Geneva Convention of 1864, seems to have acted. It had been shown by the Vaughn and Shakespeare Board that nearly 65 per cent, of the typhoid fever of that war was transmitted by contact of man with man, and was not water borne. Hence sanitation could only reduce typhoid to a cer¬ tain level and not eradicate it. The introduction of compulsory typhoid inoculation in the army has prac¬ tically eradicated the disease. Following the work of the English medical corps in the Boer War, a United States Army surgeon, F. F. Russell, made possible the practical application of this method in the U. S. Army and proved conclusively that typhoid fever could be completely controlled. The American Army Medical Corps has, in the recent war, discovered the trans- missibility of trench fever by body lice, and thus has shown the means of prevention of this new disease which, while killing no one, rendered thousands of men useless for weeks and ineffective for fighting. This discovery came to save thousands of men for the fighting lines at a time when they were urgently needed. Influenza, rrieasles and pneumonia, in the respira¬ tory group, still stand as baffling problems, and their control has not been accomplished. Measles appeared and spread until it no longer had material on which to spread, as one attack confers immunity to a second. Pneumonia, following influenza or originating as a primary disease, still eludes control. But the know¬ ledge which we have gained in this war of the meth¬ ods of its spread, of the various infectious organisms which produce it, and their various types and varying virulence, of its occurrence as a secondary complica¬ tion to measles and influenza, has enormously increased. The value of the facts thus learned are incalculable, and belief is justified that the problem is better understood than ever before, and that we soon shall see the solution of these problems. p ob e s The occurrence in the camps of meningitis, another disease of the respiratory group, as far as its portal of infection is concerned, has been forty-five times as fre¬ quent in the army as its occurrence in civil life among the same age group. This has been due to overcrowd¬ ing and the diminution of air space allowed the indi¬ vidual soldier in badly ventilated barracks. The responsibility for these sanitary sins rests on the Gen¬ eral Staff and not on the Medical Corps. Gorgas, who seized this great opportunity and transformed a Studying comparatively the diseases of the Amer¬ ican armies during the Civil War, Spanish-American War and the recent war, we find that malaria was one of the chief causes of disability in both the Civil War and the Spanish-American War, though it caused but 6 per cent, of the deaths in the Civil War and but 10 per cent, in the Spanish-American War. But in the recent war malaria has caused such a small number of Downloaded From: http://jama.jamanetwork.com/ by a University of Manitoba User on 06/04/2015 Downloaded From: http://jama.jamanetwork.com/ by a University of Manitoba User on 06/04/201 pesthole of tropical diseases into a healthy and safe ter¬ rain, that the engineering genius of the United States Army might be free to construct the canal. The French under De Lesseps had failed because of the epidemic and tropical diseases which were at that time uncontrollable. Disease had defied and overcome engineering skill and genius. Preventive medicine controlled and conquered. the young men, remain as yet to be controlled, but they are not of great import in the armies in war. The disabling type of disease coming under the head of venereal disease has, in this war, been so controlled that the number of cases brought from civil life was greater than the number occurring in the American Expeditionary Forces in France, which was reduced to twenty-two per thousand per year, a rate only one eighth as high as the incidence among recruits coming from civil life, and only one third as high as the best that ever had been accomplished in the army before. pesthole of tropical diseases into a healthy and safe ter¬ rain, that the engineering genius of the United States Army might be free to construct the canal. The French under De Lesseps had failed because of the epidemic and tropical diseases which were at that time uncontrollable. Disease had defied and overcome engineering skill and genius. Preventive medicine controlled and conquered. conquered. Ten years ago the practical application of the knowledge gained from the study of the epidemic of typhoid fever of the Spanish-American War brought about the compulsory inoculation against typhoid in the United States Army. MODERN CONTROL OF DESTRUCTIVE DISEASES Medical science has today, therefore, within its grasp the power to control the diseases which, in for¬ mer times, decimated warring armies and spread out from these armies among the noncombatant popula¬ tions. Formerly, when war broke out, it was almost inevitably followed by some dread pestilence among the civil populations of the countries in which the war was waged. By proper sanitation and preventive inoculation, dysentery and cholera can be abolished ; by vaccination armies can be protected against small¬ pox. Body lice disseminate typhus, recurrent fever, and trench fever, and by proper disinfestation of these vermin these diseases cease to occur. Through sani¬ tation and preventive inoculation, typhoid fever, the scourge of the two previous wars, is absolutely con¬ trolled, and this includes also paratyphoid, which has been recognized as a separate entity only since the Spanish-American War. In the Spanish-American War, 60.5 per cent, of all deaths were caused by typhoid, and in the present war 85 per cent, were caused by pneumonia. The typhoid of the Spanish- American War was due to local causes and local epi¬ demics. The pneumonia of this war was beyond con¬ trol, and was part of a world-wide epidemic that swept over both hemispheres, and the morbidity and mortal¬ ity of some of the cities of this country exceeded those of the camps. Subtracting the death rate caused by pneumonia from the total death rate by disease in the recent war, we have 2.2 per thousand for the entire army on both sides of the water, which is practically a peace-time death rate. Meningitis has caused, in this war, ten times as many deaths as typhoid fever; pneumonia has caused two hundred times as many. Mumps and scarlet fever, of the infectious diseases of Downloaded From: http://jama.jamanetwork.com/ by a University of Manitoba User on 06/04/2015 NEED OF IMPROVED ORGANIZATION One very important duty to be performed soon is the reorganization of the Medical Reserve Corps and the rearrangement of the Medical Reserve officers and of the medical officers of the National Guard into one National Reserve Corps. This must be done when a realization that the medical profession in the regular Medical Corps and in the Reserve and National Guard Corps are all members of one and the same profession, united in desire to serve and obtain a single objective. Those in the Regular Corps have specialized in the study of medicine in its application to military require¬ ments. The Reserve and Guard Corps have specialized in clinical medicine, with sufficient knowledge of mili¬ tary requirements to permit of their early adaptation to military environment when war comes. Equal ethical responsibilities rest on all alike, since all are called together for the common purpose of caring for the sick and wounded of the army; but the amount of practical responsibility must be unevenly distributed among individuals, that proper organization may be perfected. responsibilities. In the mobilization of the industrial forces of the nation by the Council of National Defense, the health of the nation and the protection of both nation and its armies was regarded of such importance that it demanded direct representation of the medical profes¬ sion on this board. This is also true of the navy, for its Medical Department is represented on the Gen¬ eral Board. Oddly enough, the anachronism still exists that in the General Staff of the United States Army the Medical Department is regarded as an out¬ sider. The safeguarding of the health and fighting vigor of an army, the salvage of its wounded, the saving of man power through protection from disease are still regarded as foreign to staff organization. The medical and sanitary formations are still regarded as noncombatants, although those serving with the troops often go forward and mingle with them in the combats, that the morale of the men may be better sustained. Duty demands it, and they have shown themselves willing, in this war, to be unarmed combatants, not noncombatants. The ratio of the medical officers killed and dying of wounds has been exceeded only by that of the infantry and artillery, which branches neces¬ sarily bear the brunt of the battles. ^EDUCATIONAL NEEDS OF THE MEDICAL PROFESSION One lesson of the war which stands out with great distinctness is the necessity for the American Medical Association to continue its unceasing struggle to raise the standards of medical education in this country. Such are the increasing demands made on the medical profession that the young men entering it today must realize that the broad and excellent education obtain¬ able is none too good. It is not asking too much to require that all medical schools which are permitted to continue should soon be raised to the A Class. i d i There is another urgent educational need in this country that should be taken up immediately : that is, increase in the postgraduate opportunities for medical study. The opportunities that are presented in this country are practically undeveloped. It is for the profession to develop them, and every member of the Medical Corps of the army should be given an oppor¬ tunity to avail himself, for a certain number of weeks each year, of the chance to study some branch of medi¬ cine or surgery at some medical center—not required to do it at his own expense, but detailed by the govern¬ ment to take up, for a definite number of weeks, his chosen branch of study. Physicians acquire their knowledge best by daily contact with opportunities which broaden their experiences. The opportunity to do this at short intervals, rather than at intervals of two, three or five years, would produce better results. NEED OF IMPROVED ORGANIZATION The pro rata death rate of the medical officers has exceeded that of aviators and of engineers. i perfected. Authority of the individual must always equal his responsibilities. Military authority is always expressed by rank and cannot be separated from it. Hence rank, authority, and amount of responsibility must coincide. The Reserve and Guard Corps should not be discriminated against in rank, as they have been in the past, because it invariably prevents authority from equaling responsibility, and thus cripples effi¬ ciency. One solution would be to have all Reserve Corps officers of equal rank, such as captain, and have the office held bestow the authority in proportion to the responsibilities contained therein. The adminis¬ trator of each hospital unit must always have supreme local authority, but there should be an appointed group of clinical consultants in the different specialized branches of medicine, surgery and sanitation for the proper correlation of clinical procedures, that there may be uniformly good treatment and care given equally to all sick and wounded in all hospitals. Medical and surgical specialization was developed in the army for the first time in this war, and beyond question it must remain permanently. These con¬ sultants should be utilized as medical and surgical advisers on clinical subjects to the Chief Sur¬ geon, with direct access to him without intervening mechanism of departmental heads. It is axiomatic that these consultants must have sufficient rank and authority to equal their responsibilities, and necessarily higher rank than the commanding officers of the hos¬ pitals under their supervision. These are but sugges¬ tions, and it is unnecessary to go further into details at this time, but some solution of this problem must be found soon. g This subject is a matter for congressional action, but the profession of this country, while the experi¬ ences of this war are still vivid in its mind, must turn to the Congress, must make an intelligent exposition of these facts, and must bring about, by legal enact¬ ment, an adequate representation of the Medical Department on the General Staff of the army. VALUE OF LESSONS LEARNED IN DETERMINING ACTION IN FUTURE What then are the lessons that we can draw for future action? There is no question but that the salvage of human beings, the protection of troops from disease in an army, renews and saves the fighting forces. Until recently, until medical science could control disease during war time, armies had been more decimated and injured by disease than through battle casualties. Now that, except for epidemic spread of respiratory diseases, the communicable and epidemic spreading diseases can practically be controlled, the medical corps of an army has become an essential part of the fighting organization. Whole nations must now go to war. No longer can they mobilize a selected por¬ tion of volunteers and send them to fight the war and defend the nation. Since all the youth of the nation must mobilize and turn to war, it becomes the duty of a general staff to save its man power and to salvage it to the greatest extent possible. The history of the Crimean War, of our Spanish-American War, and our experience in the recent war have clearly shown that only through proper representation on the general staff by those men trained in such salvage, and by experts in such knowledge of sanitation, can this duty be performed. When the General Staff of the United States Army comes to realize this fully; one cannot conceive that it will fail to give proper representation in its councils and organization to the Medical Depart¬ ment. The practical necessity for this was finally recognized in the A. E. F. by General Pershing and three medical officers were detailed at General Head¬ quarters as substantive members of the General Staff. Responsibility and authority cannot be separated, and only by such organization can adequate authority equal the inevitable responsibilities. NEED OF IMPROVED ORGANIZATION Downloaded From: http://jama.jamanetwork.com/ by a University of Manitoba User on 06/04/2015 RELATION OF RED CROSS TO ARMY f Through the Red Cross Research Society, whose mem¬ bership consisted of all members of the Medical Corps in France, this research committee furnished a forum in which was discussed and given out the knowledge of medicine and surgery of the war, gathered in the years just previous to our entry. It proved to be the intellectual center for the medical portion of the Amer¬ ican Expeditionary Forces, and here, discussion by the Medical Corps of the British and French armies in its meetings, gave to the members of the American Medi¬ cal Corps the knowdedge gained in the hard and cruel experiences of the three years previous to our entrance. It was by this means that the American Medical Corps started with the medical knowledge of 1917 instead of with the knowledge of 1915 or 1916. It thus trained and prepared thousands of officers by reenforcing their practical experience with knowledge of the experiences to come. Another real contribution by the Red Cross, in administrative matters in the war, was the founding of the base hospital organizations on the advice of Gen. J. R. Kean. These organizations brought together from various hospitals groups of medical and sur¬ gical men and nurses who were accustomed to work together and who knew each other's ideas and ways of work, and had them fully equipped and prepared for service before war broke out. The Spanish-American War had shown how difficult it was to gather men quickly into efficient organizations with no previous acquaintance which accustomed them to work together. The base hospital units produced a homogeneous struc¬ ture instead of a heterogeneous mass thrown together by haphazard, and even when members of the hospitals were taken out later and sent as leaders of other units and teams, there still remained the basic continuity, which proved of the greatest value. It is of the utmost importance for future preparedness that the Red Cross should have these base hospital groups ready to go at all times, and nothing should be allowed to stand in the way of this or of some similar plan. As is known, the Red Cross gave up these units to the army as soon as they were called to active duty. RELATION OF RED CROSS TO ARMY f The relationship of the Red Cross and the army is not generally understood. The Red Cross is not a private society supported by private contributions, but is a governmental body incorporated by Congress, with a definite function, that of giving voluntary aid to the soldiers and sailors of the army and navy during war. It differs from other governmental functions in that it is not supported by congressional appropriations, but by voluntary contributions. Many of its functions and their limitation are defined in international treaties with other nations. Originally conceived to give aid to the Downloaded From: http://jama.jamanetwork.com/ by a University of Manitoba User on 06/04/201 sick and wounded in battle, and to place them and the attending medical and nursing personnel safely into a special noncombatant group, its functions have broad¬ ened and grown until they ramify, in war time, among civil and military populations alike. furnished, on request, any information regarding scien¬ tific subjects. It thus supplied medical knowledge, and prevented medical stagnation and deterioration through lack of knowledge. o edge One of the unforeseen but logical sequences of the Red Cross Research Society was to establish a liaison with the medical corps of our Allies. Instead of a slow and gradual acquaintance, there arose a rapid amalga¬ mation and a rapid fusion into a frank and trusting friendship between the medical men of France, Eng¬ land and America. This proved to be one of the really valuable contributions which the Red Cross made to the war. Another contribution of great value was the making and furnishing to the army of the nitrous oxid fqr general anesthesia. Researches with this gas showed that, in the seriously wounded suffering from surgical shock, it did not increase this shock, as chloro¬ form and ether did, nor did it tend to send into shock the seriously wounded. The death rate, with this gen¬ eral anesthetic, was 20 per cent, less among the collapsed and seriously wounded than with the other anesthetics. The Red Cross brought over to France a plant to manufacture this gas ; it manufactured it and placed it up in the front hospitals of the advanced zone. The practical proof that such an anesthetic, in huge cylinders, could be carried forward to the advanced hospitals, and used in practical abundance back of the battle lines, was a successful accomplishment. RELATION OF RED CROSS TO ARMY f The lives saved justified the expenditures, for its advan¬ tages were so definite that its use meant the purchasing of the lives of our wounded. military populations When, two years ago, the war began with us, the popular idea in the army, among the majority of medi¬ cal and line officers alike, of the full extent of Red Cross duties, seemed to be that the Red Cross workers were to be kept as far in the rear as possible, to hold the little hot hand of the homesick convalescent soldier, and, on off moments, make comfort bags for soldiers and sailors. The idea of the average person eager to go into Red Cross work was to make as many surgical dressings as possible and, with armfuls of these, keep as far forward in the advanced zone as possible, ready to rush on the field of battle and stem the hemorrhages of the wounded and gasping soldiers. Stern reality soon effected a compromise, and time only permits here of a short reference to some of the medical activities in their relation to the army. y The war has shown that the Red Cross has proved an excellent stop-gap for supplies, and a source of all kinds of emergency and surgical supplies and relief, even to complete and extensive hospitalization, when the situation called for them. No more satisfactory and cordial relationships could have existed between two departments than did exist in France between the Med¬ ical Department of the Red Cross and the Chief Sur¬ geon's Office of the American Expeditionary Forces. Because of this relationship, the Red Cross was able to supply opportunities to the medical men in France which could not otherwise have been obtained. Through the broad-minded policy of Major Grayson M. P. Mur¬ phy, Red Cross Commissioner to Europe, there was formed a research committee, with American, English and French medical men, which fostered research and secured progress in medicine even during the war. The discoveries of the origin of trench fever and its transmission through body lice was the direct result of this. The standardization of blood transfusion, the striking progress of surgery of the chest, and the con¬ tinuous study of surgical shock are other examples of work accomplished by this same committee. Downloaded From: http://jama.jamanetwork.com/ by a University of Manitoba User on 06/04/2015 NATIONAL CONTROL OF PREVENTABLE DISEASE CHARLES A. KOFOID, Ph.D., Sc.D. (Berkeley, Calif.) Major, Sanitary Corps, U. S. Army NATIONAL CONTROL OF PREVENTABLE DISEASE I desire to draw but one more deduction from the medical lessons of this great war, and that in reality is the climax toward which everything points. That is, if this nation, through its present medical knowl¬ edge, has within its grasp the power to control com¬ municable, and hence preventable, diseases, there must be established a nation-wide controlling organization for this purpose, a National Department of Health. Over 33 per cent, of our young men were disqualified from the draft for physical defects. There is need of wider supervision of our growing boys and girls to build up a more robust nation, and it is especially urgent in rural districts. If we are to have some form of universal military service, the very necessity of its universality demands some general supervision of the health of the youth of the nation, through protection against the transmissible diseases, and direction over the giving of health to the people as we now give edu¬ cation. This war has taught that there remains eco¬ nomic value in the maimed and wounded, and it is our duty to develop this value to its fullest extent. The maiming and injury of our workers, in the everyday work of industry, far exceeds each year the battle casualties of this war, and there is an economic neces¬ sity and duty to be performed in the salvage and recon¬ struction of the industrially injured. SIDNEY I. KORNHAUSER, Ph.D. (Evanston, Ill.) Second Lieutenant, Sanitary Corps, U. S. Army AND J. T. PLATE, A.B. (Elizabeth, N. J.) Second Lieutenant, Sanitary Corps, U. S. Army NEW YORK SIDNEY I. KORNHAUSER, Ph.D. (Evanston, Ill.) Second Lieutenant, Sanitary Corps, U. S. Army AND J. T. PLATE, A.B. (Elizabeth, N. J.) Second Lieutenant, Sanitary Corps, U. S. Army NEW YORK The intermingling of men from the tropics and of troops which had seen service in regions where amebic dysentery is widely endemic with our own troops on the western front during the war has opened possibili- ties of increased infections by Endamoeba dysenteriae and other intestinal parasites. EXAMINATIONS FOR INTESTINAL PARASITES Examinations of 1,200 men of the United States army who had been in overseas services, and of 300 men from home service troops, have made possible the following preliminary account of the relative degrees of infection in these two groups of men. This work has been carried on at the Army Laboratory, Port of Embarkation, New York, since Dec. 28, 1918. The overseas troops examined were sick and wounded sol¬ diers in transit through Debarkation Hospital No. 3, New York City. They included men who had seen service in Flanders, Château Thierry, the Argonne and Toul regions, as well as quite a number from France who never reached the front. They are drawn from 584 different regiments, etc., and are therefore fairly representative of our overseas troops. They come from every state in the Union and constitute approxi¬ mately a fair sample of our population. Only a small fraction of them saw service on the Mexican border. It is obviously impossible to determine what proportion of the infections detected in them were acquired over¬ seas and which ones are of home origin. Th h dust a y injured. Malaria still prevents the use of large areas of our southern states, and saps the energy of a large portion of the population. Typhoid fever still rests as a blot on the rural hygiene of this country. The control of epidemics between states is already in the hands of the Public Health Service, and within states, if state authorities request aid. Quarantine from outside infection is also under federal control. There are many other federal activities partially supervising health and disease through the various departments of the federal government. But it all lacks the efficient power of central correlation, and there remain many public health activities that should be undertaken by central action, from some of the problems of infant mortality to the problems of the increase of degenera¬ tive diseases of late middle life. It is the duty of the American Medical Association, and of each member of each state association, 1?o urge on Congress the estab¬ lishment of a National Department of Health. origin. The home service troops are mainly cooks, bakers and food handlers from the port of debarkation and principally from the Medical Department. From the U. S. Army Laboratory, Port of Embarkation, New York City, E. H. Schorer, Major, M. C., U. S. Army, officer-in-charge. For an amplification of this subject we would refer the reader to an article giving further details of the work done at the New York Army Laboratory, namely: Kofoid, C. A.; Kornhauser, S. I., and Swezy, Olive: Criterions for Distinguishing the Endameba of Amebiasis from Other Organisms, Arch. Int. Med., to be published. In the names of the organisms mentioned in this article, I have followed the best biologic usage on the following points: (1) Dysen- teriae in place of histolytica as the specific name for the ameba of human amebiasis. This usage will shortly be sanctioned by the Com- mittee on Protozoology of the National Research Council, has recently been approved by the British Academy of Medicine, and is the current French usage. I shall use it in my report to the National Research Council. (2) Trichuris trichiura for Trichocephalus dispar. The latter is displaced by the former in all the best recent parasitologies, and is the legal name for the species. (3) Giardia for Lamblia, for the last- mentioned reasons. NATIONAL CONTROL OF PREVENTABLE DISEASE This is especially possi- ble in cases of dust-borne, fly-borne or water-borne infections in which ova or cysts from the stools of infected men find their way into the food or water of troops in the trenches or in the even more exposed conditions attending a rapidly advancing army. Downloaded From: http://jama.jamanetwork.com/ by a University of Manitoba User on 06/04/2015 RELATION OF RED CROSS TO ARMY f y y It is a question, and one that should be fully dis¬ cussed, whether or not it would be advisable for the Red Cross to retain control of the base hospitals in the rear zone of the army. This has been done in Italy, by the Italian Red Cross, with pronounced success, and the chain of hospitals continued up even into the advanced zone ; but where they touched the advanced zone they left the control of the Red Cross and pro¬ ceeded under the control of the army. The Red Cross can often obtain its supplies quicker than the army, and can often act independently in emergencies in which the army must proceed along more slowly acting established lines. The American Red Cross has shown The Red Cross also published a medical journal, a digest of all war articles of the Allied countries, and disseminated these broadcast in the American Expedi¬ tionary Forces and among the medical corps of our Allies. It further disseminated knowledge by means of a library and a medical intelligence department that conclusively in France that, with its own or with army personnel, it can furnish, equip, and efficiently run hospitals in the advanced zone, or in the rear as base hospitals. This has proved advantageous in an emer¬ gency. Would it not prove equally advantageous as an established policy? From the U. S. Army Laboratory, Port of Embarkation, New York City, E. H. Schorer, Major, M. C., U. S. Army, officer-in-charge. F lifi ti f thi bj t ld f h EXAMINATIONS FOR INTESTINAL PARASITES Of the total of 300 men examined, eighty-two, or 27 per cent., bear foreign names suggestive of Russian, Polish, Italian Hull (England) After-Care Colony for the Tuberculous — This colony was opened in April, 1918. The first report detailing the history of the movement and work done to Dec. 31, 1918, has just been issued. The colony is intended for such persons as have previously suffered from tubercu¬ losis, in whom the disease is arrested, and who are certified to be noninfectious and able to perform at least six hours of hard work daily. Every applicant undertakes to remain for at least one year, during which time the individual is brought to full earning capacity by a gradual process of training. The work consists of market gardening, fruit growing, intensive horticulture, sheep, poultry and pig rais¬ ing. The women colonists also assist with light household duties. The rearing of medicinal herbs is also to be attempted during the coming year. The colonists pay a certain sum for their maintenance, but they receive pay for their work. From: http://jama.jamanetwork.com/ by a University of Manitoba User on 06/04/2015 Downloaded From: http://jama.jamanetwork.com/ by a University of Manitoba User on 06/04/201
https://openalex.org/W4213363440	https://zenodo.org/records/6651361/files/MatSciEng_Amare_EASN2021.pdf	English	null	Innovative test methodology for shelf life extension of carbon fibre prepregs	IOP conference series. Materials science and engineering	2,022	cc-by	5,299	To cite this article: Constance Amare et al 2022 IOP Conf. Ser.: Mater. Sci. Eng. 1226 012101 To cite this article: Constance Amare et al 2022 IOP Conf. Ser.: Mater. Sci. Eng. 1226 012101 View the article online for updates and enhancements. Zhenyu Han, Shouzheng Sun, Yunzhong Fu et al. This content was downloaded from IP address 92.184.108.250 on 16/06/2022 at 08:19 Publication title Innovative test methodology for shelf life extension of carbon fibre prepregs Authors Constance Amare, Olivier Mantaux, Arnaud Gillet, Matthieu Pedros, Eric Lacoste Issue Date 15 February 2022 Publisher IOP Conference Series: Materials Science and Engineering Type of publication Paper Acknowledgement This project has received funding from the Clean Sky 2 Joint Undertaking (JU) under grant agreement No 887104 — MANIFICA Recycling — H2020-CS2-CFP10-2019-01. The JU receives support from the European Union’s Horizon 2020 research and innovation programme and the Clean Sky 2 JU members other than the Union Disclaimer The content of this article reflects only the authors’ view. The Clean Sky 2 Joint Undertaking is not responsible for any use that may be made of the information it contains. IOP Conference Series: Materials Science and Engineering PAPER • OPEN ACCESS PAPER • OPEN ACCESS Innovative test methodology for shelf life extension of carbon fibre prepregs To cite this article: Constance Amare et al 2022 IOP Conf. Ser.: Mater. Sci. Eng. 1226 012101 You may also like Effect of carbon nanotubes on the electrical, thermal, mechanical properties and crystallization behavior of continuous carbon fiber reinforced polyether-ether- ketone composites Liang Qiao, Kaili Zhu, Hongsheng Tan et al. - Fabrication and characterization of aerosol-jet printed strain sensors for multifunctional composite structures Da Zhao, Tao Liu, Mei Zhang et al. - Multiscale analysis of the correlation of processing parameters on viscidity of composites fabricated by automated fiber placement Zhenyu Han, Shouzheng Sun, Yunzhong Fu et al. - You may also like You may also like Effect of carbon nanotubes on the electrical, thermal, mechanical properties and crystallization behavior of continuous carbon fiber reinforced polyether-ether- ketone composites Liang Qiao, Kaili Zhu, Hongsheng Tan et al. - Fabrication and characterization of aerosol-jet printed strain sensors for multifunctional composite structures Da Zhao, Tao Liu, Mei Zhang et al. - Multiscale analysis of the correlation of processing parameters on viscidity of composites fabricated by automated fiber placement Zhenyu Han, Shouzheng Sun, Yunzhong Fu et al. - constance.amare@u-bordeaux.fr Abstract. The aerospace industry makes extensive use of composite materials in the form of fibre fabrics pre-impregnated with thermosetting resin, called prepregs. In order to minimize the resin polymerization before curing, prepregs must be stored at -18°C (0°F). There are therefore expiration dates for prepregs before use. Although manufacturers try to minimize storage time, offcuts and time out of the freezer, it is estimated that 30% to 40% of the prepregs are not used [1]. Today, recertification of expired materials is still complex and expensive, therefore it is generally chosen to send expired prepregs to landfill. The purpose of this work is to correlate physicochemical measurements with the loss of mechanical performance in order to point out and measure the real aging effects during excessive storage time. Processability, physicochemical and mechanical tests were performed in order to understand which tests are truly representative of ageing. This study was illustrated by testing on unidirectional Hexcel carbon/epoxy prepreg. Different expiry dates of this material were studied and the properties were compared. It was shown that the main observed degradation was the processability of the prepreg while mechanical performance was minimally degraded after the expiry date. This study could lead to a simpler measurement of the actual expiry rate of prepregs, which could be useful to speed up recertification procedures or to propose new scenarios to extend the shelf-life of expired prepregs [2]. Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Published under licence by IOP Publishing Ltd 1 aded from IP address 92.184.108.250 on 16/06/2022 at 08:19 This content was downloaded from IP address 92.184.108.250 on 16/06/2022 at 08:19 IOP Publishing onf. Series: Materials Science and Engineering 1226 (2022) 012101 doi:10.1088/1757-899X/1226/1/0121 1.1. Context High performance composites can be manufactured by implementation of pre-impregnated reinforcements, called prepregs. These semi-products stored at an intermediate cured state limit variation in fibre/resin ratios and simplify manufacturing. Prepregs have to be stored at low temperature (-18°C) in order to freeze the polymerization reaction. Two dates are considered to ensure that the reaction progress remains sufficiently low and acceptable for the implementation and the characteristics of the future composite: • Storage time/Shelf life: Prepreg manufacturers define a shelf date, corresponding to maximum storage time at -18°C in a waterproof bag. This period is usually close to 1 year. • Storage time/Shelf life: Prepreg manufacturers define a shelf date, corresponding to maximum storage time at -18°C in a waterproof bag. This period is usually close to 1 year. • Out time: Material exposure time to temperatures above -18°C which can accelerate polymerization reaction. During a too long storage at -18°C, or during exposition > -18°C, the polymerization reaction may progress, with a possible impact on the prepreg processability or on the properties of the final composite. Finally, the material may no longer be usable for production of aeronautical certified parts. When one of these two dates has passed, the material is then considered expired. As the use of these semi-products increases, the amount of scrap generated could lead to important waste. There are several reasons for exceeding expiry dates: Minimum batch orders: Customer must purchase a minimum quantity of prepreg in order to • Minimum batch orders: Customer must purchase a minimum quantity of prepreg in order to be supplied. Sometimes, this minimum quantity remains higher than the total quantity required • Minimum batch orders: Customer must purchase a minimum quantity of prepreg in order to be supplied. Sometimes, this minimum quantity remains higher than the total quantity required be supplied. Sometimes, this minimum quantity remains higher than the total quantity required be supplied. Sometimes, this minimum quantity remains higher than the total quantity req 1 IOP Publishing IOP Conf. Series: Materials Science and Engineering 1226 (2022) 012101 doi:10.1088/1757-899X/1226/1/012101 to produce the entire series of composite parts. Some material will therefore be left unused in the freezer until expiry. • Short expiry date: Out time of some prepregs can be very short (depending on the resin grade). This may be very close to the manufacturing time of complex parts. 1.1. Context Indeed, a production incident can then lead to a production stop and thus exceeding the out time. • Shifts of production: Exceptional events, such as the Covid19 crisis, can lead to delays or shifts in production of several months. This has led to expiries of prepregs stored awaiting production. • Shifts of production: Exceptional events, such as the Covid19 crisis, can lead to delays or shifts in production of several months. This has led to expiries of prepregs stored awaiting production. Today, two solutions are available for aeronautical manufacturers to deal with expired prepregs: i) recertify the material to use it for the same application after expiration. ii) send it to landfill. Recertification is possible but time consuming and expensive to perform because it is equivalent to a complete certification procedure. Many tests must be performed and it is often more expensive than the material itself. Recertification is possible but time consuming and expensive to perform because it is equivalent to a complete certification procedure. Many tests must be performed and it is often more expensive than the material itself. 1.2. Proposed method The objective of this work, carried out in the frame of the MANIFICA European project, is to propose a simplified procedure to extend the shelf life of prepregs. Then, new reuse scenarios will be developed to avoid landfilling. The current physicochemical recertification tests were implemented and compared with the measurement of mechanical properties drop. Tests were performed on prepregs at different expiry dates. It could be shown that some tests are redundant or unnecessary. As a consequence, only representative tests showing a possible loss of property could be retained to simplify recertification procedure. In the absence of proven loss of property, an extension of the shelf life can be imagined. 1.3. Prepreg manufacturing & aging effect 1.3. Prepreg manufacturing & aging effect All of these effects lead to a reduction in the reactivity of the resin and degrade the processability and the properties of the final composite. quantity. All of these effects lead to a reduction in the reactivity of the resin and degrade the processability and the properties of the final composite. 2.1. Material A unidirectional Hexcel carbon/epoxy prepreg (reference) was used to initiate this study. Its shelf life was 1 year. Three different rolls manufactured in 2012, 2015 and at the beginning of the year 2019 are used. These three materials were therefore considered to be outdated since this work was conducted at the end of 2020. 2. Experimental measurements This section presents the first results obtained on the physicochemical and mechanical tests selected. This section presents the first results obtained on the physicochemical and mechanical tests selected. 2.2. Mechanical tests To carry out mechanical tests, composite plates were manufactured by draping 11 unidirectional plies of prepreg (Standard for the plate lay-up is NF ISO 1268-4 [4]). Two plates of each material were necessary to obtain specimens with specific dimensions (two different thicknesses). Characteristics of the plates and corresponding tests are displayed on Table 1. Prepreg plies were applied and rolled over successively. Plies were stacked on a PTFE substrate and under vacuum bag. The plate was then covered, with 1) a tear away fabric, 2) a perforated release film and 3) an absorption film. The self-adhesive seal and the vacuum bag were used to create a sealed vacuum during the entire process. Plates were then cured using a heating press (plate 1) or into an autoclave (plate 2) at 180°C during 2 hours with a 7-tonns applied pressure. Plate Length (mm) Width (mm) Thickness (mm) Plies number Orientation Tests 1 300 300 2 11 0° Tensile 0°, ILSS 2 450 300 4 21 0° Pure bending Table 1 : Characteristics of the prepreg plates Table 1 : Characteristics of the prepreg plates Table 1 : Characteristics of the prepreg plates (b) Measurement of the interlaminar shear strength (ILSS) The ILSS tests were performed with a 3-point bending system with close supports fitted on the 3R Syntech machine. The length between supports was 10 mm and the diameter of the loading pin were 3 mm according to the standard test EN 2563 [6]. 1.3. Prepreg manufacturing & aging effect The fabric reinforcement is impregnated continuously with a thermosetting resin (only epoxy resins will be considered in the frame of MANIFICA). After impregnation, the polymerization reaction is initiated in an oven and the material is then in an unstable intermediate polymerization state called stage B [3]. The advance of the polymerization reaction during a too long or improper storage of the prepreg is called chemical aging. This is by far the most important effect on a B-stage thermoset resin. At the beginning of the curing reaction, g , Step 1) Macromolecules with various structures are formed. g Step 1) Macromolecules with various structures are formed. g Step 1) Macromolecules with various structures are formed. Step 2) The average molecular weight of the molecules increases with time until the size of the chain is equal to the size of the system, forming a network. Step 3) The remaining molecules react with the network creating further cross-links. Step 4) The cross-linking rate grows until the system reaches the end of the chemical reaction The initial “pre-cure” of the prepreg will promote polymerization. During step 2, cross-linking generates a decrease of the molecular mobility and an increase of the resin viscosity. This results in low resin flow, which affects processability (draping of complex shapes and compacting of plies). Depending on the out-time period, cross-linking can increase beyond limits that will hamper the molecular mobility during the final curing process and isolate active groups without any connection. The initial “pre-cure” of the prepreg will promote polymerization. During step 2, cross-linking generates a decrease of the molecular mobility and an increase of the resin viscosity. This results in low resin flow, which affects processability (draping of complex shapes and compacting of plies). Depending on the out-time period, cross-linking can increase beyond limits that will hamper the molecular mobility during the final curing process and isolate active groups without any connection. To understand the chemical aging of prepregs, some physical parameters can be studied as the rheological behavior, the degree of cure, the glass-transition temperature or the resin composition and To understand the chemical aging of prepregs, some physical parameters can be studied rheological behavior, the degree of cure, the glass-transition temperature or the resin composi 2 IOP Publishing IOP Conf. Series: Materials Science and Engineering 1226 (2022) 012101 g doi:10.1088/1757-899X/1226/1/012101 quantity. (a) Tensile test This test was performed according to the standard EN 2561 [5]. The Young’s modulus and the tensile strength of the composite were determined with 250 mm long and 20 mm wide samples. The machine was a 3R Syntech. An extensometer was used in the elastic range and removed before the plastic range and fracture. (d) “Tack” test The “tack” corresponds to the ability of a prepreg to adhere to a substrate or on itself. This represents the ability of the prepreg to be stacked. This characteristic is essential to maintain the ability of the plies to be compacted. Without tack, plies cannot adhere to each other or to the mould. To compare prepregs, many parameters have to be considered, such as the weight per unit area and viscosity of the resin or the organization of the reinforcement [8]. The 1993 tack test is currently used and has been standardized for the evaluation of prepreg tack [9]. A method described by NCAMP standards can also be used [10]. In both cases, these tests are qualitative and only a "grade" of tack can be determined. For both standards, the test consists in draping a prepreg to a previously cleaned metal substrate and then adding another prepreg of the same size on top. The metal substrate was then placed vertically and a visual control of the adhesion of the prepregs was performed after 30 minutes (NCAMP standards) or 1 hour (NF L17-461 standard). In this study, the test was derived from these 2 standards and both types of tack classes were presented. 1 Stiff and brittle 2 Dry but adheres slightly 3 Holds to itself but not to the substrate 4 Holds to itself and to the substrate 5 Sticks to fingers and gloves without resin transfer 6 Wet with resin transfer Table 2: example of Standard NCAMP rating after 30 minutes [10] Class I II III IV V Prepreg adheres to itself ✔ ✔ ✔ ✖ ✔ Prepreg can be separated from itself after draping without damage. ✔ ✖ ✔ ✔ ✖ Prepreg adheres to the substrate ✔ ✔ ✖ ✖ ✖ Table 3: Example of Standard EN rating after 1 hour [9] (e) Thermic measurements by DSC During the curing of a thermoset system with a DSC, three different events can be identified [11]: III Prepreg adheres to itself (c) Analysis of the compression behaviour by pure bending test (c) Analysis of the compression behaviour by pure bending test Pure bending test consists in analysing the compression behaviour of a specimen loaded in pure bending system with a specific specimen (Figure 1). A dumbbell shape specimen with a large thinning radius makes it possible to localise the fracture in the centre while keeping a quasi-homogeneous and uniaxial stress. It was then possible to study the compressive behaviour by simultaneously measuring the bending moment and the tensile and compressive strains at the centre of the specimen [7]. (c) Analysis of the compression behaviour by pure bending test Pure bending test consists in analysing the compression behaviour of a specimen loaded in pure bending system with a specific specimen (Figure 1). A dumbbell shape specimen with a large thinning radius makes it possible to localise the fracture in the centre while keeping a quasi-homogeneous and uniaxial stress. It was then possible to study the compressive behaviour by simultaneously measuring the bending moment and the tensile and compressive strains at the centre of the specimen [7]. Pure bending test consists in analysing the compression behaviour of a specimen loaded in pure bending system with a specific specimen (Figure 1). A dumbbell shape specimen with a large thinning radius makes it possible to localise the fracture in the centre while keeping a quasi-homogeneous and uniaxial stress. It was then possible to study the compressive behaviour by simultaneously measuring the bending moment and the tensile and compressive strains at the centre of the specimen [7]. 3 11TH-EASN 11TH-EASN IOP Publishing IOP Conf. Series: Materials Science and Engineering 1226 (2022) 012101 doi:10.1088/1757-899X/1226/1/012101 doi:10.1088/1757-899X/1226/1/012101 Figure 1: Pure bending specimens’ parameters 2 3 P bilit & Ph i h i l t t Figure 1: Pure bending specimens’ parameters 2.3. Processability & Physicochemical tests Prepreg adheres to itself Prepreg can be separated from itself aft without damage. g Prepreg adheres to the substrate (e) Thermic measurements by DSC ❖ The glass transition temperature of the uncured system (Tg0) ❖ The glass transition temperature of the cured system (Tg) ❖ The exothermic peak of the polymerisation reaction 4 4 11TH-EASN IOP Publishing 11TH-EASN IOP Conf. Series: Materials Science and Engineering 1226 (2022) 012101 IOP Publishing doi:10.1088/1757-899X/1226/1/012101 IOP Conf. Series: Materials Science and Engineering 1226 (2022) 012101 doi:10.1088/1757-899X/1226/1/012101 The enthalpy of the polymerization reaction was determined by integrating the area under the exothermic reaction peak. In the case of prepreg, this value corresponds to a raw enthalpy value, which must be recalculated according to the mass proportion of the resin, since carbon fibres remain inert below 450°C. The area under the curve was determined with the software Proteus developed by Netzsch-Gerätebau GmbH. The enthalpy of the matrix (∆𝐻𝑚𝑎𝑡𝑟𝑖𝑥) was calculated with the equation 1: ∆𝐻𝑚𝑎𝑡𝑟𝑖𝑥= ∆𝐻𝑝𝑟𝑒𝑝𝑟𝑒𝑔× 100 %𝑚𝑟𝑒𝑠𝑖𝑛 (𝐸𝑞1) (𝐸𝑞1) A temperature cycle was set up: the peak of the polymerization reaction was detected during a 1st temperature rise and the reaction’s enthalpy and the Tg of the cross-linked system were determined by a 2nd temperature rise. In this study, the glass transition was determined with the software Proteus. (f) Measurement of the volatile content The volatile content of an uncured prepreg is an important indicator of the progress of the polymerization reaction (the presence of volatiles is proportional with the amount of gas emitted during polymerisation). Moreover, this amount must remain below a limit in order to avoid porosity in the final composite material. This test is standardized according to the European standard EN 2558 [12]. This test consists in measuring the mass of an uncured prepreg before and after its cure cycle. The mass of the samples must be taken at room temperature, after cooling in a desiccator. However, it is impossible to determine the nature of the volatile material. This test is part of the acceptance test of prepregs in the industry. The volatile content was determined with the equation 2: %𝑣𝑜𝑙𝑎𝑡𝑖𝑙𝑒𝑠= 𝑚𝑏𝑒𝑓𝑜𝑟𝑒−𝑚𝑎𝑓𝑡𝑒𝑟 𝑚𝑏𝑒𝑓𝑜𝑟𝑒 × 100 (𝐸𝑞2) %𝑣𝑜𝑙𝑎𝑡𝑖𝑙𝑒𝑠= 𝑚𝑏𝑒𝑓𝑜𝑟𝑒−𝑚𝑎𝑓𝑡𝑒𝑟 𝑚𝑏𝑒𝑓𝑜𝑟𝑒 × 100 (𝐸𝑞2) (𝐸𝑞2) (a) Tack and processability Production year NCAMP EN 2012 1 IV 2015 4 II 2019 5 I New material 5 I able 4 introduces results of tack test realized on prepregs and the desired result on a new materia Production year NCAMP EN 2012 1 IV 2015 4 II 2019 5 I New material 5 I Table 4: Tack results on 2012, 2015 and 2019 rolls Table 4: Tack results on 2012, 2015 and 2019 rolls To be properly draped, a material must be class I on the EN rating and at least class 4 on the NCAMP rating. These rankings were achieved for the material of 2019. The 2015 material was draped well but 5 11TH-EASN IOP Conf. Series: Materials Science and Engineering 1226 (2022) 012101 IOP Publishing doi:10.1088/1757-899X/1226/1/012101 IOP Publishing IOP Conf. Series: Materials Science and Engineering 1226 (2022) 012101 doi:10.1088/1757-899X/1226/1/012101 the plies can be separated from each other after stacking. On the other hand, it was very complicated to drape the 2012 material because it was completely dry and brittle. Tack deteriorates with time and aging of the resin. the plies can be separated from each other after stacking. On the other hand, it was very complicated to drape the 2012 material because it was completely dry and brittle. Tack deteriorates with time and aging of the resin. These tests clearly highlight the influence of polymerization advancement. The limitation of this test is that it is not quantitative. The determination of the tack classification is only based on the qualitative observations of the handler. To correct this uncertainty, different operators could perform this test two times for example. (b) Thermic events measurements (b) Thermic events measurements Figure 2 summarizes the results obtained during the DSC measurements on the different prepregs. Figure 2: DSC measurements results: a) Polymerization enthalpy evolution as function of time; b) Glass temperature of the cured sample evolution gure 2 summarizes the results obtained during the DSC measurements on the different prepregs. DSC measurements on the different prepregs. on enthalpy evolution as function of time; b) Glass Figure 2: DSC measurements results: a) Polymerization enthalpy evolution as function of time; b) Glass temperature of the cured sample evolution It can be shown in Figure 2a) that the enthalpy of reaction did not show a significant difference from 1 year of expiry to 5 years of expiry (loss less than 15%). However, after 9 years, the enthalpy has decreased by 60%. This test therefore shows an effect of aging of the resin during long storage; moreover, the enthalpy measurement results was consistent with the results of the tack test. The longer the prepreg is in the freezer, the lower its enthalpy. It shows that less energy was needed to complete polymerization, which means that the reaction has advanced. It can be shown in Figure 2a) that the enthalpy of reaction did not show a significant difference from 1 year of expiry to 5 years of expiry (loss less than 15%). However, after 9 years, the enthalpy has decreased by 60%. This test therefore shows an effect of aging of the resin during long storage; moreover, the enthalpy measurement results was consistent with the results of the tack test. The longer the prepreg is in the freezer, the lower its enthalpy. It shows that less energy was needed to complete polymerization, which means that the reaction has advanced. p y , Regarding the Tg (Figure 2b), the determination might be difficult but the main observation is that Tg increases as the resin ages. Indeed, Tg2012 is 154,5 °C while Tg2020 is 135,8°C. This increase of Tg can be explained by i) increase of the polymer molecular weight and ii) development of inter-chain links that form a structural 3D polymer (crosslinking). These two phenomena induce a decrease of the molecular mobility and an increase of Tg. This test is coherent with tack test and reaction enthalpy measurement but might be useless because of its lack of precision. (b) Compressive behaviour Finally, Table 5 shows that the ultimate compressive strain at 0° do not vary with aging. As for the tensile strengths value, only the results of the 2012 and 2019 samples are presented. There is no difference of the compressive properties between 2012 and 2019 samples. This test does not seem to be useful for recertification of expired prepregs. 3.2. Parameters not evolving with aging 3.2. Parameters not evolving with aging rameters not evolving with aging Tensile modulus (GPa) Tensile strength (MPa) ILSS (MPa) Utimate bending strain (%) 2019 162.9 ± 5.0 1697.9 ± 137.7 98.4 ± 5.7 1.49 ± 0.8 2015 168.5 ± 11.9 / 76.10 ± 1.8 / 2012 163.1 ± 3.0 1713.7 ± 129.1 78.26 ± 3.8 1.46 ± 0.1 Table 5: Results of mechanical tests Table 5: Results of mechanical tests (c) Volatile content (c) Volatile content Figure 3 shows the results of the volatile content obtained on the 3 tested materials. ( ) Figure 3 shows the results of the volatile content obtained on the 3 tested materials Figure 3 shows the results of the volatile content obtained on the 3 tested materials. he results of the volatile content obtained on the 3 tested materials. Figure 3: Volatile content of the rolls from 2019, 2015 and 2012 Figure 3: Volatile content of the rolls from 2019, 2015 and 2012 6 11TH-EASN IOP Publishing 11TH-EASN IOP Conf. Series: Materials Science and Engineering 1226 (2022) 012101 IOP Publishing doi:10.1088/1757-899X/1226/1/012101 IOP Conf. Series: Materials Science and Engineering 1226 (2022) 012101 doi:10.1088/1757-899X/1226/1/012101 doi:10.1088/1757-899X/1226/1/012101 It can be noted an important decrease in the volatile content between 1 year peremption (2019) and 5 years expiry (2015). After 5 years of peremption, the volatile content did not seem to be reduced. As the polymerization reaction had advanced, the amount of uncured resin in the prepreg had decreased. Therefore, there was fewer organic molecules emitted during the curing process. This test is very easy to set and give meaningful results. It can be noted an important decrease in the volatile content between 1 year peremption (2019) and 5 years expiry (2015). After 5 years of peremption, the volatile content did not seem to be reduced. As the polymerization reaction had advanced, the amount of uncured resin in the prepreg had decreased. Therefore, there was fewer organic molecules emitted during the curing process. This test is very easy to set and give meaningful results. (d) Interlaminar shear strength Table 5 shows the evolution of the ILSS results with time. It shows a decrease in interlaminar shear strength with time, even if the 2012 result is slightly higher than the 2015 result. ILSS is decreased by about 20% between the 2012 and 2019 samples. It is possible to understand that the aging of the resin leads to poor processability and lack of adhesion between plies. These effects can give to samples a greater sensitivity to delamination. This test is very representative of the loss of mechanical performance of the prepregs during aging. (a) Tensile properties at 0° Then Table 5 depicts the calculated young modulus for the 2012, 2015 and 2019 rolls. For comparison, the prepreg datasheet indicated a Young's modulus of 178 GPa. The measured values are all lower than this reference but they are very close for the three prepregs. This small deviation may be due to differences in the alignment of the fibres. Considering the average values obtained for the 3 years, it seems that the Young's modulus at 0° in the fibre’s direction does not vary with aging. This result supports the idea that the fibres properties are not affected during aging and that only the matrix properties undergo aging. The same observations are made with the tensile strength values (The tensile strength results of the 2015 samples are not presented due to a technical problem). So, 0° tensile test does not seem to be useful for recertification of expired prepregs. 4. Conclusion & Perspectives Several new scenarios could then be considered following simplified recertification. We could imagine to keep the originally defined application, applications in other domains (non-structural aerospace applications or non aerospace applications) or even the integration of the carbon fibre recycling chain p p p y g g g Several new scenarios could then be considered following simplified recertification. We could imagine to keep the originally defined application, applications in other domains (non-structural aerospace applications or non-aerospace applications) or even the integration of the carbon fibre recycling chain implemented by the MANIFICA project. The final objective of this study is to reduce waste as much as possible. Complete results shall be published in 2022. 4. Conclusion & Perspectives This paper presents the first results of a study with the objective of developing a simplified method for expired prepregs recertification. Today, these tests are derived from standards already used for quality control of the virgin prepregs. Some of these tests may not be useful and other tests may be simplified in the future. During these studies, it was noticed that one of the major problems is the difficult processability of the prepreg induced by the aging of the resin. Thus, compaction of plies, curvatures may become difficult to manufacture, introducing defects that can weaken the final part, especially if it is a complex part. 7 11TH-EASN IOP Publishing 11TH-EASN IOP Conf. Series: Materials Science and Engineering 1226 (2022) 012101 IOP Publishing doi:10.1088/1757-899X/1226/1/012101 IOP Conf. Series: Materials Science and Engineering 1226 (2022) 012101 doi:10.1088/1757-899X/1226/1/012101 It has also been shown that some parameters do not vary with time and that simplifying the procedure is possible. As a matter of fact, all tests related to the fibres could be useless as only the resin really ages. The comprehension of the aging mechanisms could show that some parameters can be linked together because they give the same information. To simplify the recertification as much as possible, considerate could be proposed to perform only the tests measuring the real ageing of the resin with time. It has also been shown that some parameters do not vary with time and that simplifying the procedure is possible. As a matter of fact, all tests related to the fibres could be useless as only the resin really ages. The comprehension of the aging mechanisms could show that some parameters can be linked together because they give the same information. To simplify the recertification as much as possible, considerate could be proposed to perform only the tests measuring the real ageing of the resin with time. It has also been shown that some parameters do not vary with time and that simplifying the procedure is possible. As a matter of fact, all tests related to the fibres could be useless as only the resin really ages. The comprehension of the aging mechanisms could show that some parameters can be linked together because they give the same information. To simplify the recertification as much as possible, considerate could be proposed to perform only the tests measuring the real ageing of the resin with time. References [1] K. Pannkoke, M. Oethe et J. Busse, «Efficient prepreg recycling at lowtemperatures,» Cryogenics , vol. 38, n° %11, 1998. [2] Carbon Composte e.V, «Composites Market Report 2019: The global CF- and CC-Market 2019,» 2019. [En ligne]. Available: https://composites-united.com/media/3988/eng_ccev_market- report_2019_short-version.pdf. [3] J. P. Martins de Silva Luis, «Effect of out-time aging in composite prepreg material,» Lisbonne, 2014. [4] AFNOR, «Plastiques renforcés de fibres - Méthodes de fabrication de plaques d'essai - P moulage des préimprégnés,» NF ISO 1268-4, 2006. [5] AFNOR, «Série aérospatiale - Plastiques renforcés de fibres de carbone - Stratifiés unidirectionnels - Essai de traction parallèlement à la direction des fibres,» NF EN 2561, 1996. [6] AFNOR, «Série aérospatiale - Plastiques renforcés de fibres de carbone. Stratifiés unidirectionnels. Détermination de la résistance apparente au cisaillement interlaminaire,» NF EN 2563, 1997. [7] C. Bois, O. Montagnier et C. Hochard, «Caracterisation of compression behavior of composite materials using a bending test,» JNC15. [8] K. Gautier, Etude du tack des préimprégnés, Brest: 24ème Congrès Français de Mécanique, 2019. AFNOR, Série aérospatiale - Préimprégnés - Détermination de la pégosité, NF L17-461, 1993. [10] NCAMP standard, «Low intial temperature Vacuum bag only,» Wichita state university, 2017. [11] L. K. Grunenfelder, «Defect control in vacuum bag only processing of composites prepreg,» Thesis of the South Carolina University, 2012. [12] AFNOR, «Série aérospatiale - Préimprégnés de fibres de carbone. Détermination de la teneur en matières volatiles,» 1997. 8 8
https://openalex.org/W4283590736	https://univ-angers.hal.science/hal-03708030/document	English	null	Evaluation of two commercial kits and two laboratory-developed qPCR assays compared to LAMP for molecular diagnosis of malaria	Malaria journal	2,022	cc-by	7,414	Bouzayene et al. Malaria Journal (2022) 21:204 https://doi.org/10.1186/s12936-022-04219-1 Bouzayene et al. Malaria Journal (2022) 21:204 https://doi.org/10.1186/s12936-022-04219-1 Malaria Journal Open Access Evaluation of two commercial kits and two laboratory‑developed qPCR assays compared to LAMP for molecular diagnosis of malaria Azza Bouzayene1, Rizwana Zaffaroullah1, Justine Bailly2, Liliane Ciceron1, Véronique Sarrasin1,2, Sandrine Cojean1, Nicolas Argy1,2, Sandrine Houzé1,2 and Valentin Joste1,2 on behalf of the French National Malaria Reference Centre study group © 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. 1 National Malaria Reference Centre, AP-HP, Hôpital Bichat - Claude Bernard, 46 Rue Henri Huchard, 75018 Paris, France Full list of author information is available at the end of the article Abstract Background: Malaria is an infectious disease considered as one of the biggest causes of mortality in endemic areas. This life-threatening disease needs to be quickly diagnosed and treated. The standard diagnostic tools recommended by the World Health Organization are thick blood smears microscopy and immuno-chromatographic rapid diagnostic tests. However, these methods lack sensitivity especially in cases of low parasitaemia and non-falciparum infections. Therefore, the need for more accurate and reliable diagnostic tools, such as real-time polymerase chain reaction based methods which have proven greater sensitivity particularly in the screening of malaria, is prominent. This study was conducted at the French National Malaria Reference Centre to assess sensitivity and specificity of two commercial malaria qPCR kits and two in-house developed qPCRs compared to LAMP. Methods: 183 blood samples received for expertise at the FNMRC were included in this study and were subjected to four different qPCR methods: the Biosynex Ampliquick® Malaria test, the BioEvolution Plasmodium Typage test, the in-house HRM and the in-house TaqMan qPCRs. The specificity and sensitivity of each method and their confidence intervals were determined with the LAMP-based assay Alethia® Malaria as the reference for malaria diagnosis. The accuracy of species diagnosis of the Ampliquick® Malaria test and the two in-house qPCRs was also evaluated using the BioEvolution Plasmodium Typage test as the reference method for species identification. Results: The main results showed that when compared to LAMP, a test with excellent diagnostic performances, the two in-house developed qPCRs were the most sensitive (sensitivity at 100% for the in-house TaqMan qPCR and 98.1% for the in-house HRM qPCR), followed by the two commercial kits: the Biosynex Ampliquick® Malaria test (sensitivity at 97.2%) and the BioEvolution Plasmodium Typage (sensitivity at 95.4%). Additionally, with the in-house qPCRs we were able to confirm a Plasmodium falciparum infection in microscopically negative samples that were not detected by commercial qPCR kits. This demonstrates that the var genes of P. falciparum used in these in-house qPCRs are more reliable targets than the 18S sRNA commonly used in most of the developed qPCR methods for malaria diagnosis. Conclusion: Overall, these results accentuate the role molecular methods could play in the screening of malaria. This may represent a helpful tool for other laboratories looking to implement molecular diagnosis methods in their routine Correspondence: azza.bouzayenne@aphp.fr 1 National Malaria Reference Centre, AP-HP, Hôpital Bichat - Claude Bernard, 46 Rue Henri Huchard, 75018 Paris, France Full list of author information is available at the end of the article Background Malaria is an infectious disease caused by a mosquito- transmitted parasite of the genus Plasmodium. According to the World Malaria Report of 2021, 241 million malaria cases were estimated in 2020 in 85 malaria endemic countries [1]. Even though the mortality rate of this dis- ease has reduced globally through the years over the period 2000–2019, it is still considered as one of the big- gest causes of mortality with an estimated 627,000 deaths in 2020 [1]. In fact, in 2020 malaria deaths increased by 12% compared to 2019 with an estimated 47,000 (68%) of the additional 69,000 deaths that were caused by ser- vice disruptions during the COVID-19 pandemic [1]. More importantly, it is one of the leading causes of death for children under five and is problematic for pregnant women in endemic countries [1]. Since the late 1980s, several polymerase chain reac- tion (PCR) based methods were developed for malaria detection. These techniques represented a significant improvement to light microscopy and other conventional diagnostic tools because of the superior limit of detection (LOD) [8]. Most of the methods developed had a com- mon target: the Plasmodium 18S SSU RNA gene, includ- ing the loop-mediated isothermal amplification (LAMP), nested, semi-nested and real-time PCRs [16]. Neverthe- less, the specific identification of the different Plasmo- dium species has remained problematic since it requires multiplexing, which can cause primer competition and thus failure to detect species with lower parasite densities in mixed infections. It could also be either time consum- ing or expensive. Six common Plasmodium species are known to be responsible for the majority of human infections: Plas- modium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium knowlesi and Plasmodium ovale, which is divided in two genetically distinct sympatric species P. ovale curtisi and P. ovale wallikeri [2]. However, recent advances in molecular diagnosis and genotyping have shown that other primate malaria species can also cause human infections including Plasmodium brasi- lianum [3], Plasmodium simium [4] and Plasmodium cynomolgi [5]. In metropolitan France, most reported cases are imported from sub-Saharan Africa. Based on the reports of the French National Malaria Reference Centre (FNMRC), 2895 malaria cases were declared in 2019, but over 5000 imported cases have been estimated in France. Symptomatic patients were mainly migrants, travellers or military staff [6]. © 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. Bouzayene et al. Malaria Journal (2022) 21:204 Page 2 of 10 analysis, which could be essential for the detection and treatment of malaria carriers and even for the eradication of this disease. rds: Malaria, Plasmodium, P. falciparum, Molecular diagnosis, qPCR, LAMP, Bio-Evolution, Biosynex such as the histidine-rich protein II (HRP2) synthesized by P. falciparum and the Plasmodium specific lactate dehydrogenase (pLDH) or p-aldolase usually synthesized during the erythrocytic cycle and therefore common to all malaria species [10]. Recently, some of these RDT’s have included the P. vivax-specific lactate dehydrogenase (pvLDH) allowing the detection of P. vivax [11]. While these tests can detect approximately 100 parasites/µL (0.002% parasitaemia) [12], their interpretation can be tricky. In fact, the major restrictions of RDTs are: cases when the test is falsely interpreted as positive due to the persistence of HRP2 in the blood even several days after parasite clearance and malaria recovery [13], false nega- tives caused by gene deletions and decreased sensitivity for non-falciparum infections [14, 15]. Background The LAMP methodology was first published in the year 2000 and is based on the isothermal amplification of DNA by using the high strand displacement activity of the Bacillus stearothermophilus (Bst) DNA polymerase and specific sets of inner and outer primers identifying distinct regions of the targeted DNA [17]. This generates loop formations and inverted repeats of target sequences permitting a highly efficient DNA amplification under isothermal conditions in less than one hour with a LOD as few as six copies [17]. f In practice, accurate diagnosis of this disease is a very important tool for an effective treatment. The micro- scopic examination of Giemsa-stained thick blood smears has always been the “gold standard” for malaria diagnosis in many endemic areas [7]. This method is inexpensive and ensures the identification of Plasmodium species and parasite densities. However, it is limited due to the inter- observer variability especially with low parasitaemia and mixed or non-falciparum infections [8]. Therefore, it requires well-trained experts and microbiologists. In addition to light microscopy, the World Health Organiza- tion (WHO) recommends the use of immuno-chromato- graphic rapid diagnostic tests (RDTs) as a routine tool for malaria diagnosis [9]. These tests detect parasite antigens When quantitative real-time PCR (qPCR) technology was first introduced, it was considered revolutionary for the molecular diagnosis of malaria. It is in fact more sen- sitive than other conventional PCR methods (LOD < 0.1 parasites/µL) [18] and easier to execute with no post-PCR manipulations. In practice, two main types of qPCRs exist: the ones using fluorescent dyes such as SYBR green Bouzayene et al. Malaria Journal (2022) 21:204 Bouzayene et al. Malaria Journal (2022) 21:204 Page 3 of 10 which intercalates with nonspecific double-stranded DNA and the ones with specific fluorescent probes such as TaqMan probes [19]. A new method has been recently added to the molecular detection and genotyping of parasites, real-time qPCR coupled with high resolution melting (HRM) curve analysis. This technique is based on detecting the differences of nucleotide sequences in targeted fragments of a gene generating different melting temperatures (Tm) by amplifying the region of interest in the presence of a specialized DNA binding dye and grad- ual denaturation of the amplicons producing character- istic melting profiles [19]. This method has already been used for both Plasmodium diagnosis and distinction and was proven to be very effective by Chua et al. and Joste et al. Background [19, 20].h non-interventional research and therefore only requires the non-opposition of the patient during sampling (per article L1211-2 of the public health code). The data col- lected were anonymized before use. LAMP methodh The commercial LAMP based assay Alethia® Malaria is a qualitative isothermal molecular test allowing the direct detection of Plasmodium spp, without species identifi- cation, by targeting segments of its mitochondrial DNA after lysis of whole blood samples. It was performed on the Illumipro-10™ (Meridian Bioscience, Ohio, US) auto- mated isothermal amplification and detection system fol- lowing the test procedure provided by the manufacturer. The present study was carried out at the French National Malaria Reference Centre (FNMRC) with the aim to assess the sensitivity and specificity of a new com- mercial malaria qPCR kit, the Ampliquick® Malaria test (Biosynex), and two in-house qPCRs developed at the FNMRC with the LAMP Alethia® Malaria (Meridian Bioscience) as the reference method for positivity. The LAMP technology has shown ≥ 95% pooled sensitivity and specificity for the detection of Plasmodium infec- tions and is, therefore, deemed to be a test with an excel- lent diagnostic performance [21]. This test is also the first screening option used in some medical laboratories in France. For those reasons, the commercial LAMP-based assay Alethia® Malaria was considered as the gold stand- ard for sensitivity during this study. However, for the identification of malaria species, the three tested qPCR methods were compared with the TaqMan qPCR Plas- modium Typage (Bio-Evolution), another commercial kit available in France. DNA extraction DNA was extracted from a sample of 200 µL of whole blood and eluted in 100 µL of buffer using Magnapure® (Roche diagnosis, Bale, Switzerland) following the manu- facturer’s instructions. DNA was then stored at − 20 °C until analyses. Plasmodium Typage qPCR methodh The Plasmodium Typage (Bio-Evolution, Île-de-France, France) real-time qPCR kit is a TaqMan based diagnostic test allowing the detection and the simultaneous identifi- cation of P. falciparum, P. ovale, P. vivax, P. malariae and P. knowlesi. This test was routinely used for malaria diag- nosis at the FNMRC. Two reaction wells are necessary to detect the five Plas- modium species each one containing 15 µL of either the Master Mix 1 or the Master Mix 2, prepared following the procedure provided by the manufacturer, and 5 µL of extracted DNA. Ready to use positive and negative con- trols are also provided with the qPCR kit. Primers target- ing the human beta actin are used in the Master Mix 1 to control DNA extraction. This qPCR was performed using the Viia7™ (Thermo Fisher Scientific, Massachusetts, US) thermal cycler following the thermal program: 30 s at 95 °C then 40 cycles of 15 s at 95 °C and 45 s at 60 °C and finally a cooling phase of 1 s at 37 °C. In‑house HRM and TaqMan qPCR assays developmenth A total of 183 samples were included but only 147 were analysed with the LAMP Alethia® Malaria method, the gold standard for positivity in this study, to determine whether there was a Plasmodium infection or not (Addi- tional file 1). The results showed 104 LAMP positive samples from which only 86 were positive by microscopy, and 43 negatives. The primers used for both the HRM and TaqMan assays, described by Schindler et al. [22], target two independ- ent Plasmodium genes: the Pan-Plasmodium 18S rRNA sequence (Pspp 18S) and the P. falciparum-specific acidic terminal sequence of the var genes (PfvarATS). The HsR- NaseP human gene was used as an internal control (Ci) to rule out extraction failure (Table 1). For the remaining 36 Plasmodium spp. positive sam- ples included in this study, but not tested with the LAMP method, DNA extracts were still used to compare species diagnosis and to correlate the cycle thresholds (Ct) and the parasite density for the different qPCR methods. The HRM and TaqMan qPCR assays were performed on the Viia7™ Real-Time PCR System (Thermo Fisher Scientific, Massachusetts, US). The thermal profile for the HRM-qPCR was as following: initialization step at 95 °C for 10 min, 40 cycles of 15 s at 95 °C, 1 min à 60 °C and an HRM phase of 10 s at 95 °C and 1 min at 60 °C then 15 s at 95 °C and 15 s at 60 °C. The one for the TaqMan-qPCR was: 15 min at 95 °C, 45 cycles of 15 s at 95 °C and 1 min at 55 °C. Clinical samples For this study, blood samples of patients suspected with malaria and received for expertise at the FNMRC from January 2019 to January 2020 were retrospectively included. These blood samples were collected into EDTA tubes and, after reception, were subjected to routine bio- logical diagnosis: microscopy on stained thin and thick blood films and DNA extraction. Ampliquick® Malaria qPCR method The Ampliquick® Malaria kit is a real-time TaqMan qPCR diagnostic test. This technique depends on the gene amplification of a specific region of the 18S RNA gene of Plasmodium spp (Pan) and P. falciparum. This kit can be used in two ways: directly from a whole blood sample following the manufacturer’s instructions or after DNA extraction. The whole blood sample direct detec- tion protocol was not tested in this study. i There was no need for specific consent from the patients since all the data was collected from the FNM- RC’s database and analysed in accordance with the com- mon public health mission of all the National Reference Centres of France. Everything was coordinated with the ‘Santé Publique France’ organization for malaria surveil- lance and care. According to the article L1221-1.1 of the public health code in France, the study of biological sam- ples obtained from routine medical care is considered as qPCR experiments were performed using the 7500 Fast Real-Time PCR System (Thermo Fisher Scientific, Bouzayene et al. Malaria Journal (2022) 21:204 Bouzayene et al. Malaria Journal (2022) 21:204 Page 4 of 10 Kappa coefficient [23]. Ct values were compared using the Mann–Whitney U-test and the linear regression was evaluated using the F-test. Massachusetts, US) following the thermal program: 3 min at 95 °C then 50 cycles of 10 s at 94 °C and 20 s at 60 °C. Each reaction well contained 8 µL of sample or control and 12 µL of the lyophilized mix in microtubes provided by the manufacturer. Average melting curve peak (Tm) values for Plasmodium spp. and P. falciparumh The in-house HRM-qPCR assay was able to identify the presence of a Plasmodium spp. or a P. falciparum infec- tion. Plasmodium spp infections displayed a specific Pan melting curve peak value (Tm1), which was reproducible no matter the species or the association of species. In case of a P. falciparum infection, a second Tm value spe- cific to P. falciparum (Tm2) was also present (Table 2). As The reaction wells for the HRM assay contained 10 µL of the 1 × MeltDoctor™ HRM Master Mix (Thermo Fisher Scientific, Massachusetts, US), 0.3 µM of each primer (Pspp18S and PfvarATS) (Table 1) and 5 µL DNA. The reaction wells for the TaqMan assay contained 10 µL of the 1 × Luna® Universal Probe qPCR Master Mix (New England BioLabs Inc, Massachusetts, US), 0.1 to 0.4 µM of the probes and primers of each target (Pspp18S, Pfvar- ATS and HsRNaseP) (Table 1) and 6 µL DNA. Table 2 Average Tm values and their Standard Deviations (SD) for P. falciparum and Plasmodium spp other than P. falciparum Table 2 Average Tm values and their Standard Deviations (SD) for P. falciparum and Plasmodium spp other than P. falciparum Accuracy of species diagnosis Table 3 Sensitivity and Specificity of microscopy and the different qPCR methods studied compared to LAMP Alethia® Malaria Microscopy Plasmodium Typage (Bio- Evolution) Ampliquick® Malaria In-house HRM In-house TaqMan + − + − + − + − + − LAMP + (N = 104) 86 18 99 5 101 3 102 2 104 0 LAMP − (N = 43) 0 43 0 43 0 43 0 43 0 43 Specificity 100% 100% 100% 100% 100% Sensitivity 95% CI 85.2% [74–89.4] 95.4% [89.1–98.4] 97.2% [91.8–99.4] 98.1% [93–99.8] 100%[100–100] Kappa 95% CI 0.74 [0.63–0.85] 0.92 [0.85–0.99] 0.95 [0.9–1] 0.97 [0.92–1] 1 [1–1] Table 3 Sensitivity and Specificity of microscopy and the different qPCR methods studied compared to LAMP Alethia® Malaria Microscopy Plasmodium Typage (Bio- Evolution) Ampliquick® Malaria In-house HRM In-house TaqMan + − + − + − + − + − LAMP + (N = 104) 86 18 99 5 101 3 102 2 104 0 LAMP − (N = 43) 0 43 0 43 0 43 0 43 0 43 Specificity 100% 100% 100% 100% 100% Sensitivity 95% CI 85.2% [74–89.4] 95.4% [89.1–98.4] 97.2% [91.8–99.4] 98.1% [93–99.8] 100%[100–100] Kappa 95% CI 0.74 [0.63–0.85] 0.92 [0.85–0.99] 0.95 [0.9–1] 0.97 [0.92–1] 1 [1–1] Table 4 Accuracy of Plasmodium species identification with the different qPCR methods studied compared to Plasmodium Typage (Bio-Evolution) Plasmodium Typage (Bio-Evolution) Ampliquick® Malaria In-house HRM qPCR In-house TaqMan qPCR P. falciparum (n = 73) Pf+/Pan+ 73 Pf+/Pan+ 73 Pf+/Pan+ 71 Pf+ 2 P. ovale (n = 36) Pan+ 36 Pan+ 36 Pan+ 36 P. vivax (n = 8) Pan+ 8 Pan+ 8 Pan+ 8 P. malariae (n = 15) Pan+ 15 Pan+ 15 Pan+ 14 Pf+/Pan+ 1 P. falciparum + P. malariae (n = 2) Pf+/Pan+ 2 Pf+/Pan+ 2 Pf+/Pan+ 2 P. falciparum + P. malariae + P. ovale (n = 1) Pf+/Pan+ 1 Pf+/Pan+ 1 Pf+/Pan+ 1 Table 4 Accuracy of Plasmodium species identification with the different qPCR methods studied compared to Plasmodium Typage (Bio-Evolution) Plasmodium Typage (Bio-Evolution) Ampliquick® Malaria In-house HRM qPCR In-house TaqMan qPCR P. falciparum (n = 73) Pf+/Pan+ 73 Pf+/Pan+ 73 Pf+/Pan+ 71 Pf+ 2 P. ovale (n = 36) Pan+ 36 Pan+ 36 Pan+ 36 P. vivax (n = 8) Pan+ 8 Pan+ 8 Pan+ 8 P. malariae (n = 15) Pan+ 15 Pan+ 15 Pan+ 14 Pf+/Pan+ 1 P. falciparum + P. malariae (n = 2) Pf+/Pan+ 2 Pf+/Pan+ 2 Pf+/Pan+ 2 P. Statistical analysish microscopy (n = 86) were also positive after analysis with the four tested qPCR methods. Statistical analysish The sensitivity, specificity and Kappa coefficient with 95% confidence intervals (CI) of each qPCR method were cal- culated compared to the LAMP method. The R software was used to perform statistical tests and calculate the Table 1 The mix of primers and probes used in this study, adapted from Schindler et al. [22] Species Oligo name Target region Oligo final concentrations (TaqMan assay) (µM) Oligo final concentrations (HRM assay) (µM) Supplier Pan-Plasmodium Pspp18S fwd 18S rRNA 0.4 0.3 Eurogentec Pspp18S rev 18S rRNA 0.4 0.3 Eurogentec Pspp18S probe 18S rRNA 0.2 – Eurogentec P. falciparum PfvarATS fwd varATS 0.4 0.3 Eurogentec PfvarATS rev varATS 0.4 0.3 Eurogentec PfvarATS probe varATS 0.25 – Eurogentec H. sapiens (Ci) HsRNaseP fwd Rnasep gene 0.2 – Eurogentec HsRNaseP rev Rnasep gene 0.2 – Eurogentec HsRNaseP probe Rnasep gene 0.1 – Eurogentec Table 1 The mix of primers and probes used in this study, adapted from Schindler et al. [22] Bouzayene et al. Malaria Journal (2022) 21:204 Page 5 of 10 Fig. 1 Derivative Melt curve peaks (Tm) for P. falciparum and Plasmodium spp. after HRM phase. A Tm peaks for both P. falciparum and Plasmodium spp. targets in case of P. falciparum carriage. B Tm peak for the Plasmodium spp. target in case of Plasmodium spp. other than P. falciparum carriage. X axis represents the temperature (°C). Y axis represents the fluorescence (derivative melt curve) Fig. 1 Derivative Melt curve peaks (Tm) for P. falciparum and Plasmodium spp. after HRM phase. A Tm peaks for both P. falciparum and Plasmodium spp. targets in case of P. falciparum carriage. B Tm peak for the Plasmodium spp. target in case of Plasmodium spp. other than P. falciparum carriage. X axis represents the temperature (°C). Y axis represents the fluorescence (derivative melt curve) Fig. 1 Derivative Melt curve peaks (Tm) for P. falciparum and Plasmodium spp. after HRM phase. A Tm peaks for both P. falciparum and Plasmodium spp. targets in case of P. falciparum carriage. B Tm peak for the Plasmodium spp. target in case of Plasmodium spp. other than P. falciparum carriage. X axis represents the temperature (°C). Y axis represents the fluorescence (derivative melt curve) Bouzayene et al. Malaria Journal (2022) 21:204 Page 6 of 10 shown in Fig. 1, the Tm of P. falciparum can be clearly distinguished from that of Plasmodium spp. (Pan). Accuracy of species diagnosis On each included sample, microscopy diagnosis and species identification by qPCR were systematically done (Additional file 2). The Bio-Evolution Plasmodium Typage is a routinely used qPCR kit for malaria diagno- sis at the FNMRC. It allows the simultaneous identifica- tion of P. falciparum, P. ovale, P. vivax, P. malariae and P. knowlesi and was therefore considered as the reference method for the Plasmodium species identification when the sample was positive by this method. The 147 isolates tested with LAMP were assessed with the four different qPCR methods. The 43 LAMP negative samples were also negative by all four methods, which gives them all 100% specificity. Of the 104 positive sam- ples, five were not detected by Plasmodium Typage (Bio- Evolution), three by Ampliquick® Malaria and two by the HRM-qPCR but all with the in-house TaqMan-qPCR (Table 3). Compared to LAMP, the in-house TaqMan and HRM qPCRs were the most sensitive (sensitivity = 100%, 95% CI [100–100] and 98.1%, 95% CI [93–99.8] respectively), followed by the two commercial kits: the Ampliquick® Malaria test (sensitivity = 97.2%, 95% CI [91.8–99.4]) and the Bio-Evolution Plasmodium Typage (sensitiv- ity = 95.4%, 95% CI [89.1–98.4]). Microscopic tech- niques had a sensitivity of 85.2% (95% CI [74–89.4]). Kappa’s coefficient shows that the qPCRs tested are highly concordant with the LAMP method, in particu- lar the in-house qPCRs (Table 3). All positive samples by Analysis with the Bio-Evolution kit showed 135 posi- tive and 48 negative samples. Of the 135 positive samples: 73 were identified as P. falciparum, 36 were identified as P. ovale spp, 8 were identified as P. vivax, 15 were identi- fied as P. malariae, 2 mixed infections by P. falciparum and P. malariae and one mixed infection P. falciparum, P. malariae and P. ovale. These findings were compared with the other tech- niques (Table 4). The results for P. falciparum, P. ovale, P. vivax and the mixed infections were correlated no mat- ter the qPCR method used. Additionally, out of the 15 P. Discussion To control malaria, accurate and rapid diagnostic tools are very essential not only for the establishment of an effective treatment but also for surveillance and epide- miological monitoring. The quality and sensitivity of the diagnosis are very important criteria to avoid misdiagno- sis which could lead to severe health problems, recrudes- cence, drug resistance and possibly death [24].h The in-house developed qPCRs target the var genes for P. falciparum detection, a multigenic family with approx- imately sixty copies in each P. falciparum genome [26]. It may probably explain the better sensitivity compared to the two commercial kits that target the 18S rRNA gene, present in four to eight copies in each Plasmodium genome. This was also highlighted when three samples were only positive for the var genes target after the in- house TaqMan qPCR. These samples were either micro- scopically negative or had very low parasitaemia implying that the var genes of P. falciparum are more sensitive tar- gets than the 18S rRNA (Pan), despite no statistical dif- ference in Ct values. Moreover, the same DNA extracts were evaluated with the four qPCR methods implying that the different sensitivities are proper to each method and not due to the quality of DNA extraction. The implication of the molecular diagnosis of malaria has been highlighted in many studies. As reported in the literature, PCR and qPCR methods displayed high sen- sitivity compared to other conventional methods such as microscopy or RDTs [8, 13, 24]. In the present study, the sensitivity and specificity of the commercial malaria qPCR kit Ampliquick® Malaria and two in-house devel- oped qPCRs were assessed by comparing them with the highly sensitive LAMP method Alethia® Malaria for sensibility evaluation and with the Plasmodium typage (Bio-Evolution) qPCR method for species diagnosis eval- uation. Unlike other LAMP methods that can differenti- ate Plasmodium spp., P. falciparum or P. vivax infections [25], Alethia® Malaria does not allow species diagnosis and requires, for that reason, an equally sensitive species PCR. It is very necessary to differentiate the Plasmodium species to identify P. ovale or P. vivax infections because they require specific treatment with primaquine, but also P. knowlesi infections which can lead to severe illness and are commonly misidentified with conventional methods. Compared to Plasmodium Typage (Bio-Evolution), the major limitation of the other techniques is the absence of species identification. Accuracy of species diagnosis Malaria Journal (2022) 21:204 Page 8 of 10 Page 8 of 10 Correlation between parasite density and qPCR cycle threshold (Ct) valuesh The main results of this study showed that when com- pared to LAMP, the gold standard for positivity through- out this study, the Bio-Evolution Plasmodium Typage was the least sensitive at 95.2% and the in-house TaqMan- qPCR was the most sensitive at 100%. When it comes to commercial kits, it is very clear that Ampliquick® Malaria is better at detecting Plasmodium infections than the Bio-Evolution Plasmodium Typage. While analysing the data, the Bio-Evolution kit results were compared to microscopy results and when the initial microscopy diagnosis is positive for a Plasmodium infection, the subsequent species identification by this method is accu- rate. Mixed infections were even distinguished in some cases. However, when compared to negative microscopy results it is clear that this qPCR method is not as reliable as the others in detecting false negatives. Indeed, out of the 61 negatives with microscopy, 48 were negative with Plasmodium Typage (Bio-Evolution) while 43 were con- sidered truly negative after the LAMP and the in-house TaqMan qPCR analysis. Of the 48 negatives one was identified as a Plasmodium spp. infection and four were identified as P. falciparum mono-infections. These find- ings show that the in-house TaqMan qPCR was able to detect P. falciparum infections that were not detected by the commercial kit.h The median Pan-Plasmodium Ct of the Ampliquick® Malaria (median Ct = 23.40), the species Ct of the Bio- Evolution Plasmodium typage (median Ct = 27) and the undifferentiated Ct of the in-house HRM-qPCR (median Ct = 22.88) and the in-house TaqMan-qPCR (median Ct = 23.58) were calculated. The data show that the in- house qPCRs and the commercial Ampliquick® Malaria have lower Ct values than the Bio-Evolution Plasmodium Typage (pooled Ct for all species) (p < 0.001, Mann– Whitney U-test), concordant with a better sensitivity. For the in-house TaqMan-qPCR, Ct of the var genes and the 18S rRNA targets were not different (24.2 vs 23.5, p = 0.31, Mann–Whitney U-test). As shown in Fig. 2, comparison of the parasite density determined by microscopy and the Ct values of the 135 positive samples with the different qPCR methods shows a clear correlation between the four methods and the parasite density (all linear regression p-values are < 0.001 with the F-test). Accuracy of species diagnosis falciparum + P. malariae + P. ovale (n = 1) Pf+/Pan+ 1 Pf+/Pan+ 1 Pf+/Pan+ 1 Table 4 Accuracy of Plasmodium species identification with the different qPCR methods studied compared (Bio-Evolution) Page 7 of 10 Bouzayene et al. Malaria Journal (2022) 21:204 Bouzayene et al. Malaria Journal (2022) 21:204 Table 5 Correlation between Alethia® Malaria (LAMP) and the different qPCRs for the discordant samples with Bio-Evolution Alethia® Malaria (LAMP) Plasmodium Typage (Bio- Evolution) Ampliquick® Malaria In-house HRM qPCR In-house TaqMan qPCR Positive 5 Negative 5 Negative 3 Negative 2 Negative 0 Positive 0 Pf+/Pan+ 2 Pf+/Pan+ 1 Pf+/Pan+ 3 Pf+ 1 Pf+ 1 Pan+ 1 Pan+ 1 in-house TaqMan-qPCR analysis, which correlated with the initial LAMP results (Table 5). in-house TaqMan-qPCR analysis, which correlated with the initial LAMP results (Table 5). Tables 4 and 5 show that for the in-house TaqMan- qPCR, the 18S rRNA target (Pan) was positive in 96% (74/77) of P. falciparum mono-infections. The three remaining mono-infections were only positive for the var genes target (P. falciparum). malariae positive samples by the Bio-Evolution kit, one turned out to be a mixed infection highlighted by the in- house TaqMan since this assay confirmed the presence of P. falciparum as well (Table 4).hi Tables 4 and 5 show that for the in-house TaqMan- qPCR, the 18S rRNA target (Pan) was positive in 96% (74/77) of P. falciparum mono-infections. The three remaining mono-infections were only positive for the var genes target (P. falciparum). The five negative isolates with the Bio-Evolution Plas- modium typage but positive with the LAMP Alethia® Malaria turned out positive (four P. falciparum and one Plasmodium spp. other than P. falciparum) after the Fig. 2 qPCR Ct values compared to parasite density. Linear regression between the parasite density and Pan-Plasmodium Ct of the Biosynex Ampliquick® Malaria (red), the species Ct of the Bio-Evolution Plasmodium typage (green), the undifferentiated Ct of the in-house qPCR-HRM (blue) and the in-house qPCR-TaqMan (purple). 95% confidence interval (gray) and coefficient of determination R2 are indicated Fig. 2 qPCR Ct values compared to parasite density. Linear regression between the parasite density and Pan-Plasmodium Ct of the Biosynex Ampliquick® Malaria (red), the species Ct of the Bio-Evolution Plasmodium typage (green), the undifferentiated Ct of the in-house qPCR-HRM (blue) and the in-house qPCR-TaqMan (purple). 95% confidence interval (gray) and coefficient of determination R2 are indicated Bouzayene et al. Malaria Journal (2022) 21:204 Bouzayene et al. Discussion The other downside is, with it being a commercial kit, the Ampliquick® Malaria test is more expensive and thus cannot be systematically used in developing countries for the diagnosis of malaria. How- ever, the TaqMan and HRM in-house qPCRs adapted from Schindler et al. [22] showed both high sensitivity and cost effectiveness. Bouzayene et al. Malaria Journal (2022) 21:204 Bouzayene et al. Malaria Journal (2022) 21:204 Page 9 of 10 Page 9 of 10 Conclusionh Eric Dannaoui (Hôpital Européen Georges Pompidou Hospital, Paris), Françoise Botterel (Henri Mondor Hospital, Créteil), Guillaume Desoubeaux (Tours), Gha- nia Belkadi (Tenon Hospital, Paris), Isabelle Salimbeni (Cannes), Jean Philippe Lemoine (Angers), Luce Landraud (Louis-Mourrier Hospital, Colombes), Louise Basmaciyan (Dijon), Loic Favennec (Rouen), Marie Fleur Durieux (Limoges), Marie Laure Darde (Limoges), Milene Sasso (Nîmes), Marc Thellier (Hospital Pitié Salpétrière, Paris), Naima Dahane (Hospital Cochin, Paris), Nathalie Fauchet (Créteil), Nathalie Bourgeois (Montpellier), Odile Eloy (Versailles), Odile Fenneteau (Hospital Robert Debré, Paris), Pascale Penn (Le Mans), Pauline Caraux Paz (Villeneuve Saint George), Roseanne Lavergne (Nantes), René Nabias (Poissy), Sorya Belaz (Rennes), Sylvain Mermond (La Rochelle), Samia Hamane (Hospital Lariboisière, Paris), Sébastien Larréché (Begin), Sylvain Clauser (Boulogne-Billancourt), Stéphane Lastere (Polynésie Française), Yaye Senghor (Hospital Saint Joseph, Paris), Yohann Le Govic (Amiens). All authors read and approved the final manuscript. This paper represents a comparative study in which four qPCR methods for malaria diagnosis were evaluated to provide information about their sensitivities and accu- racy. This could be a helpful tool for other laboratories looking to implement molecular diagnosis methods in their routine analysis. Compared to the LAMP assay, the four tested qPCR methods showed varying sensitivities with the in-house TaqMan qPCR being the most sensi- tive. Although it does not enable the detection of the five common malaria human infecting species, the in- house TaqMan has a comparable sensitivity to that of the LAMP assay with the advantage of identifying P. fal- ciparum infections, which is the most common and life- threatening species. Availability of data and materials All data generated or analysed during this study are included in this published article and its additional files. Funding This study was in part funded by ‘Santé Publique France’ through financial sup- port of the French National Malaria Reference Centre. The Biosynex Group pro- vided the Ampliquick® Malaria kits tested throughout this study. They did not however participate in this study nor had any impact on the data generated. Taken together, the results demonstrate the role molecular methods could play in the screening of malaria and infectious diseases in a rapid and effective way by providing critical information for the clinical context and the epidemiological survey. Consent for publication Not applicable. Additional file 2. Results of the microscopic diagnosis and molecular assays for each included sample. A table detailing the results of: micro- scopic diagnosis (species identification and parasite densities), LAMP Alethia® Malaria and the four different qPCR methods tested in this study for the 183 included samples. Competing interests The authors declare that they have no competing interests. References 1. WHO. World malaria report 2021. Geneva: World Health Organization. 2021. https://apps.who.int/iris/handle/10665/350147. Accessed 13 Jan 2022. 1. WHO. World malaria report 2021. Geneva: World Health Organization. 2021. https://apps.who.int/iris/handle/10665/350147. Accessed 13 Jan 2022. 2. Sutherland CJ, Tanomsing N, Nolder D, Oguike M, Jennison C, Pukrit- tayakamee S, et al. Two nonrecombining sympatric forms of the human malaria parasite Plasmodium ovale occur globally. J Infect Dis. 2010;201:1544–50. 2. Sutherland CJ, Tanomsing N, Nolder D, Oguike M, Jennison C, Pukrit- tayakamee S, et al. Two nonrecombining sympatric forms of the human malaria parasite Plasmodium ovale occur globally. J Infect Dis. 2010;201:1544–50. Author details 1 l l 1 National Malaria Reference Centre, AP-HP, Hôpital Bichat - Claude Bernard, 46 Rue Henri Huchard, 75018 Paris, France. 2 University of Paris Cité, IRD, MERIT, 75006 Paris, France. 1 National Malaria Reference Centre, AP-HP, Hôpital Bichat - Claude Bernard, 46 Rue Henri Huchard, 75018 Paris, France. 2 University of Paris Cité, IRD, MERIT, 75006 Paris, France. Ethics approval and consent to participate The online version contains supplementary material available at https://doi. org/10.1186/s12936-022-04219-1. This study is a non-interventional retrospective study following the relevant guidelines and regulations stated by the French Health Public Law (CSP Art L1121-1.1). It does not include the identification of patients or the use of their information and is, therefore, exempted from informed consent requirement or approval by an ethics committee. Additional file 1. The different methods used to evaluate the included samples. A flow-chart showing the number of samples included and the different methods we used to analyze these samples. Acknowledgements The authors thank the Biosynex Group for kindly providing the Ampliquick® Malaria kits tested throughout this study and the hospital correspondents for sending Plasmodium samples and filling in the online patient forms. Adela Angoulvant, Anne Pauline Bellanger, Antoine Huguenin, Anthony Marteau, Agnes Durand, Céline Tournus, Céline Nourrisson, Céline Malassigne, Cécile Garnaud, Caroline Lohmann, Edith Mazars, Emilie Sitterle, Eric Dannaoui, Françoise Botterel, Guillaume Desoubeaux, Ghania Belkadi, Isabelle Salimbeni, Jean Philippe Lemoine, Luce Landraud, Louise Basmaciyan, Loic Favennec, Marie Fleur Durieux, Marie Laure Darde, Milene Sasso, Marc Thellier, Naima Dahane, Nathalie Fauchet, Nathalie Bourgeois, Odile Eloy, Odile Fenneteau, Pascale Penn, Pauline Caraux Paz, Roseanne Lavergne, René Nabias, Sorya Belaz, Sylvain Mermond, Samia Hamane, Sébastien Larréché, Sylvain Clauser, Stéphane Lastere, Yaye Senghor, Yohann Le Govic. 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Lalremruata A, Magris M, Vivas-Martínez S, Koehler M, Esen M, Kempaiah P, et al. Natural infection of Plasmodium brasilianum in humans: man and monkey share quartan malaria parasites in the Venezuelan Amazon. EBioMedicine. 2015;2:1186–92. 4. Brasil P, Zalis MG, de Pina-Costa A, Siqueira AM, Júnior CB, Silva S, et al. Outbreak of human malaria caused by Plasmodium simium in the Atlantic Forest in Rio de Janeiro: a molecular epidemiological investigation. Lancet Glob Health. 2017;5:e1038–46. 5. Ta TH, Hisam S, Lanza M, Jiram AI, Ismail N, Rubio JM. First case of a naturally acquired human infection with Plasmodium cynomolgi. Malar J. 2014;13:68. Page 10 of 10 Bouzayene et al. Malaria Journal (2022) 21:204 Bouzayene et al. Malaria Journal (2022) 21:204 6. Centre national de référence du Paludisme. Rapport annuel d’activité 2020, Année d’exercise 2019. https://cnr-paludisme.fr/activites-dexpe rtise/rapports-dactivites/. Accessed 13 Jan 2022. 7. Mbanefo A, Kumar N. Evaluation of malaria diagnostic methods as a key for successful control and elimination programs. Trop Med Infect Dis. 2020;5:102. 8. Khairnar K, Martin D, Lau R, Ralevski F, Pillai DR. Multiplex real-time quan- titative PCR, microscopy and rapid diagnostic immuno-chromatographic tests for the detection of Plasmodium spp: performance, limit of detec- tion analysis and quality assurance. Malar J. 2009;8:284. tion analysis and quality assurance. Malar J. 2009;8:284. y y 9. WHO. Guidelines for the treatment of malaria. 3rd ed. Geneva: World Health Organization; 2015. 10. Ugah UI, Alo MN, Owolabi JO, Okata-Nwali OD, Ekejindu IM, Ibeh N, et al. Evaluation of the utility value of three diagnostic methods in the detec- tion of malaria parasites in endemic area. Malar J. 2017;16:189. 11. van Dijk DP, Gillet P, Vlieghe E, Cnops L, van Esbroeck M, Jacobs J. Author contributions • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: 25. Nolasco O, Infante B, Contreras-Mancilla J, Incardona S, Ding XC, Gamboa D, et al. Diagnosis of Plasmodium vivax by loop-mediated isothermal amplification in febrile patient samples from Loreto. Perú Am J Trop Med Hyg. 2020;103:1549–52. yg 26. Rask TS, Hansen DA, Theander TG, Gorm Pedersen A, Lavstsen T. Plas- modium falciparum erythrocyte membrane protein 1 diversity in seven genomes—divide and conquer. PLoS Comput Biol. 2010;6: e1000933. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: Author contributions Evalu- ation of the Palutop+4 malaria rapid diagnostic test in a non-endemic setting. Malar J. 2009;8:293. g 12. Moody A. Rapid diagnostic tests for malaria parasites. Clin Microbiol Rev. 2002;15:66–78. 13. Kamaliddin C, Joste V, Hubert V, Kendjo E, Argy N, Houze S. Evaluation of PCR to monitor Plasmodium falciparum treatment efficacy in a nonende- micity setting. J Clin Microbiol. 2019;58:e01080-19. 14. Thomson R, Parr JB, Cheng Q, Chenet S, Perkins M, Cunningham J. Preva- lence of Plasmodium falciparum lacking histidine-rich proteins 2 and 3: a systematic review. Bull World Health Organ. 2020;98:558–68. 15. Joste V, Bailly J, Hubert V, Pauc C, Gendrot M, Guillochon E, et al. Plasmodium ovale wallikeri and P. ovale curtisi infections and diagnostic approaches to imported malaria, France, 2013–2018. Emerg Infect Dis. 2021;27:372–84. 16. Alemayehu S, Feghali KC, Cowden J, Komisar J, Ockenhouse CF, Kamau E. Comparative evaluation of published real-time PCR assays for the detec- tion of malaria following MIQE guidelines. Malar J. 2013;12:277. 17. Notomi T. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000;28:63e–63. 18. Kamaliddin C, Sutherland CJ, Houze S, Cottrell G, Briand V, Castaneda Mogollon D, et al. The role of ultrasensitive molecular methods for detect- ing malaria—the broader perspective. Clin Infect Dis. 2021;73:e1387–90. 19. Chua KH, Lim SC, Ng CC, Lee PC, Lim YAL, Lau TP, et al. Development of high resolution melting analysis for the diagnosis of human malaria. Sci Rep. 2015;5:15671. 20. Joste V, Kamaliddin C, Kendjo E, Hubert V, Argy N, Houzé S. Distinction of Plasmodium ovale wallikeri and Plasmodium ovale curtisi using quantita- tive polymerase chain reaction with high resolution melting revelation. Sci Rep. 2018;8:300. 21. Morris U, Aydin-Schmidt B. Performance and application of commercially available loop-mediated isothermal amplification (LAMP) kits in malaria endemic and non-endemic settings. Diagnostics. 2021;11:336. 22. Schindler T, Robaina T, Sax J, Bieri JR, Mpina M, Gondwe L, et al. Molecular monitoring of the diversity of human pathogenic malaria species in blood donations on Bioko Island, Equatorial Guinea. Malar J. 2019;18:9. 23. Wickham H, Henry L. Tidyr: easily tidy data with “spread ()” and “gather ()” functions. R package version 0.8.1. 2018. https://CRAN.R-project.org/ packageExternal=tidyr. Accessed 25 Jan 2022. 24. Berzosa P, de Lucio A, Romay-Barja M, Herrador Z, González V, García L, et al. Comparison of three diagnostic methods (microscopy, RDT, and PCR) for the detection of malaria parasites in representative samples from Equatorial Guinea. Malar J. 2018;17:333. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations.
https://openalex.org/W4210500075	https://ojs.library.queensu.ca/index.php/pocus/article/download/15342/2022-07-kidney-p24-26-pdf	English	null	POCUS Allows for Rapid Elucidation of Acute Kidney Injury in a Patient with Progressive Multiple Myeloma	POCUS journal	2,022	cc-by	1,801	Case File Case File FEB 2022 vol. 07 Kidney \| POCUS J \| 24 POCUS Allows for Rapid Elucidation of Acute Kidney Injury in a Patient with Progressive Multiple Myeloma Liann Abu Salman, MD; Nathaniel Reisinger, MD Liann Abu Salman, MD; Nathaniel Reisinger, MD Division of Renal, Electrolytes and Hypertension, University of Pennsylvania, Philadelphia, PA, USA Renal, Electrolytes and Hypertension, University of Pennsylvania, Philadelphia, PA, USA Abstract A 63-year-old man with past history of multiple myeloma recently started on a regimen of daratumumab, carfilzomib, and dexamethasone was referred to our emergency department for a rapidly rising serum creatinine as high as 10 mg/ dL. He complained of fatigue, nausea, and poor appetite. Exam revealed hypertension, but no edema or rales. Labs were consistent with AKI without hypercalcemia or evidence of hemolysis or tumor lysis. Urinalysis and urine sediment were bland without proteinuria, hematuria, or pyuria. Initial concern was for hypovolemia or myeloma cast nephropathy. POCUS revealed no overt evidence of volume overload or depletion, instead revealing bilateral hydronephrosis. Bilateral percutaneous nephrostomies were placed with resolution of the AKI. Ultimately, referral imaging revealed interval progression of bulky retroperitoneal extramedullary plasmacytomas compressing the ureters bilaterally related to the underlying multiple myeloma. hydronephrosis. (Video S4-6 and Figure 1-4). Physical exam demonstrated moist mucous membranes and good skin turgor. Jugular veins were not well- visualized. Axillary sweat was present. Pulmonary crackles and lower extremity edema were absent. There was no costovertebral angle tenderness. On ultrasound, hydronephrosis appears as confluent anechoic areas representing urine in a dilated renal pelvis and calyces [1]. In mild hydronephrosis, the renal pelvis and calyces are dilated but the cortex and pelvicalyceal pattern is unaffected. As the hydronephrosis increases in severity, the medullary pyramids flatten and the pelvicalyceal system dilates leading to outpouching of the calyces. In severe hydronephrosis, the renal pelvis may appear ballooned and the cortico-medullary differentiation is lost leading to a thin cortex [2]. On ultrasound, hydronephrosis appears as confluent anechoic areas representing urine in a dilated renal pelvis and calyces [1]. In mild hydronephrosis, the renal pelvis and calyces are dilated but the cortex and pelvicalyceal pattern is unaffected. As the hydronephrosis increases in severity, the medullary pyramids flatten and the pelvicalyceal system dilates leading to outpouching of the calyces. In severe hydronephrosis, the renal pelvis may appear ballooned and the cortico-medullary differentiation is lost leading to a thin cortex [2]. Initial labs confirmed elevated blood urea nitrogen (91mg/dl) and creatinine levels (10.76 mg/dl). Other labs were notable for serum sodium of 138 mmol/dL, potassium elevated at 5.5 mmol/dL, bicarbonate low at 17 mmol/dL, calcium normal at 8.7 mg/dL (with normal albumin), and phosphate elevated at 8.7 mg/dL. Lactate dehydrogenase and uric acid were only mildly elevated at 200 units per liter and 10.8 mg/dL respectively. Complete blood count revealed white blood cells of 4400 per µL, hemoglobin of 10.3 g/dL, and platelets of 6300 per µL. Urinalysis was negative for heme, albumin, and leukocyte esterase with bland sediment. Quantitative serum kappa free light chains were 203.8 mg/L and quantitative serum lambda free light chains were 6.5 mg/ L with kappa to lambda ratio of 31.4 roughly unchanged from the week prior. Repeat immunoglobulin levels and serum protein electrophoresis revealed immune paresis with a stable M spike in the gamma region consistent with known IgG kappa band and stable from prior. On review of the patient’s prior imaging, he was noted to have retroperitoneal extramedullary plasmacytomas on a computed tomography scan done six weeks prior. He had interval increase of the plasmacytomas despite initiation of chemotherapy leading to obstruction. Case and joint pain. He denied any urinary complaints and noted no change in urine output. He noted some loose stools. He had weekly outpatient labs and was noted to have a rising serum creatinine (Cr) from 1.6 mg/dL to 3.8 mg/dl one week prior to presentation. Repeat labs the day of presentation demonstrated a Cr of 9.3mg/dL and the patient was instructed to present to the emergency department for further evaluation. A 63-year-old man with past medical history of advanced IgG Kappa multiple myeloma presented with worsening fatigue, nausea, and loss of appetite. He had progression of disease despite completion of multiple prior therapies for myeloma including autologous stem cell transplant and three weeks prior to presentation was started on combination therapy with daratumumab, carfilzomib, and dexamethasone. Subsequently, he had waxing and waning levels of energy and decreased oral intake. He denied lightheadedness, abdominal pain, vomiting, rash, On arrival, the patient’s vitals were: blood pressure 176/109 mmHg, heart rate 64 beats per minute, oxygen saturation 99% on room air, and temperature of 36.9C. Figure 1. Confirmatory referral kidney ultrasound by radiology in sepia tone demonstrating hypoechoic 5.2 cm retroperitoneal lesions likely representing extramedullary plasmacytomas compressing the proximal ureter. Left: right kidney long axis. Right: right kidney short axis. Figure 2. Doppler imaging of right kidney, long axis demonstrating absence of flow in the dilated renal pelvis, proximal ureter, and masses in the retroperitoneum at the inferior pole. Figure 2. Doppler imaging of right kidney, long axis demonstrating absence of flow in the dilated renal pelvis, proximal ureter, and masses in the retroperitoneum at the inferior pole. Figure 1. Confirmatory referral kidney ultrasound by radiology in sepia tone demonstrating hypoechoic 5.2 cm retroperitoneal lesions likely representing extramedullary plasmacytomas compressing the proximal ureter. Left: right kidney long axis. Right: right kidney short axis. Figure 1. Confirmatory referral kidney ultrasound by radiology in sepia tone demonstrating hypoechoic 5.2 cm retroperitoneal lesions likely representing extramedullary plasmacytomas compressing the proximal ureter. Left: right kidney long axis. Right: right kidney short axis. 25 \| POCUS J \| FEB 2022 vol. 07 Kidney Figure 4. Doppler imaging of left kidney in long axis demonstrating lack of fast flow in the area of suspected plasmacytomas. Figure 3. Referral kidney ultrasound of left kidney in long axis demonstrating suspected plasmacytomas 3.7 cm in maximum diameter at the inferior pole. Left: left kidney long axis. Right: left kidney short axis. Case Figure 4. Doppler imaging of left kidney in long axis demonstrating lack of fast flow in the area of suspected plasmacytomas. Figure 3. Referral kidney ultrasound of left kidney in long axis demonstrating suspected plasmacytomas 3.7 cm in maximum diameter at the inferior pole. Left: left kidney long axis. Right: left kidney short axis. hydronephrosis. (Video S4-6 and Figure 1-4). References chemotherapeutic and immunotherapy agents used [3]. Acute kidney injury may also arise from side effects of the chemotherapeutic agents which often include nausea, vomiting, and decreased oral intake leading to pre-renal insults or even acute tubular necrosis. Given the bland nature of the urine sediment and lack of physical exam or ultrasonographic evidence of extremes of volume overload our initial differential diagnosis included myeloma cast nephropathy, obstructive nephropathy, and thrombotic microangiopathy due to carfilzomib therapy. 1. O'Neill, W.C., Renal relevant radiology: use of ultrasound in kidney disease and nephrology procedures. Clin J Am Soc Nephrol, 2014. 9 (2): p. 373-81. 2. Koratala, A., D. Bhattacharya, and A. Kazory, Point of care renal ultrasonography for the busy nephrologist: A pictorial review. World J Nephrol, 2019. 8(3): p. 44-58. 3. Hutchison, C.A., et al., The pathogenesis and diagnosis of acute kidney injury in multiple myeloma. Nat Rev Nephrol, 2011. 8(1): p. 43- 51. 4. Damaj, G., et al., Features of extramedullary and extraosseous multiple myeloma: a report of 19 patients from a single center. Eur J Haematol, 2004. 73(6): p. 402-6. 5. Igel, T.C., et al., Renal plasmacytoma: Mayo Clinic experience and review of the literature. Urology, 1991. 37(4): p. 385-9. Extramedullary plasmacytomas rarely occur during the course of multiple myeloma with an incidence rate of around 4% [4]. When present, they are most frequently seen along the respiratory tract, lymph nodes and skin and soft tissue. Involvement of the urinary tract is quite uncommon with seven cases involving the renal parenchyma and only two cases of urinary tract obstruction reported in the literature [5, 6]. When evaluating acute kidney injury, a wide differential should be sought, and obstructive nephropathy should be considered. As POCUS is inexpensive and readily available, it should be used in the evaluation of AKI as prompt intervention can lead to marked improvement and even resolution of kidney injury. 6. Bigé, N., et al., Urinary tract obstruction due to extramedullary plasmacytoma: report of two cases. NDT Plus, 2009. 2(2): p. 143-6. 6. Bigé, N., et al., Urinary tract obstruction due to extramedullary plasmacytoma: report of two cases. NDT Plus, 2009. 2(2): p. 143-6. Patient Consent The authors certify that informed consent was obtained for the clinical information and images reported. hydronephrosis. (Video S4-6 and Figure 1-4). Despite the temporal association between our patient starting chemotherapy and the development of acute kidney injury, it was not in fact a causal relationship. The use of POCUS revealed bilateral hydronephrosis and guided our management. We recommended bilateral percutaneous nephrostomy (PCN) insertion as both a diagnostic and therapeutic maneuver. Within 12 hours of PCN placement, his Cr started to decline and within 72 hours his renal function returned to his prior baseline. Ultimately repeat computed tomography demonstrated interval increase in size of retroperitoneal extramedullary plasmacytomas (Figure 5, 6). Point of care ultrasound including focused cardiac, lung, and bilateral kidney ultrasound was undertaken. Lung included diffuse A-line pattern in all visualized fields and no pleural effusions (Video S1). Focused cardiac assessment revealed a normal-sized inferior vena cava that collapsed less than 50% with inspiration (Video S2). The right internal jugular vein was undistended 2 centimeters above the sternal angle with the patient in the supine position. (Video S3). Kidney ultrasound revealed enlarged kidneys bilaterally with bilateral When evaluating acute kidney injury (AKI) in multiple myeloma patients the differential diagnosis is wide. Most commonly, myeloma cast nephropathy is suspected. Other causes include deposition diseases, proximal tubulopathy, or direct nephrotoxicity from the various FEB 2022 vol. 07 Kidney \| POCUS J \| 26 Figure 5. Computed tomography, coronal view in the abdomen demonstrating interval placement of bilateral percutaneous nephrostomies. On the left the plasmacytomas measure 5.3 cm in maximum diameter. Figure 6. Computed tomography, coronal view in the abdomen demonstrating bilateral kidneys with nephrostomies in place and bulky plasmacytomas at inferomedial border of kidneys bilaterally. Figure 6. Computed tomography, coronal view in the abdomen demonstrating bilateral kidneys with nephrostomies in place and bulky plasmacytomas at inferomedial border of kidneys bilaterally. Figure 5. Computed tomography, coronal view in the abdomen demonstrating interval placement of bilateral percutaneous nephrostomies. On the left the plasmacytomas measure 5.3 cm in maximum diameter. 1. O'Neill, W.C., Renal relevant radiology: use of ultrasound in kidney disease and nephrology procedures. Clin J Am Soc Nephrol, 2014. 9 (2): p. 373-81. Disclosures View the online article: https://doi.org/10.24908/pocus.v7iKidney.15342 6. Bigé, N., et al., Urinary tract obstruction due to extramedullary plasmacytoma: report of two cases. NDT Plus, 2009. 2(2): p. 143-6.
https://openalex.org/W3005030780	http://journal.uinjkt.ac.id/index.php/refleksi/article/download/11272/7041	Indonesian	null	Kritik Matan Hadis dengan Pendekatan Al-Qur’an: Studi Pemahaman Muḥammad Al-Ghazālī dan Jamāl Al-Bannā	Refleksi	2,019	cc-by	10,018	223 223 Agung Abdillah dan Rizal Alwi Mampa, Kritik Matan Hadis dengan Pendekatan Al-Qur’an \| 223 Kata Kunci: Kritik, Matan, Pendekatan, al-Qur’an, Hadis. Kritik Matan Hadis dengan Pendekatan Al-Qur’an: Studi Pemahaman Muḥammad Al-Ghazālī dan Jamāl Al-Bannā Agung Abdillah dan Rizal Alwi Mampa Universitas Muhammadiyah Jakarta agungabdillah77@gmail.com; alwimampa90@gmail.com Abstract: Matan ḥadīth criticism is not new in the scientific world of ḥadīth, criticism of matan ḥadīth has been done since the time of the Prophet and the companions and then continued until the present. The existence of criticism of traditions in the history of traditions must have been born along with the number of persons who misuse the function of ḥadīth to the rise of counterfeiting of traditions to achieve irresponsible interests. The figures to be discussed are figures that are considered controversial for some people, because these two figures reject the authentic hadith that contradicts its meaning with the Qur’an. Therefore the author here feels the need for a review of the understanding of these two figures related to their understanding of the traditions, hadith aḥad, and the methodology of understanding the traditions with the Qur’anic approach. Keywords: Criticism, Matan, Approach, Qur’an, Ḥadīth. Keywords: Criticism, Matan, Approach, Qur’an, Ḥadīth. Abstrak: Kritik matan hadis bukanlah hal yang baru dalam dunia keilmuan hadis, kritik matan hadis sudah dilakukan sejak masa Rasululah masih hidup dan pada masa para sahabat kemudian berlangsung hingga masa sekarang. Adanya kritik matan hadis dalam kesejarahan hadis tentunya lahir seiring dengan banyaknya oknum-oknum yang menyalahgunakan fungsi hadis hingga maraknya pemalsuan- pemalsuan terhadap hadis guna mencapai kepentingan-kepentingan yang tidak bertanggung jawab.Tokoh yang akan dibahas merupakan tokoh yang dianggap kontroversial bagi sebagian kalangan, dikarenakan pemahaman kedua tokoh ini yang menolak hadis sahih yang bertentangan maknanya dengan al-Qur’an, oleh sebab itu penulis di sini merasa perlu adanya pengkajian kembali pemahaman kedua tokoh ini terkait pemahaman mereka tentang hadis, hadis aḥad, dan metodologi pemahaman matan hadis dengan pendekatan al-Qur’an. Kata Kunci: Kritik, Matan, Pendekatan, al-Qur’an, Hadis. Kata Kunci: Kritik, Matan, Pendekatan, al-Qur’an, Hadis. DOI: 10.15408/ref.v18i2.11272 DOI: 10.15408/ref.v18i2.11272 224 \| REFLEKSI,Volume 18, Nomor 2, Oktober 2019 Pendahuluan Hadis1 merupakan satu dari sumber utama hukum Islam setelah al-Qur’an, yang juga merupakan penjelasan dari al-Qur’an tersebut.2 Tidak hanya sekedar penjelas bagi al-Qur’an, hadis pun merupakan bagian dari tuntunan Nabi Muhammad saw. dalam kehidupannya. Jika al-Qur’an mempunyai jaminan akan kebenaran dan kemurniannya, maka lain hal nya dengan hadis yang sama sekali tidak mempunyai jaminan akan kebenaran dan kemurniannya dari Allah swt,3 seperti halnya terdapat hadis yang benar-benar dijamin kesahihannya dan terdapat pula hadis yang tidak dapat dijamin akan kebenarannya atau yang biasa disebut dengan hadis ḍa’if. Hadis ṣaḥīḥ adalah hadis yang dianggap benar dan layak dijadikan pe- doman oleh perhitungan para ulama, dengan syarat bahwa hadis tersebut di- riwayatkan oleh orang yang thiqah dan rangkaian riwayatnya bersambung ke- pada Nabi Muhammad saw.,4 sementara hadis ḍa’if merupakan kebalikan dari hadis sahih, hadis ḍa’if adalah hadis yang dianggap tidak layak untuk dijadikan pedoman, tergantung pada bagaimana para ulama dalam menilai sebuah hadis dengan metodologinya masing-masing. Perihal mengenai hadis ṣaḥīḥ dan ḍa’if, terdapat dua faktor yang sangat mempengaruhinya, yaitu sanad atau jalur periwayatan dan Matan atau isi periwayatan atau yang biasa disebut dengan sanad hadis dan matan hadis. Jika dalam suatu hadis terdapat sanad dan matannya baik maka hadis itu bisa jadi dapat diterima, namun jika salah satunya terdapat kecacatan maka hadis itu ada kemungkinan untuk ditolak, kecacatan hadis bisa terjadi pada sanad hadis dan bisa juga terjadi pada matan hadis, atau bisa terjadi pada keduanya, sanad mau- pun matan hadis. Ditinjau dari unsur sanad, ada dua hal yang harus dimiliki oleh Periwa-yat, yaitu adil dan ḍabiṭ, terdapat pula beragam ilmu yang memfokuskan kajian sanad. Di antaranya, ilmu rijāl al-ḥādīth, tarīkh rūwat al-ḥādīth, dan al-jarḥ wa ta’dil. Sedangkan dari unsur matan, sanagat sedikit ilmu yang secara khusus membahas mengenai kesahihan matan hadis, namun bukan berarti para ulama melupakannya begitu saja. Para ulama memberikan beberapa metodologi dalam memahami matan hadis, di antaranya adalah dengan membandingkan isi matan hadis dengan al-Qur’an. Baik ulama klasik maupun kontemporer sepakat den- gan metode perbandingan ini, dan di antara ulama kontemporer yang meng- gunakan metode tersebut adalah Muḥammad al-Ghazālī dan Jamāl al-Bannā. Muḥammad al-Ghazālī dan Jamāl al-Bannā adalah dua ulama kontem- porer Mesir yang juga ahli dalam bidang Fiqh, al-Qur’an dan juga Hadis, hal itu bisa dilihat dari berbagai karya mereka yang membahas secara khusus ten-tang DOI: 10.15408/ref.v18i2.11272 Agung Abdillah dan Rizal Alwi Mampa, Kritik Matan Hadis dengan Pendekatan Al-Qur’an \| 225 225 Fiqh, al-Qur’an, maupun hadis. Pendahuluan Jamāl al-Bannā dalam bukunya Manifesto Fiqh Baru jilid 2 (terjemahan dari Nahwa Fiqh Jadīd: 2) banyak memuat peno-lakan terhadap hadis-hadis yang matannya diduga bertentangan dengan nilai-nilai universal al-Qur’an sekalipun hadis tersebut telah dinilai shahih oleh seba-gian ulama, menolak hadis Ahad dan memahami makna Hadis dan Sunnah dengan pengertian yang sempit, yaitu hanya kepada apa yang disandarkan kepa-da Nabi Muhammad saw, sedangkan apa yang disandarkan kepada Sahabat maupun Tabi’in, beliau menolaknya sebagai hadis/suannah maupun hujjah5 dalam hal ini Jamāl al-Bannā memahami makna hadis yang diselaraskan dengan wawasan al-Qur’an sehingga menimbulkan beberapa pemahaman yang meno-lak matan hadis meskipun sahih secara Sanad. Mengenai pengertian Hadis, al-Bannā mendefinisikan hadis sebagai berikut: “Pergeseran definisi hadis, pengertian hadis yang selama ini dipahamioleh mayoritas umat muslim tidak hanya sebatas apa yang dinisbahkan kepada Nabi, melainkan juga apa yang dinisbahkan kepada sahabat maupun Tabiin, selain itu jumhur ulama juga menerima definisi sunnah yang disinonimkan dengan hadis, Khabar, dan atsar.6Menurut jamal, menilai ucapan atau fatwa sahabat dan tabiin tidaklah layak untuk dijadikan Hujjah. Ini karena Allah hanya mengutus seorang Nabi untuk dijadikan rujukan atau hujjah. Umat islam tidak diperintahkan untuk menjadikan selain Rasulullah sebagai teladan dan rujukan.”7 “Pergeseran definisi hadis, pengertian hadis yang selama ini dipahamioleh mayoritas umat muslim tidak hanya sebatas apa yang dinisbahkan kepada Nabi, melainkan juga apa yang dinisbahkan kepada sahabat maupun Tabiin, selain itu jumhur ulama juga menerima definisi sunnah yang disinonimkan dengan hadis, Khabar, dan atsar.6Menurut jamal, menilai ucapan atau fatwa sahabat dan tabiin tidaklah layak untuk dijadikan Hujjah. Ini karena Allah hanya mengutus seorang Nabi untuk dijadikan rujukan atau hujjah. Umat islam tidak diperintahkan untuk menjadikan selain Rasulullah sebagai teladan dan rujukan.”7 Dari pengertian di atas dapat disimpulkan bahwa al-Bannā memahami hadis dalam ruang lingkup yang lebih kecil.8Perihal mengenai hadis aḥad, al- Bannā menilai bahwa hadis aḥad masih mengandung beberapa kemungkinan, dan sesuatu yang mengandung kemungkinan itu tidak dapat disebut yaqīn.9 Al- Bannā berpendapat bahwa keboehan mengamalkan hadis ahad tidaklah meng- ubah faedah hadis ahad yang ẓannī tersebut. DOI: 10.15408/ref.v18i2.11272 Pendahuluan Bagi al-Bannā hadis aḥad yang sesuai dan dapat diterima oleh akal adalah lebih baik daripada hadis mutawattir yang justru bertentangan dengan akal.10 Salah satu faktor mengapa al-Bannā mempunyai fokus perhatin terhadap hadis ahad adalah, Banyaknya hadis aḥad yang mewarnai al-Kutūb al-Ṣiḥḥah, salah satu faktor yang mengakibatkan munculnya hadis ḍa’if dan mawḍū’ adalah karena kebolehan meriwayatkan hadis melalui riwayah bi al-ma’nā.11 Mengenai Muḥammad al-Ghazālī yang juga merupakan salah satu ulama modern yang memusatkan perhatiannya kepada hadis, yang juga merupakan sosok pendakwah agama yang kritis dan mempunyai komitmen yang tinggi terhadap perjuangan agama.12Al-Ghazālī lebih berkeinginan agar setiap hadis DOI: 10.15408/ref.v18i2.11272 226 \| REFLEKSI,Volume 18, Nomor 2, Oktober 2019 dipahami di dalam kerangka makna-makna yang ditunjukkan oleh al-Qur’an, baik secara langsung ataupun tidak.13 Pendapat al-Ghazālī dalam memahami hadis, menurutnya tidak setiap yang dinisbahkan kepada Rasulullah saw. itu berarti Sunnah yang dapat di- terima. Tidak setiap yang valid penisbatannya itu pasti benar pemahamannya.14 Agar terciptanya hadis yang sesuai dengan apa yang akan dijadikkan hujjah oleh para ulama, maka dari itu al-Ghazālī merumuskan kriteria kesahi-han hadis, yaitu (1) setiap periwayatan dalam sanad hadis harus dilakukan oleh orang yang kuat hafalannya, (2) di samping kuat hafalannya juga penting untuk memiliki kepribadian yang baik, kemudian (3) mataan hadis tidak boleh me-ngandung shādh, maupun cacat.15 Al-Ghazālī dalam bukunya al-Sunnah an-Nabawiyah: Baina Ahl Fiqh wa Ahl al-Ḥadīth) pun banyak menuai kecaman, di antaranya dari ulama Saudi bernama Rabī’ bin Hādi al-Makhdalī. Menurut al-Makhdalī bahwa buku kara- ngan Muḥammad al-Ghazālī ini banyak memuat kontroversi seputar penola- kannya terhadp beberapa hadis yang dianggap sahih oleh kebanyakan para ulama yang kemudian dilemahkan/ditolak oleh al-Ghazālī dikarenakan banyak hadis- hadis yang diduga bertentangan dengan al-Qur’an dalam segi makna, seperti halnya hadis tentang siksaan bagi mayit dikarenakan ratapan atau tangi-san dari keluarganya. Seperti Jamāl al-Bannā, Muḥammad al-Ghazālī juga memahami hadis aḥad bahwa hadis aḥad harus dimundurkan apabila bertentangan dengan naṣ al-Qur’ān atau kebenaran ilmiah ataupun fakta historis.16 Muḥammad al-Ghazālī meragukan kualitas hadis ahad dengan alasan, pertama, periwayat yang sendirian terkadang tidak luput dari kesalahan lupa atau kesalahan dalam meriwayatkan hadis, kedua, perihal tentang jumlah kesak-sian terhadap hadis aḥad.17 Dengan alasan inilah Muḥammad al-Ghazālī lebih memilih hadis mutawattir dalam menjadikan rujuklan atau pijakan ketimbang harus meyakini hadis ahad yang masih dalam keraguan dalam benaknya. Pendahuluan Kedua tokoh yang akan dibahas dalam penelitian ini merupakan tokoh yang dianggap kontroversial bagi sebagian kalangan, dikarenakan pemahaman kedua tokoh ini yang menolak hadis sahih yang bertentangan maknanya dengan al-Qur’an, oleh sebab itu penulis di sini merasa perlu adanya pengkajian kem-bali pemahaman kedua tokoh ini terkait pemahaman mereka tentang hadis, hadis aḥad, dan metodologi pemahaman matan hadis dengan pendekatan al-Qur’an. DOI: 10.15408/ref.v18i2.11272 DOI: 10.15408/ref.v18i2.11272 Agung Abdillah dan Rizal Alwi Mampa, Kritik Matan Hadis dengan Pendekatan Al-Qur’an \| 227 DOI: 10.15408/ref.v18i2.11272 Biografi dan Karir Intelektual 1. Muḥammad al-Ghazālī Muḥammad al-Ghazālī dilahirkan pada 22 september 1917 dari keluarga miskin yang taat beragama di Dessa Nakhla al-Inab, Provinsi Buhairah,18 Mesir. Muḥammad al-Ghazālī adalah anak pertama dari tujuh bersaudara19 al-Ghazālī tumbuh dan dibesarkan dalam lingkungan keluarga yang religious. Ayahnya seorang pedagang yang saleh, seorang penghafal al-Qur’an yang juga menyukai tradisi tasawwuf yang selalu mengarahkan anak-anaknnya pada pendidikan agama. Pada usia 5 Tahun, ia sudah dimasukkan ke al-Kuttāb (tempat untuk menghafal al-Qur’an). Di sana ia mulai menghafal al-Qur’an dan berhasil menghafal al-Qur’an 30 juz pada usia 10 tahun, dan sedikit mengetahui kaidah- kaidah imla dan sebagian ilmu hisab. Al-Kuttāb menjadi tempat pertama kali penempaan dirinya. Setelah menuntaskan pendidikan ditempat tersebut, ia me- lanjutkan studinya di Ma’had al-Azharī di kota Buhairah, dan kemudian pindah ke Iskandariah. Di kota inilah ia menghabiskan masa mudanya untuk melanjut- kan dan menyelesaikan pendidikan dari sekolah dasarnya hingga sekolah me- nengah umum di Ma’had al-Dini al-Azhari. Pemahaman Filosof dan teolog al-Ghazālī banyak belajar dari buku-buku karangan Abū Ḥāmid al-Ghazālī yaitu Taḥāfut al-Falāsifah dan kitab karya Ibnu Rūshd Taḥāfut al-Taḥāfut. Dari kedua tokoh yang saling bertentangan itu ia banyak belajar darinya oleh karena itu, tidak heran jika ayahnya yang juga penggila ilmu tasawwuf lantas memberikan nama al-Ghazālī kepada anaknya. Pada tahun 1937 Muḥammad al-Ghazālī melanjutkan pendidikan di al- Azhar Kairo Fakultas Ushuluddin dan kemudian lulus pada tahun 1941. Di sini pemikirannya mulai dipengaruhi oleh guru pribadinya yaitu Syeikh Maḥmūd Shaltūt dan Syaikh ‘Abd al-Adhīm al-Zarqānī. Tahun ini pula Muḥammad al- Ghazālī berjumpa dengan Ḥasan al-Bannā (kakak kandung dari Jamāl al-Bannā), pendiri gerakan Islam Ikhwān al-Muslimīn dan bergabung bersama mereka. Dari sinilah ia mulai menghadapi berbagai perubahan dan peristiwa penting dalam perjalanan pemikiran dan aktivitas ijtihadnya. Biografi dan Karir Intelektual 1. Muḥammad al-Ghazālī Setelah lulus S-1 pada fakultas Ushuluddin Universitas al-Azhar, ia kemu- dian melanjutkan studinya pada jenjang S-2 dan lulus pada tahun 1943 dengan nilai yang baik dengan gelar Master pada Fakultas Adab di Universitas yang sama.20 Muḥammad al-Ghazālī juga pernah diangkat sebagai wakil kementerian Wakaf Mesir, juga pernah menjadi Imam dan khatib di masjid al-Taubah al- Khaṭrah di kota kairo dan selanjutnya diangkat menjadi pemimpin umum seluruh masjid serta ketua dewan dakwah pada tanggal 2 Juli tahun 1971.21 DOI: 10.15408/ref.v18i2.11272 228 \| REFLEKSI,Volume 18, Nomor 2, Oktober 2019 228 Muḥammad al-Ghazālī juga pernah menjabat sebagai dosen di Uni-versitas ‘Umm al-Qurrā makkah dan Universitas King ‘Abd al-Azīz Jeddah, Arab Saudi, kemudian juga pernah menjadi dosen pada Fakultas Syari’ah, Dirasat Islamiah, dan Tarbiah, di Universitas al-Azhar, serta pernah pula men-jadi tenaga pengajar di fakultas Syariah Universitas Qatar.22 Al-Ghazālī juga pernah dianugerahkan bintang kehormatan tertinggi di Mesir pada tahun 1988. Juga pernah mendapatkan penganugerahan medali al- Itsr di Aljazair, bintang kehormatan tertinggi dalam bidang dakwah dari peme- rintahan Aljazair, juga pernah mendapatkan penghargaan Internasional dari Raja Faishal dari kerajaan Saudi Arabia dalam bidang pengabdian kepada Islam.23Al- Ghazālī juga pernah diangkat menjadi anggota dewan penasihat pada International Institute of Islamic Though, yang bermarkas di Washington.24 Muḥammad al-Ghazālī wafat pada usia 78 tahun pada hari Sabtu tanggal 9 Syawal 1416 H, bertepatan dengan tanggal 6 Maret 1996, di Riyadh Arab Saudi, beliau mendadak terkena penyakit serangn jantung ketikas saat mengha- diri sebuah seminar. Jenazahnya dimakamkan di pekunuran Baqi, hanya bebe- rapa meter dari makam Rasulullah saw. di Madinah.25 Al-Ghazālī merupakan ulama yang sangat produktif dalam menulis, beberapa karya yang penah ia tulis adalah, Fiqh al-Sirah, yaitu buku yang meng- ulas tentang sejarah kerasulan Nabi Muhammad saw., kondisi masyarakat Arab pada masa Nabi dan berbagai peristiwa penting pada zamam itu. al-Islām wa al- Awdha’ī al-Iqtiṣādiyyah, yaitu buku yang mengulas tentang kritikan terhadap kebijakan-kebijakan para penguasa Mesir pada saat itu, al-Sunnah al-Nabāwiy- yah bayna Ahl-Fiqh wa Ahl al-Ḥādīth, yaitu karya yang membahas mengenai persoalan-persoalan Hadis Nabi, beberapa karya lainnya adalah Dustūr Wahdah al-Tsaqafiyyah bain al-Muslimin, Min Huna Na’im, al-Islām wa al-Manāhij al- Isytirakiyyah, al-Islām al-Muftara ‘Alai bain al-Syuyuiyyun, Fann al-Dhikir wa al- Dhu’a ind Khatam al-Anbiyā, dan masih banyak lagi karyanya. 2. Jamāl al-Bannā Nama lengkapnya adalah Aḥmad Jamāl al-Dīn Aḥmad ‘Abd Raḥmān, lahir pada tanggal 15 Desember 1920 di desa Mahmudiyyah26yang berjarak se-kitar 50 kilometer dari kota wisata Alexandria, Propinsi Bukhairah, Mesir. Ia akrab dengan dunia tulis-menulis dan jurnalistik di usia muda. Jamāl merupa-kan adik kandung dari pendiri Ikhwanul Muslimin, yaitu Imam al-Syahid Ḥassan al- Bannā (1906-1949). Ayahnya adalah ‘Abd al-Raḥmān bin Muḥam-mad al-Bannā al-Sa’atī, pengarang kitab al-Fatḥ al-Rabbānī fī Tartīb al-Musnad al-Imām Aḥmad bin Ḥanbāl al-Syaibānī sebanyak 24 jilid. Ayahnya merupakan sosok pengagum DOI: 10.15408/ref.v18i2.11272 Agung Abdillah dan Rizal Alwi Mampa, Kritik Matan Hadis dengan Pendekatan Al-Qur’an \| 22 229 Jamāl al-Dīn al-Afghānī, maka tidak heran jika menamakan putra bungsunya “Jamāl”.27 Ibunya bernama Ummu Sa’ad Ṣadr. Pendidikan Jamāl al-Bannā dimulai dari tingkat sekolah dasar yang dija- laninya di salah satu sekolah di daerah Ismailiyah, di mana kakak tertuanya, yaitu Ḥasan al-Bannā, mengajar di sana. Setelah menamatkan jenjang tersebut ia pun melanjutkan pendidikan Tsanawiyahnya di Khadyawiyah, salah satu sekolah favorit di Kairo saat itu.28 Jamal sendiri merupakan seorang aktifis di berbagai organisasi, salah satu organisasi yang didirikannya yaitu Partai Buruh Nasional-Sosialis (Ḥizb al- ‘Ummāl al-Watānī al-Ijtimā’ī), organisasi ini dikenal sebagai pembela hak-hak kaum buruh, yang selama ini kurang mendapatkan perhatian dari pemerintah. Pada tahun 1953 M. ia mendirikan Jam’iyah Miṣriyah untuk melindungi hak- hak tahanan dan keluarganya. Pada tahun 1956 M. ia mulai memberikan ceramah-ceramah perihal hak buruh di Ma’had Niqabiah di daerah Dokki-Kairo yang berlangsung hingga 1993 M. atau sekurang-kurangnya 30 tahun lamanya. Pada tahun 1953, Jamal mendirikan The Egyptian Society for the Care of Prisoners and Their Families (Masyarakat Mesir untuk Perawatan Tahanan dan Keluarganya). Jamal juga merintis dan menjadi presiden pertama atas Konfede- rasi Buruh Islam Internasional pada tahun 1981 di Jenewa, di mana organisasi tersebut menghimpun sejumlah perserikatan dagang dari berbagai penjuru dunia Islam. Jamal juga pernah mengajar di Cairo Institute of Trades Union Studies selama 30 tahun (1963-1993). Selanjutnya pada tahun 1991, bersama saudara perempuannya (Fawziyah al-Bannā), ia mendirikan Fawziyah and Gamal al- Bannā Foundation for Islamic Culture and Information, sebuah yayasan yang memiliki koleksi lengkap tentang informasi kebudayaan Islam. Yang ter-akhir, pada tahun 2000. Di Mesir ia juga mendirikan Da’wah al-Iḥyā’ al-Islāmī sebagai seruan untuk menghidupkan kembali nilai-nilai Islam. Karir politik Jamal pun berkelanjutan selepas itu, Pada tahun 1953 Jamal mendirikan Asosiasi Mesir untuk Bantuan Narapidana. 2. Jamāl al-Bannā Tahun 1981 mendiri- kan Persatuan Buruh Islam Internasional dengan persatuan-persatuan buruh di Jordania, Maroko, Pakistan, Sudan, Bangladesh, yang kantornya di Geneva, ke- mudian pindah ke Rabat, Maroko. Selama tahun-tahun dari 50-an hingga 80-an Jamāl al-Bannā aktif di LSM perserikatan buruh. Menulis berbagai buku panduan, hingga menerjemahkan buku-buku asing (Inggris) mengenai perseri- katan buruh di dunia la, Jamal terpilih menjadi dewan pengurus pada perseri- katan buruh dan Pada tahun 2004, bersama koleganya, Sa’d al-Dīn Ibrāhīm, Jamāl mulai aktif berkecimpung di Ibn Khaldun Center, sebuah organisasi yang bertujuan melakukan reformasi keagaamaan (Islam). DOI: 10.15408/ref.v18i2.11272 230 \| REFLEKSI,Volume 18, Nomor 2, Oktober 2019 230 Sebagai seorang pemikir, ia sangat produktif menulis buku, dalam kon-teks fiqh beliau menulis Nahwa Fiqh Jadīd (Manifesto Fiqh Baru) dalam tiga jilid. Dalam kajian al-Qur’an beliau menggagas Taṣwīr al-Qur’ān (Revolusi al- Qur’an). Dalam bidang Tafsir beliau menggagas Tafsīr al-Qur’ān al-Karīm Bain al-Qudama wa al-Muḥaddīthīn (Tafsir al-Qur’an: Antara Ahli Tafsir Lama dengan Pembaharu). Dalam bidang Hadis beliau menggagas al-Aṣlāni al- Adzimānī: Ru’yah Jadīdah (Dua Pondasi Agung: al-Qur’an dan al-Sunnah, se- buah pandangan baru). Dalam bidang kebebasan beliau menggagas Maṭlabuna al-Awwal Huwa al-Ḥurriyyah (Kebebasan adalah Pertama dan Utama). Dalam wacana Pluralitas beliau menggagas al-Ta’addudiyah fī al-Mujtama’ al-Islāmī’ (Pluralitas dalam Masyarakat Islam). Untuk merespon perdebatan Islam dan Terorisme beliau menggagas al-Jihād. dalam konteks islam dan kekuasaan beliau menggagas al-Islām Dīn wa Ummah, wa Laisa Dīn wa Dawlah (Islam adalah Agama dan Umat, bukan Agama dan Negara). Mawqifuna Min al-Maniyah, al- Qawmiyyah, al-Istirakiyyah, al-Uṣūl al-Fikriyah lid-Dawlah Islāmī-yah, Mas’ūliyyah Fashalid-Dawlah al-Islāmīyah (Tanggung jawab Kegagalan Negara Islam), al-Daulah al-‘Aṣriyyah, Kamsatu Ma’ayir li Miṣdaqiyyāt al-Ḥukmi al- Islāmī. Masih banyak lagi karya yang belum disebutkan di sini. Di samping menulis, Jamāl al-Bannā juga aktif menerjemahkan buku-buku asing ke dalam bahasa arab, yaitu al-Niqabat fī al-Wilyat al-Muttaḥidah (1962), al-Niqabat fī al-Mamlakah al-Muttaḥidah (1962), Al-Niqabat fī al-Ittiḥād al- Sufyitī (1962), al-Niqabat fī al-Suwaydī (1962), al-Niqābat fī al-Burmā (1962), Muqratiyyah al-Niqabiyyah (1969), Taws’iyat al-‘Amāl al-Dawliyyah (1971), dan al-Barnamij al-‘Alāmi li al-‘Umalah (1971).29 Teori dan Aplikasi Kritik Matan Muḥammad al-Ghazālī dan Jamāl al-Bannā Kedua tokoh ini merupakan beberapa cendekiawan muslim Mesir yang banyak mempengaruhi pemikiran-pemikiran pada masa mereka hingga pada masa kekinian, kedua ualama yang juga ahli di bidang al-Qur’an dan hadis ini juga dikenal sebagai ulama yang getol dalam bidang dakwah islam juga menjadi aktivis pada jamannya, ide-ide mereka dalam berdakwah banyak dituangkan dalam karya-karya mereka, hingga pada akhirnya karya mereka mempunyai pe- ngaruh dalam dunia keisalaman, khususnya dalam bidang al-Qur’an dan hadis. a. Model Pemahaman Hadis Sebelum masuk ke dalam model pemahaman hadis Muḥammad al- Ghazālī, alangkah baiknya mengetahui bagaimana posisi Sunnah di dalam al- DOI: 10.15408/ref.v18i2.11272 DOI: 10.15408/ref.v18i2.11272 Agung Abdillah dan Rizal Alwi Mampa, Kritik Matan Hadis dengan Pendekatan Al-Qur’an \| 231 231 Qur’an menurut al-Ghazālī, Model pemahaman hadis Muḥammad al-Ghazālī, dalam karyanya Fiqh al-Sirah, menjelaskan tentang kedudukan hadis atau Sunnah terhadap al-Qur’an dengan menguraikan; “al-Qur’an adalah undang- undang Islam dan Sunnah adalah pelaksananya. Seorang muslim diwajibkan menghormati pelaksana (sunnah) sebagaimana ia diwajibkan menghormati undang- undang (al-Qur’an) itu sendiri. Allah Swt telah memberikan pada Nabi-Nya hak untuk diikuti apapun yang diperintah dan dilarangnya. Karena semua itu (perintah dan larangan) tidak muncul dari dirinya melankan Karena arahan Allah swt. Dengan demikian, taat pada Nabi saw. berarti taat pada Allah swt. Tidak ada ketundukan yang lebih buta kepada siapapun, kecuali kepada Nabi saw.”30 Qur’an menurut al-Ghazālī, Model pemahaman hadis Muḥammad al-Ghazālī, dalam karyanya Fiqh al-Sirah, menjelaskan tentang kedudukan hadis atau Sunnah terhadap al-Qur’an dengan menguraikan; “al-Qur’an adalah undang- undang Islam dan Sunnah adalah pelaksananya. Seorang muslim diwajibkan menghormati pelaksana (sunnah) sebagaimana ia diwajibkan menghormati undang- undang (al-Qur’an) itu sendiri. Allah Swt telah memberikan pada Nabi-Nya hak untuk diikuti apapun yang diperintah dan dilarangnya. Karena semua itu (perintah dan larangan) tidak muncul dari dirinya melankan Karena arahan Allah swt. Dengan demikian, taat pada Nabi saw. berarti taat pada Allah swt. Tidak ada ketundukan yang lebih buta kepada siapapun, kecuali kepada Nabi saw.”30 Kesimpulan yang dapat diambil dari pernyataan al-Ghazālī di atas adalah, pertama, Sunnah adalah bagian dari pelaksanaan al-Qur’an, kedua, Sunnah adalah bagian dari apa yang keluar dari arahan Allah swt dalam al-Qur’an, ketiga, ketaatan kepada Nabi saw. juga berarti ketaatan kepada Sunnahnya yang diidentikkan dengan ketaatan kepada Allah swt. dan al-Qur’annya. Namun demikian, al-Ghazālī menyimpulkan sunnah bahwa tidak setiap yang dinisbahkan kepada Rasulullah saw. itu berarti Sunnah yang dapat di- terima. Tidak setiap yang valid penisbatannya (kepada Rasulullah saw) itu pasti benar pemahamannya.31 Lebih lanjut mengenai kriteria kesahihan sanad hadis oleh al-Ghazālī tidak jauh berbeda dengan apa yang menjadi kriteria ulama hadis lainnnya,32 yaitu: pertama, setiap periwayatan sanad hadis harus dilakukan oleh orang yang kuat hafalannya dan mampu mengingat dengan sangat baik apa yang didengar-nya, kemudian ia mampu mentransfernya kepada periwayat di bawahnya juga dengan baik. Kedua, di samping kuat hafalan dan cerdas ingatan, seorang peri-wayat harus memiliki tingkah laku dan kepribadian yang baik. DOI: 10.15408/ref.v18i2.11272 a. Model Pemahaman Hadis Ketiga, kedua kriteria tersebut diatas harus dimiliki oleh masing-masing periwayat dalam se-luruh rangkaian para periwayat hadis. Jika salah satu tidak dipenuhi oleh peri-wayat maka hadis itu tidak dapat dikategorikan sahih. Keempat, setelah meneliti rangkaian sanad, selanjutnya adalah penelitian terhadap matan hadis, matan hadis tidak boleh mengandung shādh, dan ilat. Tidak hanya sampai pada pembahasan tentang kesahihan sanad hadis, hadis aḥad pun tidak luput dari perhatiannya. Pandangan al-Ghazālī terhadap hadis aḥad adalah bahwa hadis aḥad tidak bisa diterima begitu saja karena ia hanya menghasilkan dugaan atau pengetahuan yang bersifat dugaan, dan hadis aḥad hanya berlaku pada bidang-bidang syariah, bukan dalam bidang uṣūl al-dīn, dengan pandangan al-Ghazālī33 di antaranya: pertama, periwayat yang sen-dirian kadang-kadang lupa atau salah dalam meriwayatkan hadis, karena ia adalah DOI: 10.15408/ref.v18i2.11272 232 \| REFLEKSI,Volume 18, Nomor 2, Oktober 2019 232 manusia biasa. Kemungkinan terjadinya lupa atau salah dalam riwayat aḥad adalah hal yang tak dapat diragukan. Kedua, dalam urusan dunia saja, kita hanya mengesahkan kesaksian dua orang laki-laki yang adil. Al-Ghazālī dalam memahami hadis tidak terpaku pada persyaratan-per- syaratan yang dibuat oleh ulama hadis dalam menilai sebuah hadis Nabi. Bagi- nya, ada yang lebih penting dari sekadar metode, yaitu maslaḥāt al-Muslimīn (kepentingan umat Islam).34 Al-Ghazālī dalam bukunya al-Sunnah al-Nabawiyah: Bain Ahl al-Fiqh wa Ahl al-Ḥādīth, menjadi satu dari sekian banyak bukunya yang banyak menyorot persoalan hadis, dalam buku ini al-Ghazālī mengatakan sekarang ini banyak bermunculan orang yang memberikan banyak perhatian yang berlebihan terha- dap hadis Nabi saw. dalam penetapan hukum tanpa melakukamn kritik terlebih dahulu, sehingga kesimpulan mereka sering bertentangan dengan al-Qur’an.35 Al-Ghazālī mempersoalkan status hadis ahad, beliau mengatakan bahwa beliau dalam memahami hadis aḥad bukan bermaksud untuk melemahkan suatu hadis, namun beliau hanya berkeinginan agar dalam memahami hadis dipahami dalam kerangka makna-makna yang ditunjukkan oleh al-Qur’an, baik secara langsung ataupun tidak.36 b. Teori dan Aplikasi Kritik Matan Hadis Al-Ghazālī dalam menentukan sahih atau tidaknya hadis, setidaknya ada 5 faktor yang mempengaruhinya, 3 di antaranya terkait dengan kesahihan sanad hadis dan 2 di antaranya terkait dengan matan hadis, kelima faktor tersebut adalah: a. Setiap perawi dalam sanad suatu hadis haruslah seorang yang dikenal sebagai penghafal yang cerdas dan teliti dan benar-benar memahami apa yang didengarnya. Kemudian meriwayatkannya setelah itu, tepat seperti aslinya. b. Di samping kecerdasan yang dimilikinya, ia juga harus seorang yang mantab kepribadiannya dan bertakwa kepada Allah swt, serta menolak dengan tegas setiap pemalsuan atau penyimpangan. c. Kedua kriteria di atas harus dimiliki oleh masing-masing perawi dalam seluruh rangkaian para perawi suatu hadis. Jika hal itu tak terpenuhi pada diri seorang saja dari mereka, maka hadis tersebut tidak dapat dianggap mencapai derajat ṣaḥīḥ. d. Mengenai matan hadis itu sendiri, ia harus tidak bersifat shādh. e. Hadis tersebut harus bersih dari cacat yang diketahui oleh ahli hadis. DOI: 10.15408/ref.v18i2.11272 DOI: 10.15408/ref.v18i2.11272 Agung Abdillah dan Rizal Alwi Mampa, Kritik Matan Hadis dengan Pendekatan Al-Qur’an \| 233 233 Metode yang ada di atas bukanlah hal baru yang digunakan oleh Muḥammad al-Ghazālī, secara umum kaidah di atas sudah digunakan oleh para pengkaji hadis dari dulu hingga sekarang, namun sekarang permasalahannya adalah, apakah setiap hadis yang secara sanad dianggap sahih lantas kemudian secara otomatis mensahihkan matan hadis?, al-Ghazālī sendiri mengakui adanya hadis yang sahih sanadnya, akan tetapi lemah dari sisi matannya,37 lemah dari segi matan bisa terjadi pada pertentangan matan hadis dengan matan hadis lainnya yang dianghap lebih sahih, bisa juga karena matan hadis tersebut bertentangan dengan logika/akal dan bisa juga lemahnya matan karena ber-tentangan dengan nilai-nilai yang ada didalam al-Qur’an. Al-Ghazālī menuturkan bahwa, untuk menetapkan sahihnya suatu hadis dalam matannya diperluakan ilmu yang mendalam tentang al-Qur’an, agar dengan itu semua dapat dilakukan perbandingan dan pengokohan antara yang satu dengan yang lain.38 Al-Ghazālī mendasari pijakan metodenya dalam menganalisa dan meng- kritisi matan hadis, dengan berpegang pada prinsip-prinsip umum ajaran al- Qur’an dan argumen rasional. Dengan dasar ini, al-Ghazālī juga banyak me- nolak hadis-hadis yang bila ditinjau dari segi kesahihan sanad hadis, memiliki kualitas sanad yang baik, namun cacat pada matannya. Karenanya tidak jarang hadis-hadis yang dipandang sahih oleh sebagian besar ulama, seperti al-Bukhārī, Muslim dan lain sebagainya, namun oleh al-Ghazālī dinilai ḍa’if dari segi matannya. Berikut adalah contoh kritik matan dengan pendekatan al-Qur’an yang dilakukan oleh Muḥammad al-Ghazālī yaitu dengan menggunakan al-Qur’an. b. Teori dan Aplikasi Kritik Matan Hadis Hadis tentang disiksanya mayit karena ratapan keluarganya; َِلهَْ َّحَدَّث َنَا دَاوُدُ بْنُ رُشَيْدٍ، حَد ِث َنَا إِْسَْاعِيلُ ابْنُ عُلَيَّةَ، حَدَّث َنَا أَيُّوبُ، عَنْ عَبْدِ اهللِ بْنِ أَِب ،َمُلَيْكَةَ، قَالَ: كُنْتُ جَالِسًا إَِلَ جَنْبِ ابْنِ عُمَرَ، وََنَْنُ ن َنْتَظِرُ جَنَازَةَ أُمِّ أَبَانَ بِنْتِ عُثْمَان وَعِنْدَهُ عَمْرُو بْنُ عُثْمَانَ، فَجَاءَ ا ،َبْنُ عَبَّاسٍ ي َقُودُهُ قَائِدٌ، فَأُرَاهُ أَخْب َرَهُ ِبَِكَانِ ابْنِ عُمَر َفَجَاءَ حََّتَّ جَلَسَ إَِلَ جَنِْبِ ، فَكُنْتُ ب َي ْن َهُمَا، فَإِذَا صَوْتٌ مِنَ الدَّارِ، ف َقَالَ ابْنُ عُمَر- ْكَأَنَّهُ ي َعْرِضُ عَلَى عَمْرٍو أَنْ ي َقُومَ، ف َي َن ْهَاهُم- َِ: ْس َعْتُ رَسُولَ اهللِ صَلَّى اهللُ عَلَيْهِ وَسَلَّم ِي َقُولُ: «إِنَّ الْمَيِّتَ لَي ُعَذَّبُ بِبُكَاءِ أَهْلِه َّحَدَّث َنَا دَاوُدُ بْنُ رُشَيْدٍ، حَد ِث َنَا إِْسَْاعِيلُ ابْنُ عُلَيَّةَ، حَدَّث َنَا أَيُّوبُ، عَنْ عَبْدِ اهللِ بْنِ أَِب ،َمُلَيْكَةَ، قَالَ: كُنْتُ جَالِسًا إَِلَ جَنْبِ ابْنِ عُمَرَ، وََنَْنُ ن َنْتَظِرُ جَنَازَةَ أُمِّ أَبَانَ بِنْتِ عُثْمَان وَعِنْدَهُ عَمْرُو بْنُ عُثْمَانَ، فَجَاءَ ا ،َبْنُ عَبَّاسٍ ي َقُودُهُ قَائِدٌ، فَأُرَاهُ أَخْب َرَهُ ِبَِكَانِ ابْنِ عُمَر َفَجَاءَ حََّتَّ جَلَسَ إَِلَ جَنِْبِ ، فَكُنْتُ ب َي ْن َهُمَا، فَإِذَا صَوْتٌ مِنَ الدَّارِ، ف َقَالَ ابْنُ عُمَر- ْكَأَنَّهُ ي َعْرِضُ عَلَى عَمْرٍو أَنْ ي َقُومَ، ف َي َن ْهَاهُم- َِ: ْس َعْتُ رَسُولَ اهللِ صَلَّى اهللُ عَلَيْهِ وَسَلَّم ِي َقُولُ: «إِنَّ الْمَيِّتَ لَي ُعَذَّبُ بِبُكَاءِ أَهْلِه Menceritakan kepada kami Dāwud bin Rushd, menceritakan kami Isma’īl ibn ‘Aliyah, menceritakan kami Ayyūb bin ‘Abdullāh bin Abī Mulaikah berkata: ketika aku duduk di samping Ibn ‘Umar dan kita melihat Janazah Menceritakan kepada kami Dāwud bin Rushd, menceritakan kami Isma’īl ibn ‘Aliyah, menceritakan kami Ayyūb bin ‘Abdullāh bin Abī Mulaikah berkata: ketika aku duduk di samping Ibn ‘Umar dan kita melihat Janazah DOI: 10.15408/ref.v18i2.11272 234 \| REFLEKSI,Volume 18, Nomor 2, Oktober 2019 Ummi binti ‘Utsmān dan di dekatnya ‘Umar bin ‘Uthmān, maka datang- lah Ibn ‘Abbās.... Rasulullah saw. bersabda:“Sesunggughnya Mayyit itu disiksa karena tangisan keluarganya”. (HR. Muslim)39 Ummi binti ‘Utsmān dan di dekatnya ‘Umar bin ‘Uthmān, maka datang- lah Ibn ‘Abbās.... Rasulullah saw. bersabda:“Sesunggughnya Mayyit itu disiksa karena tangisan keluarganya”. (HR. Muslim)39 Hadis ini seolah menunjukkan ketidakadilan bagi si mayyit, secara zahir (tekstual) dan sepintas lalu, bunyi hadis di atas mengesankan bahwa mayit akan disiksa akibat keluarga atau kerabatnya yang menangisi kematiannya. Namun pertanyaannya apa salah si mayit? Bagaimana ia harus bertanggungjawab terhadap apa yang tidak ia lakukan? Bukankah seseorang tidak memikul dosa orang lain? Mengapa orang disiksa akibat kesalahan yang dilakukan orang lain? b. Teori dan Aplikasi Kritik Matan Hadis Hadis ini sebenarnya telah dijelaskan (dinasakh) oleh ‘Ā’ishah, dengan hadis yang berbunyi; ِلهََِ ،َحَدَّث َنَا إِسْحَاقُ، قَالَ: حَدَّثَِنِ مَالِكٌ، عَنْ عَبْدِ اهللِ بْنِ أَِبِ بَكْرٍ، عَنْ أَبِيهِ، عَنْ عَمْرَة َأَن َّهَا أَخْب َرَتْهُ أَن َّهَا، ْسَِعَتْ عَائِشَةَ، وَذُكِرَ َلََا أَنَّ عَبْدَ اهللِ بْنَ عُمَر ُي َقُولُ: إِنَّ الْمَيِّتَ لَي ُعَذَّب ُبِبُكَاءِ اْلَْيِّ، ف َقَالَتْ عَائِشَةُ: ي َغْفِرُ اهللُ ِلَِِبِ عَبْدِ الرَّْحَْنِ، أَمَا إِنَّهُ َلَْ يَكْذِبْ، وَلَكِنَّه ،َنَسِي َ أَوْ أَخْطَأَ، إَِّنََّا مَرَّ رَسُولُ اهللِ صَلَّى اهللُ عَلَيْهِ وَسَلَّمَ، عَلَى ي " :َهُودِيَّةٍ ي ُبْكَى عَلَي ْهَا، ف َقَال إِن َّهُمْ لَيَبْكُونَ عَلَي ْهَا، وَإِن َّهَا لَت ُعَذَّبُ ِفِ ق َْبِْهَا ،َحَدَّث َنَا إِسْحَاقُ، قَالَ: حَدَّثَِنِ مَالِكٌ، عَنْ عَبْدِ اهللِ بْنِ أَِبِ بَكْرٍ، عَنْ أَبِيهِ، عَنْ عَمْرَة َأَن َّهَا أَخْب َرَتْهُ أَن َّهَا، ْسَِعَتْ عَائِشَةَ، وَذُكِرَ َلََا أَنَّ عَبْدَ اهللِ بْنَ عُمَر ُي َقُولُ: إِنَّ الْمَيِّتَ لَي ُعَذَّب ُبِبُكَاءِ اْلَْيِّ، ف َقَالَتْ عَائِشَةُ: ي َغْفِرُ اهللُ ِلَِِبِ عَبْدِ الرَّْحَْنِ، أَمَا إِنَّهُ َلَْ يَكْذِبْ، وَلَكِنَّه ،َنَسِي َ أَوْ أَخْطَأَ، إَِّنََّا مَرَّ رَسُولُ اهللِ صَلَّى اهللُ عَلَيْهِ وَسَلَّمَ، عَلَى ي " :َهُودِيَّةٍ ي ُبْكَى عَلَي ْهَا، ف َقَال إِن َّهُمْ لَيَبْكُونَ عَلَي ْهَا، وَإِن َّهَا لَت ُعَذَّبُ ِفِ ق َْبِْهَا َََِْ Menceritakan Isḥāq berkata menceritakan kepadaku Mālik dari ‘Abdullāh bin Abī Bakr dari Ayahnya dari ‘Amrah, sesungguhnya ia mengkhabarkan bahwa ‘Ā’ishah mendengar dan menyebutkannya bahwa ‘Abdullāh bin ‘Umar berkata: “Sesungguhnya mayat akan disiksa karena tangisan orang yang masih hidup.” Maka ‘Ā’ishah berkata; “Semoga Allah mengampuni Abū ‘Abd al-Raḥman. Sesungguhnya dia tidak berdusta hanya saja kemungkinan dia lupa atau salah, bahwasanya Rasulullah saw. pernah melewati seorang wanita yahudi yang minta ditangisi, maka Rasulullah bersabda: “Mereka menangisinya, padahal dia (wanita yahudi) betul-betul tengah di siksa dikuburnya.” (HR. Aḥmad bin Ḥanbal dari ‘Amrah binti ‘Abd al-Raḥman, Musnad Aḥmad ibn Ḥanbal, juz VI, hal. 107, hadits no. 23802). Bagi al-Ghazālī, hadis ini bertentangan dengan nilai-nilai yang terkan-dung di dalam al-Qur’an, yaitu QS. al-An’ām ayat 164, bahwasannya “Tidaklah seseorang menanggung dosa orang lain”.40 DOI: 10.15408/ref.v18i2.11272 DOI: 10.15408/ref.v18i2.11272 Agung Abdillah dan Rizal Alwi Mampa, Kritik Matan Hadis dengan Pendekatan Al-Qur’an \| 235 ٍقُلْ اَغَي ْرَ الل ّٰهِ اَبْغِيْ رَبًّا وَّهُوَ رَبُّ كُلِّ شَيْء ۗ َوَل ُتَكْسِب ُّكُل ٍن َفْس َّاِل عَلَي ْهَا ۗ َوَل ُتَزِر ٌوَازِرَة َوِّزْر اُخْرّٰى ۗ َُّث ّٰاَِل ْرَبِّكُم ْمَّرْجِ عُكُم ْف َي ُنَبِّئُكُم ِبَِا ْكُنْتُم ِفِيْه ََتَْتَلِفُوْن ََََِِِِٰ Artinya: “Katakanlah: "Apakah aku akan mencari Tuhan selain Allah, padahal Dia adalah Tuhan bagi segala sesuatu. b. Teori dan Aplikasi Kritik Matan Hadis Dan tidaklah seorang membuat dosa melainkan kemudharatannya kembali kepada dirinya sendiri; dan seorang yang berdosa tidak akan memikul dosa orang lain. Kemudian kepada Tuhanmulah kamu kembali, dan akan diberitakan-Nya kepadamu apa yang kamu perselisihkan" (QS. al-An’ām [6]: 164) Ayat ini secara jelas menegaskan bahwa orang yang melakukan dosa akan mendapatkan ganjaran atas dirinya sendiri, bukan pada orang lain, orang yang berbuat dosa kelak akan memikul dosanya/kemudharatannya sendiri, oleh sebab itu, orang yang berbuat dosa tidak akan memikul dosa orang lain. Ayat ini jelas bertentangan dengan hadis yang di atas, jika hadis di atas menyebutkan bahwa mayyit disiksa karena ratapan orang lain, maka ayat ini menjelakan bahwa orang lain tidak akan menanggung doa orang lain pula. Apa yang dilakukan oleh al-Ghazālī ini sebenarnya sama dengan yang dilakukan 'Ā’ishah ketika mendengar hadis yang menyatakan bahwa orang mati diadzab karena tangisan keluarganya terhadapnya. Ia menolaknya dan ber-sumpah bahwa Nabi saw., tidak pernah mengucapkan hadis tersebut. Bahkan ‘Ā’ishah kemudian mejelaskan alasan penolakannya dengan mengatakan, dike-mukakan firman Allah QS. al-An’ām ayat 164. Dengan berlandaskan sikap Aisyah yang menolak hadis tentang siksa kubur karena tangisan keluarganya, dikarenakan bertentangan dengan nila-nilai al- Qur’an. Menurut al-Ghazālī, pernyataan ‘Ā’ishah dapat dijadikan dasar untuk menguji validitas sebuah hadis yang telah berstatus sahih, dengan nash-nash al- Qur’an, kitab suci yang tidak tercampuri atau tersentuh oleh kebatilan dari arah mana saja.41 Demikianlah apa yang sudah dilakukan oleh Muḥammad al-Ghazālī ketika mengkritisi matan hadis dengan perspektif al-Qur’an yaitu dengan meng- gunakan metode yang sama dengan apa yang dilakukan oleh ‘Ā’ishah. b. Teori dan Aplikasi Kritik Matan Hadis Contoh lainnya adalah hadis tentang tangisan bayi ketika lahir dan terja- ganya Maryam dan Nabi Isya ketika Nabi Isya baru dilahirkan,42 yaitu hadis riwayat al-Bukhārī, hadis ke 3177; DOI: 10.15408/ref.v18i2.11272 DOI: 10.15408/ref.v18i2.11272 236 \| REFLEKSI,Volume 18, Nomor 2, Oktober 2019 236 ْحَدَّثَِنِ عَبْدُ اللَّهِ بْنُ ُمَُمَّدٍ حَدَّث َنَا عَبْدُ الرَّزَّاقِ أَخْب َرَنَا مَعْمَرٌ عَنْ الزُّهْرِيِّ عَنْ سَعِيدِ ب ِن ِالْمُسَيَّبِ عَنْ أَِبِ هُرَي ْرَةَ رَض ٍيَ اللَّهُ عَن ْهُأَنَّ النَِّبَِّ صَلَّى اللَّهُ عَلَيْهِ وَسَلَّمَ قَالَ مَا مِنْ مَوْلُود ََيُولَدُ إِلَّ وَالشَّيْطَانُ َيََسُّهُ حِنيَ يُولَدُ ف َيَسْتَهِلُّ صَارِخًا مِنْ مَسِّ الشَّيْطَانِ إِيَّاهُ إِلَّ مَرْي وَاب ْن َهَا ْحَدَّثَِنِ عَبْدُ اللَّهِ بْنُ ُمَُمَّدٍ حَدَّث َنَا عَبْدُ الرَّزَّاقِ أَخْب َرَنَا مَعْمَرٌ عَنْ الزُّهْرِيِّ عَنْ سَعِيدِ ب ِن ِالْمُسَيَّبِ عَنْ أَِبِ هُرَي ْرَةَ رَض ٍيَ اللَّهُ عَن ْهُأَنَّ النَِّبَِّ صَلَّى اللَّهُ عَلَيْهِ وَسَلَّمَ قَالَ مَا مِنْ مَوْلُود ََيُولَدُ إِلَّ وَالشَّيْطَانُ َيََسُّهُ حِنيَ يُولَدُ ف َيَسْتَهِلُّ صَارِخًا مِنْ مَسِّ الشَّيْطَانِ إِيَّاهُ إِلَّ مَرْي وَاب ْن َهَا ََُِنََُ “Menceritakan kepadaku ‘Abdullāh bin Muḥammad menceritakan kepada kami ‘Abd al-Razzāq mengabarkan kepada kami Ma’mar dari al-Zuhrī dari Sa’īd bin al-Musayyab dari Abī Hurayrah ra. Bahwa Nabi saw. ber-sabda: Tidak seorang bayi pun yang dilahirkan kecuali telah disentuh oleh setan sehingga ia menangis menjerit karena sentuhan setan tersebut kecuali putra Maryam dan ibunya.” Hadis ini menjelaskan mengenai perbuatan yang dilakukan oleh syaitan terhadap bayi yang dilahirkan, hadis ini menggambarkan bahwa ketika bayi baru dilahirkan kedunia dan kemudian menangis pada saat dilahirkan, perbua-tan itu tidak lain adalah karena ulah dari pada syaitan, yaitu, syaitan menyentuh setiap bayi yang baru dilahirkan yang membuat setiap bayi menangis. Hanya saja dalamhal ini putra Maryam dan ibunya mendapat pengecualian. Al-Ghazālī memahami hadis dengan membandingkan matan hadis deng- an nilai-nilai al-Qur’an, bahwa, maryam dan putranya termasuk di antara hamba- hamba Allah yang saleh. Sedangkan setan berdasarkan keterangan al-Qur’an tidak memiliki kekuasaan apapun atas diri hamba-hamba Allah, hal ini berdasarkan QS. Āli-Imrān [3]: 36; َِِّّْ ِوَإِِّنِّ ْسََّيْت ُهَا مَرْيََ وَإِِّنِّ أُعِيذُهَا بِكَ وَذُرِّي َّت َهَا مِنَ الشَّيْطَانِ الرَّجِ يم ِوَإِِّنِّ ْسََّيْت ُهَا مَرْيََ وَإِِّنِّ أُعِيذُهَا بِكَ وَذُرِّي َّت َهَا مِنَ الشَّيْطَانِ الرَّجِ يم Artinya:“. Sesungguhnya aku telah menamai dia Maryam dan aku mohon perlindungan untuknya serta anak-anak keturunannya kepada (pemelihara- an) Engkau daripada syaitan yang terkutuk" (QS. Ali-Imrān [3]: 36) َََِِِ Artinya:“. Sesungguhnya aku telah menamai dia Maryam dan aku mohon perlindungan untuknya serta anak-anak keturunannya kepada (pemelihara- an) Engkau daripada syaitan yang terkutuk" (QS. Ali-Imrān [3]: 36) an) Engkau daripada syaitan yang terkutuk" (QS. b. Teori dan Aplikasi Kritik Matan Hadis Ali-Imrān [3]: 36) Ayat ini menjelaskan tentang perlindungan untuk maryam serta perlin- dungan unuk anak-anak keturunannya (hamba-hamba yang saleh), perlindung- an dari gangguan syaitan yang terkutuk. Ayat ini secara jelas bertentangan dengan hadis di atas, jika hadis di atas dikatakan bahwa tangisan bayi yang baru lahir merupakan bagian dari gang-guan syaitan (terkecuali Maryam dan Putranya), maka dalam ayat ini dijelaskan tentang perlindungan terhadap gangguan syaitan terhadap hamba-hamba yang salih dan anak keturunan Maryam. Al-Ghazālī juga menambahkan komentar-komentar para alim ulama dalam memahami hadis yang telah disebutkan di atas, di antaranya adalah pen- dapat dari al-Baiḍawī, pengarang tafsir al-Manar, yang memahami hadis di atas (sentuhan syaitan) sebagai perumpamaan dan bukan hakiki, Rashīd Riḍā juga DOI: 10.15408/ref.v18i2.11272 DOI: 10.15408/ref.v18i2.11272 Agung Abdillah dan Rizal Alwi Mampa, Kritik Matan Hadis dengan Pendekatan Al-Qur’an \| 237 237 memahami hadis di atas dengan berupa kiasan, menurut Rashīd Riḍā, bahwa Setan tidak memiliki kemampuan untuk mengganggu hamba-hamba Allah yang Ikhlas dan terpilih apalagi Nabi dan Rasul.43 Al-Ghazālī menambahkan bahwa hadis di atas merupakan kisah-kisah Ghaib, sedangkan untuk mengimani terhadap sesuatu yang ghaib harus diperlu- kan dalil-dalil yang pasti, lantas kemudian berkenaan dengan hadis di atas me- nurut al-Ghazālī adalah hadis yang diriwayatkan secara aḥad sehingga sukar untuk dapat diterima.44 Dari kedua contoh di atas, dapat dilihat bagaimana al-Ghazālī memahami (mengkritisi) hadis dengan pendekatan al-Qur’an, di antaranya adalah: Pertama, mengikuti pendapat sahabat, hal ini bisa dilihat ketika al-Ghazālī mengikuti pendapat ‘Ā’ishah ketika mengkritisi hadis tentang disik-sanya mayit karena ratapan keluarganya. Kedua, al-Ghazālī juga menggunakan nilai-nilai yang terkandung di dalam al-Qur’an untuk mengkritisi hadis, hal ini bisa dilihat dari contoh hadis tentang disiksanya mayyit karena ratapan keluarga dan hadis tentang tangisan bayi yang baru lahir karena tusukan syaitan. Al-Ghazālī juga memahami matan hadis dengan memberikan tema pen- ting atas isi hadis tersebut, seperti dalam hal hadis yang berkaitan dengan masalah ghaib di atas, al-Ghazālī meragukan hadis tersebut karena hal itu hanyalah bersifat dugaan, yang juga merupakan hadis aḥad. a. Model Pemahaman Hadis Jamāl membedakan antara hadis dan sunnah, Sunnah menurut Jamāl adalah tata cara yang dijalankan beliau dalam ibadah, etika dan amal per- buatan.45 Jamāl menilai bahwa Sunnah lebih dekat kepada amal perbuatan Nabi saw. Namun berbeda dengan hadis yang lebih dekat kepada ucapan (sabda) Nabi saw. Namun ia tetap dapat menerima jika hadis disinonimkan dengan Sunnah. Sehingga, keduanya mencakup ucapan dan perbuatan Nabi saw. Ketika cakupan makna hadis atau sunnah diperluas yang termasuk di dalamnya fatwa sahabat, Jamāl tampak keberatan menerima hal ini. Baginya, fatwa sahabat, apalagi tabi’in tidak didapati jaminan kebenarannya. Jaminan ke- ma’sum-an yang diberikan Allah swt hanyalah milik Rasululah saw.46 Jamāl membagi Sunnah menjadi tiga bagian.47 Jamāl membagi Sunnah menjadi tiga bagian.47 1. Sunnah ‘Ibādiyyah Adalah Sunnah yang berkaitan langsung dengan ajaran-ajaran Agama. Sehingga, apabila macam sunnah ini diamalkan maka termasuk kate- Adalah Sunnah yang berkaitan langsung dengan ajaran-ajaran Agama. Adalah Sunnah yang berkaitan langsung dengan ajaran-ajaran Agama. Adalah Sunnah yang berkaitan langsung dengan ajaran-ajaran Agama. Sehingga, apabila macam sunnah ini diamalkan maka termasuk kate- Sehingga, apabila macam sunnah ini diamalkan maka termasuk kate- Sehingga, apabila macam sunnah ini diamalkan maka termasuk kate- DOI: 10.15408/ref.v18i2.11272 238 \| REFLEKSI,Volume 18, Nomor 2, Oktober 2019 238 gori ibadah. Sunnah ini tercermin dalam praktek peribadatan dalam rangka mendekatkan diri kepada Allah, baik melalui sahalat, puasa dan sebagainya. 2. Sunnah Ḥayātiyyah Sunnah ini juga bisa disebut dengsn Sunnah Ta’amuliyyah, yaitu segala sesuatu yang berkaitan dengan perbuatan Rasulullah saw. sebagai seorang ayah, suami, manusia biasa yang mengenakan pakaian, makan, mencintai, membenci dan berinteraksi dengan masyarakatnya, serta memberikan teladan yang baik tentang bagaimana seseorang harus ber- intraksi. 3. Sunnah Siyāsiyyah Sunnah ini mencakup sikap dan ketetapan-ketetapannya sebagai se- orang kepala Negara, panglima perang, pengatuir kenijakan ekonomi dan lainnya. Menurut Jamāl, tidak semua Sunah itu menjadi syariat, menurut Jamāl sunnah yang dijadikan syariat itu adalah sunnah yang berkaitan dengan agama. Sedangkan sunnah yang mencakup etika makan dan minum Rasulullah saw. Itu bukan termasuk kategori sunnah yang disyariatkan. Ketika ditemukan suatu permasalahan yang tidak ditemukannya pada masa Nabi saw. Maka umat islam harus mengembalikan solusi permasalahan itu kepada al-Qur’an dan berhak menggunakan ijtihad mereka dalam memecahkan permasalahan dan memenuhi tuntutan hidupnya. Jamāl berpendapat bahwa, ucapan atau fatwa sahabat dan tabi’in tidaklah layak dijadikan ḥujjah. a. Model Pemahaman Hadis Ini dikarenakan Allah swt hanya mengutus seorang Nabi yang dijadiakan teladan dan rujukan mengenai segala petunjuk yang datang dari Allah bagi umat Islam, sehingga, baik sahabat ataupun generasi sesudahnya tetap memiliki kewajiban mengikuti al-Qur’an dan hadis. Jamāl menilai bahwa kesahihan sanad hadis tidak menjamin kesahihan matan hadis. Dalam melakukan kajian matan hadis, Jamāl tidak ingin serta merta menerima kriteria yang telah ditetapkan para ulama, karena menurutnya, keriteria tersebut tidak memuat obyektifitas dan sangat dipengaruhi oleh kon-disi dan situasi masing-masing ulama ilmu hadis itu hidup.48 Jamāl menilai bahwa hadis yang sesuai dengan al-Qur’an sangat dimung- kinkan kebenarannya berasal dari Rasulullah saw., sedangkan hadis yang tidak sesuai dengan al-Qur’an harus dijauhkan dari penisbahan kepada Rasulullah saw. Selanjutnya, jika suatu hadis telah dipastikan kebenarannya bersumber ke-pada Rasulullah saw. Maka tidak seacara otomatis harus menjadi suatu ajaran atau syariat yang berlaku sepanjang masa. Ini dikarenakan suatu hadis itu DOI: 10.15408/ref.v18i2.11272 DOI: 10.15408/ref.v18i2.11272 Agung Abdillah dan Rizal Alwi Mampa, Kritik Matan Hadis dengan Pendekatan Al-Qur’an \| 239 239 mengandung kemungkinan kemunculannya dilatarbelakangi oleh situasi atau tuntutan tertentu, sehingga jika kondisi atau tuntutan tersebut telah tiada, maka hadis tersebut tidak dapat dipraktekkan meskipun diyakini bersumber dari Rasulullah saw.49 Pandangan Jamāl al-Bannā terhadap hadis Sahih, dalam menilai hadis Sahih, Jamāl cendderugn mengikuti pendapat para ulama terdahulu, yaitu hadis sahih termasuk kedalam tingkatan kategori kualitas hadis yang paling tinggi, mengikuti apa yang dipahami oleh al-Ḥakim, dalam memahami hadis sahih, al- Ḥakim membagi hadis-hadis sahih ke dalam lima bagian, di antaranya, Pertama, hadis sahih paling tinggi, yaitu hadis yang setidaknya terdapat lebih dari 2 orang sahabat dan tabi’in dalam periwayatannya. Kedua, kondisi seperti yang pertama, namun hanya melibatkan satu perawi dari sahabat. Ketiga, kondisi seperti yang pertama, namun hanya melibatkan satu perawi dari tabi’in. Keempat, hadis yang diriwayatkan oleh orang-orang yang tidak popular, namun terdapat sebagian ulama popular yang menceritakannya. Kelima, hadis yang diriwayatkan oleh keluarga popular dan terpercaya, walaupun sebagian sanad-nya ada yang kurang kuat.50 Pandangan Jamāl al-Bannā terhadap hadis ḥasan, hadis hasan menurut Jamāl adalah hadis yang kekuatannya tidak sekuat hadis hasan, atau dalam kata lain, hadis hasan berada di urutan kedua setelah hadis sahih yang juga hadis ini dapat diterima. Bahkan menurut Jamāl, hadis ḥasan bisa naik pangkat menjadi ṣaḥīḥ dan juga bisa menjadi ḍaif dengan beberapa ketentuan yang telah ditetapkan oleh para ulama hadis . a. Model Pemahaman Hadis Pandangan Jamāl al-Bannā terhadap hadis ḍa’if, Jamāl membagi hadis ḍa’if kedalam dua kelompok, kelompok pertama adalah hadis ḍa’if yang semula adalah hadis ḥasan, yaitu hadis ḍaif yang oleh para ulama, dikumpulkan riwayat-riwayat yang lain sehingga kualitasnya menjadi hadis ḥasan. Sedangkan kategori yang kedua adalah hadis ḍa’if yang beruah menjadi hadis mawḍū’. Yaitu hadis yang terjadinya pelonggaran terhadap para ulama yang membela hadis.51 Pandangan Jamāl al-Bannā terhadap hadis aḥad, hadis aḥad hanya bisa diamalkan jika benar-benar terbukti kebenarannya.52 Hal ini dikarenakan hadis ahad tidak memberikan keyakinan yang pasti, namun hanya menghasilkan keraguan. b. Teori dan Aplikasi Kritik Matan Hadis Jamāl meyakini bahwa para ulama telah berupaya secara maksimal dalam menetapkan suatu kriteria, kaidah dan tingkatan dalam mencapai penilaian DOI: 10.15408/ref.v18i2.11272 240 \| REFLEKSI,Volume 18, Nomor 2, Oktober 2019 240 dalam meneliti suatu hadis, namun adakalanya para ulama seringkali berbeda pendapat terhadap penilaian suatu hadis. Adanya perbedaan pendapat dikalangan ulama mengenai kesahihan hadis menurut Jamāl menunjukkan ketidakadanya tolok-ukur dalam batas-batas kriteria yang telah disepakati para ulama bersama mengenai penilaian terhadap suatu hadis. Atas dasar inilah Jamāl berpendapat bahwa hadis yang telah ditetapkan kesahihan sanad tidak mesti harus diambil, karena kesahihan dan kekuatan suatu hadis itu harus diukur dari sesuai atau tidaknya hadis tersebut dengan al-Qur’an dan nilai-nilai Islam.53 Maka dari itu Jamāl menambahkan bahwa apa-bila ditemukannya kontradiksi antara hadis dengan al-Qur’an, maka menurut Jamāl bahwa al-Qur’anlah yang harus menjadi prioritas atau yang harus lebih dikedepankan. Metode yang dipakai Jamāl dalam memahami hadis adalah dengan metode ‘Arḍ al-Ḥadīth ‘Alā al-Qur’ān, yaitu dengan membandingkan hadis dengan al- Qur’an, menurut Jamāl, hadis yang layak untuk diamalkan adalah hadis yang sejalan dengan nilai-nilai yang terkandung didalam ajaran al-Qur’an, dalam kajian ini, menurut Jamāl, salah satu metode dalam kajian kritik matan hadis yaitu dengan mongkomparasikan matan hadis dengan nilai-nilai al-Qur’an, mengingat bahwa salah satu fungsi daripada hadis adalah sebagai pen-jelas daripada al-Qur’an, oleh sebab itu, jikalau terdapat pertentangan di antara keduanya, maka al-Qur’an-lah yang harus dikedepankan. Implikasi dari terori Jamāl adalah bahwa hadis yang dinilai sahih dari segi sanadnya belum tentu bisa diterima dari segi matannya, mengingat bahwa sanad hanyalah salah satu dari syarat kesahihan hadis yang merupakan transmisi dari Rasul sampai ke periwayat akhir. Jamāl sendri menilai bahwa teori yang dikemukakan beliau sebenarnya sudah dilakukan oleh ulama terdahulu, bahkan ‘Āi’shah sudah mempraktek- kannya dikala mengomentari hadis tentang siksa kubur karena tangisan keluar- ganya, hanya saja menurut Jamāl, para ulama terdahulu meskipun mengetahui teori tersebut namun mereka tidak berani mempraktekkannya dalam mema- hami suatu hadis.54 Dari teori yang ditawarkan Jamāl mengenai ‘Arḍ al-Ḥādīth ‘Alā al-Qur’ān, dapat dirumuskan 3 model teori kritik matan hadis dengan pendekatan al- Qur’an, yaitu: Pertama, melakukan studi perbandingan antara hadis dengan teks-teks al- Qur’an. Kedua¸membandingkan hadis dengan pemahaman global ayat. b. Teori dan Aplikasi Kritik Matan Hadis DOI: 10.15408/ref.v18i2.11272 DOI: 10.15408/ref.v18i2.11272 Agung Abdillah dan Rizal Alwi Mampa, Kritik Matan Hadis dengan Pendekatan Al-Qur’an \| 24 241 Ketiga, membandingkan antara hadis dengan nilai-nilai yang terkandung di dalam al-Qur’an.55 Ketiga, membandingkan antara hadis dengan nilai-nilai yang terkandung di dalam al-Qur’an.55 Berikut adalah contoh kritik matan hadis dengan pendekatan al-Qur’an yang dilakukan oleh Jamāl al-Bannā: Hadis yang diceritakan oleh al-Tirmidhī dan Ibn Mājah. Bahwasannya Nabi pernah berkata, “saudara perempuan bila bersama anak perempuan adalah ashbah”. ِحَدَّث َنَا عَلِيُّ بْنُ ُمَُمَّدٍ حَدَّث َنَا وَكِيعٌ حَدَّث َنَا سُفْيَانُ عَنْ أَِبِ ق َيْسٍ اِلَْوْدِيِّ عَنْ اَلُْزَيْلِ بْن شُرَحْبِيلَ قَاَلََاءَ رَجُلٌ إَِلَ أَِبِ مُوسَى ٍاِلَْشْعَرِيِّ وَسَلْمَانَ بْنِ رَبِيعَةَ الْبَاهِلِيِّ فَسَأََلَُمَا عَنْ اب ْنَة ٍوَاب ْنَةِ ابْنٍ وَأُخْتٍ ِلَِبٍ وَأُمٍّ ف َقَالَ لِِلِ ب ْنَةِ النِّصْفُ وَمَا بَقِيَ فَلِْلُْخْتِ وَائْتِ ابْنَ مَسْعُود َفَسَيُتَابِعُنَا فَأَتَى الرَّجُلُ ابْنَ مَسْعُودٍ ف سَأَلَهُ وَأَخْب َرَهُ ِبَِا قَالَ ف َقَالَ عَبْدُ اللَّهِ قَدْ ضَلَلْتُ إِذًا ِوَمَا أَنَا مِنْ الْمُهْتَدِينَ وَلَكِِنِّ سَأَقْضِي ِبَِا قَضَى بِهِ رَسُولُ اللَّهِ صَلَّى اللَّهُ عَلَيْهِ وَسَلَّمَ لِِل ِب ْنَة ْالنِّصْفُ وَلِ ب ْنَةِ الِ بْنِ السُّدُسُ تَك ِمِلَةَ الث ُّلُث َنيِْ وَمَا بَقِيَ فَلِْلُْخْت ُِْنُ Menceritakan kepada kami ‘Alī bin Muḥammad menceritakan kepada kami Wakī’ menceritakan kepada Sufyān dari Abī Qais al-Audī dari Huzail bin Suraḥbīl ra, dia berkata: Abū Mūsā ra ditanya tentang kasus kewarisan seorang anak perempuan, anak perempuan dari anak laki-laki, dan seorang saudara perempuan. Abū Mūsā ra berkata: “Untuk anak perempuan setengah, untuk saudara perempuan setengah. Datanglah kepada Ibnu Mas’ūd ra, tentu dia akan mengatakan seperti itu pula.” Kemudian ditanyakan kepada Ibnu Mas’ūd ra dan dia menjawab: "Saya menetapkan berdasarkan apa yang telah ditetapkan oleh Nabi saw. Yaitu untuk anak perempuan setengah, untuk cucu perempuan seperenam sebagai pelengkap dua pertiga, sisanya untuk saudara perempuan.” (HR. al-Bukhārī, Abū Daud, Tirmidhī, dan Ibnu Mājah). Dalam pengertian ini, al-Bannā menjelaskan bahwa saudara perempuan ketika bersama anak perempuan dan tanpa adanya saudara laki-laki seperti saudara laki-laki yang mempunyai hak ‘aṣabah. Sebagaimana saudara laki-laki, mereka (saudara perempuan dan anak perempuan) berhak mendapatkan sisa warisan setelah anak perempuan mengambil haknya. Hak warisnya terhalangi bila ada saudara laki-laki.56 Menurut Jamāl, hadis ini bertentangan dengan ayat kalālah yang terdapat dalam QS. al-Nisā’ ayat 176. b. Teori dan Aplikasi Kritik Matan Hadis Karena saudara perempuan tidak mem-punyai hak waris di hadapan anak perempuan.57 DOI: 10.15408/ref.v18i2.11272 242 242 \| REFLEKSI,Volume 18, Nomor 2, Oktober 2019 ُيَسْت َفْتُونَكَ قُلِ اللَّهُ ي ُفْتِيكُمْ ِفِ الْكَِلَ لَةِ إِنِ امْرُؤٌ هَلَكَ لَيْسَ لَهُ وَلَدٌ وَلَه ُ أُخْتٌ ف َلَهَا نِصْف َمَا ت َرَكَ وَهُوَ يَرِث ُهَا إِنْ َلَْ يَكُنْ َلََا وَلَدٌ فَإِنْ كَان َتَا اث ْنَت َنيِْ ف َلَهُمَا الث ُّلُثَانِ ِمَِّا ت َرَكَ و إِنْ كَانُوا ْإِخْوَةً رِجَالً وَنِسَاءً فَلِلذَّكَرِ مِثْلُ حَظِّ اِلُْن ْث َي َنيِْ ي ُب َنيُِّ اللَّهُ لَكُم ٍأَنْ تَضِلُّوا وَاللَّهُ بِكُلِّ شَيْء ٌعَلِيم Artinya: “Mereka meminta fatwa kepadamu (tentang kalalah). Katakanlah: "Allah memberi fatwa kepadamu tentang kalalah (yaitu): jika seorang meninggal dunia, dan ia tidak mempunyai anak dan mempunyai saudara perempuan, maka bagi saudaranya yang perempuan itu seperdua dari harta yang ditinggalkannya, dan saudaranya yang laki-laki mempusakai (seluruh harta saudara perempuan), jika ia tidak mempunyai anak; tetapi jika saudara perempuan itu dua orang, maka bagi keduanya dua pertai iga dari harta yang ditinggalkan oleh yang meninggal. Dan jika mereka (ahli waris itu terdiri dari) saudara-saudara laki dan perempuan, maka bahagian seorang saudara laki-laki sebanyak bahagian dua orang saudara perempuan. Allah menerangkan (hukum ini) kepadamu, supaya kamu tidak sesat. Dan Allah Maha Mengetahui segala sesuatu” Menurut Jamāl, ayat ini secara langsung bertentangan dengan hadis kalālah di atas, karena saudara perempuan tidak mempunyai hak waris di hadapan anak perempuan, sementara dalam hadis kalālah disebutkan bahwa saudara perempuan mempunyai hak atas waris dihadapan anak perempuan. Contoh lainnya mengenai kritik matan hadis adalah hadis riwayat al- Bukhārī nomor 3017 dan al-Nasā’i nomor 4059: ََ ُحَدَّث َنَا عَلِيُّ بْنُ عَبْدِ اللَّهِ حَدَّث َنَا سُفْيَانُ عَنْ أَيُّوبَ عَنْ عِكْرِمَةَ أَنَّ عَلِيًّا رَضِيَ اللَّهُ عَنْه َحَرَّق َّق َوْمًا ف َب َلَغَ ابْنَ عَبَّاسٍ ف َقَالَلَوْ كُنْتُ أَنَا َلَْ أُحَرِّق ْهُمْ ِلَِنَّ النَِّبَِّ صَلَّى اللَّهُ عَلَيْهِ وَسَل َم َقَالَ لَ ت ُعَذِّبُوا بِعَذَابِ اللَّهِ وَلَقَت َلْت ُهُمْ كَمَا قَالَ النَِّبُِّ صَلَّى اللَّهُ عَلَيْهِ وَسَلَّمَ مَنْ بَدَّل ُدِينَه ُفَاق ْت ُلُوه58 Menceritaka kepada kami ‘Ālī bin ‘Abdillāh, menceritakan kepada kami Sufyān dari Ayyūb dari ‘Ikrimah, sesungguhnya ‘Ālī ra….. “barang siapa yang mengganti agamanya, maka bunuhlah” Secara umum hadis ini dipahami bahwa, orang yang mengganti agamanya, maka dihalalkan untuk membunuhnya, baik itu laki-laki maupun perempuan, dalam hal ini, orang tersebut dinyatakan murtad. DOI: 10.15408/ref.v18i2.11272 b. Teori dan Aplikasi Kritik Matan Hadis DOI: 10.15408/ref.v18i2.11272 DOI: 10.15408/ref.v18i2.11272 Agung Abdillah dan Rizal Alwi Mampa, Kritik Matan Hadis dengan Pendekatan Al-Qur’an \| 243 243 Jumhur ulama membedakan antara orang kafir asli dan orang yang masuk Islam, kemudian murtad. Mereka menjadikan kekafiran baru (murtad) lebih berat karena sebelumnya telah masuk Islam. Oleh karena itu, ia tetap dibunuh jika kemudian murtad. Adapun Jamāl menilai bahwa makna hadis ini sebenarnya bertentangan dengan nilai-nilai al-Qur’an,59 yaitu al-Qur’an surat al-Kahfi [18] ayat 29: َ َوَقُلِ اْلَْقُّ مِنْ رَبِّكُمْ فَمَنْ شَاءَ ف َلْي ُؤْمِنْ وَمَنْ شَاءَ ف َلْيَكْفُرْ إِنَّا أَعْتَدْنَا لِلظَّالِمِنيَ نَارًا أ َحَاط َِبِِمْ سُرَادِق ُهَا وَإِنْ يَسْتَغِيثُوا ي ُغَاثُوا ِبَِاءٍ كَالْمُهْلِ يَشْوِي الْوُجُوهَ بِئْس الشَّرَابُ وَسَاءَتْ مُرْت َفَقًا Artinya: “Dan katakanlah: "Kebenaran itu datangnya dari Tuhanmu;maka barangsiapa yang ingin (beriman) hendaklah ia beriman, danbarangsiapa yang ingin (kafir) biarlah ia kafir". Sesungguhnya Kami telah sediakan bagi orang orang zalim itu neraka, yang gejolaknya mengepung mereka. Dan jika mereka meminta minum, niscaya mereka akan diberi minum dengan air seperti besi yang mendidih yang menghanguskan muka. Itulah minuman yang paling buruk dan tempat istirahat yang paling jelek” Ayat ini menjelaskan tentang kebebasan dalam berkeyakinan dalam mengimani sesuatu, tanpa adanya hukuman di dunia, melainkan balasan di akhirat nanti, ayat ini secara tegas bertentangan dengan hadis yang telah dise- butkan di atas yaitu hukuman langsung bagi orang yang mengganti agamanya. Jamāl menambahkan bahwa, hadis-hadis yag menegaskan hukuman mati bagi orang yang murtad, apalagi sikap Nabi terhadap orang yang murtad, bahwasannya Nabi tidak menghukum orang-orang murtad, dalam hal ini Jamāl mengutip pendapat Muḥammad Dzakī Ibrāhim dalam buku yang berjudul al- Salāfiyyah al-Mu’aṣirah ilā Ayna, dalam buku tersebut dijelaskan bahwa di jaman Nabi terdapat orang yang sering berganti agama atau murtad berkali-kali. Namun tidak satupun dari mereka yang dibunuh. Bahkan salah satu juru tulis Nabi adalah orang yang murtad dan Nabi pun membiarkannya pergi.60 Begitulah pandangan Jamāl bahwa menggunakan perspektif al-Qur’an dalam mengkaji sunnah akan menjadikan al-Qur’an sebagai “hakim tunggal”, mengunakan al-Qur’an untuk menyelesaikan permasalahan merupakan langkah awal menuju terciptanya pendekatan yang objektif. Secara sederhana dapat dikatakan, yang sesuai dengan al-Qur’an itulah dari Nabi. Sementara yang tidak, hal itu bukan dari Nabi.61 Selain mengkritisi matan hadis secara langsung dengan dihadapkannya hadis dengan al-Qur’an, Jamāl juga mengkritisi kesalahan para ulama dalam memahami makna hadis dengan pendekatan al-Qur’an, di antaranya adalah di dalam kitabTabsih al-Ummah bi Ḥaqīqat al-Sunnah. b. Teori dan Aplikasi Kritik Matan Hadis Di dalam kitab ini, terda- DOI: 10.15408/ref.v18i2.11272 244 \| REFLEKSI,Volume 18, Nomor 2, Oktober 2019 244 pat hadis Imām al-Bukhārī Nomor 6556 dan Hadits Ibnu Mājah No.3954, yang artinya adalah “bila pedang dua orang Islam bertemu, kaduanya masuk neraka. Saya berkata kepada Nabi, ‘yang membunuh, iya, bagaimana dengan yang terbunuh? Nabi menjawab, ‘karena dia berusaha membunuh kawannyya.”. ِحَدَّث َنَا عَبْدُ الرَّْحَْنِ بْنُ الْمُبَارَكِ حَدَّث َنَا ْحََّادُ بْنُ زَيْدٍ حَدَّث َنَا أَيُّوبُ وَيُونُسُ عَنْ اْلَْسَن ْعَن اِلَْحْنَفِ بْنِ ق َيْسٍ قَالَذَهَبْتُ ِلَِنْصُرَ هَذَا ُالرَّجُلَ ف َلَقِيَِنِ أَبُو بَكْرَةَ ف َقَالَ أَيْنَ تُرِيدُ ق ُلْت َأَنْصُرُ هَذَا الرَّجُلَ قَالَ ارْجِ عْ ف إِِّنِّ ْسَِعْتُ رَسُولَ اللَّهِ صَلَّى اللَّهُ عَلَيْهِ وَسَلَّمَ ي َقُولُ إِذَا الْت َقَى ُالْمُسْلِمَانِ بِسَي ْفَيْهِمَا فَالْقَاتِلُ وَالْمَقْتُول ُِفِ النَّارِ ف َقُلْتُ يَا رَسُولَ اللَّهِ هَذَا الْقَاتِلُ فَمَا بَال ِالْمَقْتُولِ قَالَ إِنَّهُ كَانَ حَرِيصًا عَلَى ق َتْل ِصَاحِبِه62 Penulis dalam kitab tersebut menjelaskan bahwa hadis ini mengguncang tatanan hukum yang ada di dalam al-Qur’an, yaitu bahwa seseorang yang berbuat kejahatan juga berhak mendapatkan balasan yang setimpal, yang kemu-dian bertentangan dengan ayat al-Qur’an Surat al-Hujarāt ayat 9. Bagi Jamāl, pengarang kitab ini telah keliru dalam memahami makna hadis yang kemudian berdampak pada penolakan hadis tersebut karena diduga bertentangan dengan al-Qur’an, menurut Jamāl, makna hadis di atas adalah agar perbedaan pendapat di antara umat Islam tidak diselesaikan dengan pedang, melainkan dengan pendekatan yang baik dan sesuai dengan yang tertetra di dalam al-Qur’an,63yaitu QS. al-Ḥujarāt [49]: ayat 9: ُِن َوَإِنْ طَائِفَتَانِ مِنَ الْمُؤْمِنِنيَ اق ْتَت َلُوا فَأَصْلِحُوا ب َي ْن َهُمَا فَإِنْ ب َغَتْ إِحْدَاُهَُا عَلَى اِلُْخْرَى ف َق اتِلُوا ِالَِّتِ ت َبْغِي حََّتَّ تَفِيءَ إَِلَ أَمْر َاللَّهِ فَإِنْ فَاءَتْ فَأَصْلِحُوا ب َي ْن َهُمَا بِالْعَدْلِ وَأَقْسِطُوا إِنَّ اللَّه َُيُِبُّ الْمُقْسِطِني نُُ Artinya: “Dan jika ada dua golongan dari orang-orang mukminberperang maka damaikanlah antara keduanya. Jika salah satu dari kedua golongan itu berbuat aniaya terhadap golongan yang lain maka perangilah golongan yang berbuat aniaya itu sehingga golongan itu kembali kepada perintah Allah: jika golongan itu telah kembali, maka damaikanlah antara keduanya dengan adil dan berlaku adillah. Sesungguhnya Allah menyukai orang-orang yang berlaku adil.”. Jamāl memahami hadis di atas dengan berlandaskan al-Qur’an sehingga mendapati kesimpulan bahwa hukuman tidak berlaku untuk kedua-duanya, melainkan hanya berlaku kepada yang bersalah. Kesimpulan Pertama, baik al-Ghazālī maupun Jamāl, keduanya sama-sama meyakini bahwa, adanya hadis-hadis yang dinyatakan sahih pada sanadnya, namun cacat pada matannya, hanya saja, Jamāl dalam memahami hadis hanya membatasi pada apa yang disandarkan kepada rasul saja, berbeda dengan al-Ghazālī yang tidak hanya menerima apa yang disandarkan kepada rasul, melainkan juga menerima apa yang disandarkan kepada sahabat maupun tabi’in. Kedua, al-Ghazālī dan Jamāl dalam mengkritisi hadis dengan mengguna- kan metode perbandingan al-Qur’an, keduanya melakukan cara yang sama yang pernah dilakuan oleh ulama terdahulun, namun dengan sedkit modifikasi yang berbeda, sehingga antara keduanya dengan metode ulama terdahulu mempu-nyai perbedaan dari segi penggunaan metode pemahaman hadis. Ketiga, implikasi dari penggunaan al-Qur’an sebagai bagian dari cara me- mahami matan hadis, menghasilkan beberapa kesimpulan, di antaranya, adalah, tidak menggunakan hadis-hadis yang diduga bertentangan dengan al-Qur’an, al- Ghazālī dan Jamāl sepakat dalam hal ini. b. Teori dan Aplikasi Kritik Matan Hadis DOI: 10.15408/ref.v18i2.11272 DOI: 10.15408/ref.v18i2.11272 245 Agung Abdillah dan Rizal Alwi Mampa, Kritik Matan Hadis dengan Pendekatan Al-Qur’an \| 245 Dalam hal ini, Jamāl menunjukkan kehati-hatiannya dalam mengkritisi makna hadis, hadis yang secara lafaz dipahami bertentangan dengan al-Qur’an tidak lantas kemudian langsung dikritik dengan al-Qur’an, akan tetapi terlebih dahulu mencari makna hadis itu dengan sebenar-benarnya, sehingga tidak lantas langsung mengkritisi hadis dengan al-Qur’an. Dari ketiga contoh kritik matan hadis di atas, maka dapat dilihat bahwa Jamāl dalam mengkritisi matan hadisdengan pendekatan al-Qur’an mempunyai beberapa cara tersendiri, di antaranya, mengkritisi matan hadis dengan mem- bandingkan isi matan hadis tersebut dengan al-Qur’an, melihat matan hadisdari berbagai disiplin ilmu, seperti dalam memahami hadis tentang kalalah yang ke- mudian diperbandingkan dengan nilai-nilai yang terkandung di dalam al-Qur’an, kemudain yang terakhir, meluruskan kesalahpahaman dalam mema-hami matan hadis yaitu dengan melihat kembali maksud hadis tersebut dengan maksud dari ayat-ayat al-Qur’an. DOI: 10.15408/ref.v18i2.11272 Catatan Akhir Catatan Akhir DOI: 10.15408/ref.v18i2.11272 246 \| REFLEKSI,Volume 18, Nomor 2, Oktober 2019 246 1. Hadis merupakan terminologi dari Sunnah yang juga dapat diartikan sebagai sesuatu yang diriwayatkan dari Rasulullah saw., baik berupa perkataan, perbuatan maupun Taqrir. Lihat M. Ajjāj al-Khattib, Uṣūl al-Ḥādīth (Beirut: Dār al-Fikr, 1986), 27. 2. Bustamin dan M. Isa H. A. Salam, Metodologi Kritik Hadis (Jakarta: Rajawali Pers,2004), Cet ke-1, 1. 3. Nūr al-Dīn ‘itr, Manhaj an-Naqd fī ‘Ulūm al-Hādīth (Beirut: Dār al-Fikr, 1997), 274. 4. Bustamin dan M. Isa H. A. Salam, Metodologi Kritik Hadis (Jakarta: Rajawali Perss, 2004), Cet ke-1, 22. 5. Jamāl al-Bannā, al-Sunnah fī al- Fiqh al-Jadīd (Kairo: Dār al-Fikr al-Islāmī, t.t), 10. 6. Jamāl al-Bannā, al-Sunnah, 61. 7. Jamāl al-Bannā, al-Sunnah, 118-121. J 8. Jamāl al-Bannā, al-Ashlānī al-‘Azimanī: al-Kitāb wa al-Sunnah (Kairo: Dār al-Fikr al-Islāmī, 1978), 234. 9. Jamāl al-Bannā, al-Sunnah, 115. 10. Jamāl al-Bannā, al-Sunnah, 118 11. Jamāl al-Bannā, al-Islām Kamā Tuqaddimu Da’wa al-Iḥyā al-Islāmī (Kairo: Dār al-Fikr al- Islāmī, 2004), 86. 12. Yūsuf al-Qarḍāwī, Syeikh Muḥammad al-Ghazālī yang Saya Kenal, 7. 13. Muḥammad al-Ghazālī, Studi Kritis Atas Hadis, 26 14. Muḥammad al-Ghazālī, Fiqh al-Sirah (Kairo: Dār al-Kutūb, t.t), 38. 15. Muḥammad al-Ghazālī, al-Sunnah al-Nabawiah bain Ahl Fiqh wa Ahl al-Hādīth (Kairo: Dār al-Syurūq, 1996), 8. 16. Muḥammad al-Ghazālī, Studi Kritis Atas Hadis, 26. 17. Muḥammd al-Ghazālī, Dustur al-Wahdah al-Thaqāfiyah bain al-Muslimīn, 67-71. 18. Buhairah, atau yang biasa dikenal dengan Bahirah, salah satu kota di Mesir yang banyak melahirkan tokoh-tokoh islam terkemuka, seperti, Muḥammad ‘Abdūh, Syeikh Salīm al- Bishrī, Syaikh Ibrāhīm al-Hamrusī, Syaikh Maḥmūd Shaltūt, Syaikh Ḥasan al-Bannā, dll. Lihat. Muḥammad al-Ghazālī, Berdialog dengan al-Qur’an, terj. Masykur Hakim dan Ubaidillah (Bandung: Mizan, 1997), 5. 19. Muḥammad Syalabī, al-Syeikh al-Ghazālī wa Marakatu al-Muṣḥaf fī al-Alām al-Islāmī (Kairo: Dār al-Syahwa, 1987), Cet ke-1, 22. 20. Ahmad Muzayyin, PemikiranMuḥammad al-Ghazālī tentang Hadis Aḥad (Jakarta: UIN Jakarta, 2003), 12. 21. Muḥammad Syalabī, al-Syeikh al-Ghazālī wa Marakatu al-Muṣḥaf, 22. 22. Syeikh Muḥammad al-Ghazālī, Berdialog dengan al-Qur’an, 6. 23. Syeikh Muḥammad al-Ghazālī, Berdialog dengan al-Qur’an, 7. 24. Badri Khaeruman, Otentitas Hadis: Studi Kritis atas Kajian Hadis Kontemporer, (Bandun Rosdakara, 2004), 65 25. Syeikh Muḥammad al-Ghazālī, Berdialog dengan al-Qur’an, 9. 25. Syeikh Muḥammad al-Ghazālī, Berdialog dengan al-Qur’an, 9. 26. Jamāl al-Bannā, Manifesti Fiqh Baru (Jakarta: Erlangga, 2008), 9 27. Jamāl al-Bannā, Tajdīd al-Islām wa I’adah Ta’sisi Manzūmat al-Ma’rifah al-Islāmiyy (Kairo: Dār al-Fikr al-Islāmī, t.t). 81. 28. M. Zamzami, Pemikiran Jamāl al-Bannā tentang Relasi Agama dan Negara, 18-20. Catatan Akhir DOI: 10.15408/ref.v18i2.11272 Agung Abdillah dan Rizal Alwi Mampa, Kritik Matan Hadis dengan Pendekatan Al-Qur’an \| 247 29. Jamāl al-Bannā, Kalla Summa Kalla: Kalla li Fuqaha’ al-Taqlid wa Kalla li Du’at al-Tanwir (Kairo : Dār al-Fikr al-Islāmī,t.t), 263. 30. Muḥammad al-Ghazālī, Fiqh Sirah, 37. 31. Muḥammad al-Ghazālī, Fiqh Sirah, 38. 32. Muḥammad al-Ghazālī, al-Sunnah al-Nabawiyyah bain Ahl al-Fiqh wa Ahl-Ḥādīth, 8-19 32. Muḥammad al Ghazālī, al Sunnah al Nabawiyyah bain Ahl al Fiqh wa Ahl Ḥādīth, 8 19. 33. Muḥammad al-Ghazālī, Dustur al-Wahdah al-Tsaqafiyyah bain al-Muslimin, 67-71. 33. Muḥammad al-Ghazālī, Dustur al-Wahdah al-Tsaqafiyyah bain al-Muslimin, 67-71 34. Badri Khaeruman, Otentitas Hadis: Studi Kritis atas Kajian Hadis Kontemporer, 265 35. Badri Khaeruman, Otentitas Hadis: Studi Kritis atas Kajian Hadis Kontemporer, 271 36. Muḥammad al-Ghazālī, Studi Kritis atas Hadis Nabi saw. antara pemahaman Tekstualda Kontekstual, terjemahan Muhammad Baqir, (Bandung: Mizan, 1993), 32-33. 37. Muḥammad al-Ghazālī, Analisis Polemik Hadis: Transformasi Modernisasi, terj. Muh. Munawwir az-Zahidi (Surabaya: Dunia Ilmu, 19976), cet. Ke-1, 4. 38. Muḥammad al-Ghazālī, Analisis Polemik Hadis: Transformasi Modernisasi, 3. 39. Hadis Riwayat Muslim dari‘Abdullāh bin Abū Mulaikah, Ṣaḥīḥ Muslim, juz III, 42, hadits no. 2188. 40. Muḥammad al-Ghazālī, Analisis Polemik Hadis, h 4. (mayat itu diazab karena ratapan keluarganya) Hadis riwayat‘Abdullāh bin‘Umar, lihat shahih Bukhari, Ṣaḥīḥ al-Bukhārī (Mesir: Dār al-Kutūb al-Ilmiah, 2008), juz ke-IV, h 435. Lihat juga hadits Riwayat, Ṣaḥīḥ Muslim, juz III, hal. 42, hadits no. 2188. 41. Muḥammad al-Ghazālī, Analisis Polemik Hadis: Transformasi Modernisasi (Surabaya: Dunia Ilmu, 1997), 6. 42. Muḥammad al-Ghazālī, Analisis Polemik Hadis: Transformasi Modernisasi, 11. 43. Muḥammad al-Ghazālī, Analisis Polemik Hadis, 117. 44. Muḥammad al-Ghazālī, Analisis Polemik Hadis: Transformasi Modernisasi, 17 45. Jamāl al-Bannā, al-Sunnah, h 10. Lihat juga, Jamāl al-Bannā, Manifesto Fiqh Baru: Redefinisi dan Reposisi al-Sunnah (Jakarta: Erlangga, 2008), 2. 46. Jamāl al-Bannā, al-Sunnah, 11; Jamāl al-Bannā, al-Ashlānī al-‘Azimanī, 234. 47. Jamāl al-Bannā, al-Sunnah, 171; Jamāl al-Bannā, al-Ashlānī al-‘Azimanī, 212 48. Jamāl al-Bannā, al-Sunnah, 162-165 49. Jamāl al-Bannā, Naḥwa Fiqh Jadīd, 279 50. Jamāl al-Bannā, Manifesto Fiqh Baru: Redefinisi dan Reposisi al-Sunnah (Jakarta: Erlangga, 2008), 86. 51. Jamāl al-Bannā, Manifesto Fiqh Baru: Redefinisi dan Reposisi al-Sunnah, 94 52. Jamāl al-Bannā, Manifesto Fiqh Baru: Redefinisi dan Reposisi al-Sunnah, 98. 53. Jamāl al-Bannā, al-Ashlani al-’Azhimani; al-Kitab wa al-Sunnah, 215. Lihat juga Jamāl a Bannā, Manifesto Fiqh Baru, jilid ke-2, 224 54. Umma Farida, “Metode Komparasi Antara Hadis dengan Al-Quran: Telaah Atas Pemikiran Jamāl al-Bannā tentang Kritik Matan,” (Tesis S2 Pascasarjana UIN Syarif Hidayatullah Jakarta, 2005), h. Agung Abdillah dan Rizal Alwi Mampa, Kritik Matan Hadis dengan Pendekatan Al-Qur’an \| 247 Catatan Akhir 113. Lihat juga, Jamāl al-Bannā, Naḥwa Fiqh Jadid , jilid 3, 280 55. Jamāl al-Bannā, al-Sunnah, 248 55. Jamāl al-Bannā, al-Sunnah, 248 56. Jamāl al-Bannā, “Manifesto Fiqh Baru”, Jilid ke-2, 226 57. Jamāl al-Bannā, “Manifesto Fiqh Baru”, Jilid ke-2, 226. DOI: 10.15408/ref.v18i2.11272 248 \| REFLEKSI,Volume 18, Nomor 2, Oktober 2019 248 58. Mūsā Syahin, Taysīr Saḥīḥ al-Bukhārī (Maktabah al-Syurūq al-Dauliyah, 2003 M) jilid ke- 2, 337. 59. Jamāl al-Bannā, Tatswir al-Quran”, h. 72. Lihat juga, Ummu Farida, “Metode Komparasi Antara Hadis Dengan Al-Qur’an: Telaah atas Pemikiran Jamāl al-Bannā tentang Kritik Matan” skripsi, UIN Syarif Hidayatulllah Jakarta, 2005, 113. 60. Jamāl al-Bannā, Manifesto Fiqh Baru 3: Memahami Paradigma Fiqih Moderat, (Jakarta: Erlangga, 1997), 17. 61. Jamāl al-Bannā, Manifesto Fiqh Baru 2: Redefiinisi dan Reposisi al-Sunnah, 144 62. Musa Syahin, Taysir Saḥīḥ al-Bukhārī, 33. 62. Musa Syahin, Taysir Saḥīḥ al-Bukhārī, 33. 63. Jamāl al-Bannā, Manifesto Fiqh Baru, jilid ke-2, 212. 63. Jamāl al-Bannā, Manifesto Fiqh Baru, jilid ke-2, 212. 58. Mūsā Syahin, Taysīr Saḥīḥ al-Bukhārī (Maktabah al-Syurūq al-Dauliyah, 2003 M) jilid ke- 2, 337. Daftar Pustaka Al-Bannā, Jamāl, al-Ashlāni al-‘Azhīmāni; Al-Kitāb wa al-Sunnah, Cairo: Dār al- Fikr al-Islāmī, 1978. Fikr al-Islāmī, 1978. ---------. al-Islām kamā Tuqaddimuhu Da‘wah al-Ihyā’ al-Islāmī, Cairo: Dār al- Fikr al-Islāmī, 2004. ---------. al-Islām kamā Tuqaddimuhu Da‘wah al-Ihyā’ al-Islāmī, Cairo: Dār al- Fikr al-Islāmī, 2004. ---------. Al-Sunnah fī al- Fiqh al-Jadīd, Cairo: Dār al-Fikr al-Islāmī, t.t. ---------. Naḥwa Fiqh Jadīd, Cairo: Dār al-Fikr al-Islāmī, 1999. ---------. Tatswīr al-Qur’ān, Cairo: Dār al-Fikr al-Islāmī, t.t. Bustamin dan M. Isa H. A. Salam, Metodologi Kritik Hadis, Jakarta: Rajawali Pers, 2004. Al-Ghazālī, Muḥammad, Berdialog dengan al-Qur’an. Penerjemah Masykur Hakim dan Ubaidillah, Bandung: Mizan, 1997. ---------. al-Sunnah al-Nabawiah bain ahl Fiqh wa Ahl al-Ḥādīth, Kairo: Dār al- Syurūq, 1996. ---------. Analisis Polemik Hadis: Transformasi Modernisasi, Penerjemah Muh. Munawwir az-Zahidi, Surabaya: Dunia Ilmu, 1997. --------- Fiqh al-Sirah Kairo: Dār el-Kutūb t t ---------. Fiqh al-Sirah, Kairo: Dār el-Kutūb, t.t. Ismail, M. Syuhudi. Sunnah Nabi Menurut Pembela, Pengingkar dan Pemalsunya, Jakarta: Gema Insani, 1995. Al-Khathīb, M. Ajjāj, Uṣūl al-Ḥādīth: Ulūmuh wa Mushṭalaḥuh, Beirut: Dār al- Fikr, 1986. Khaeruman, Badri.Otentitas Hadis: Studi Kritis atas Kajian Hadis Kontemporer, Bandung: Rosdakara, 2004. Al-Qaṭṭān, Mannā‘, Mabāhith fī ‘Ulūm al-Ḥādīth, Cairo: Maktabah Wahbah, 1992. Al-Qarḍāwī, Yusuf. Bagaimana Memahami Hadis Nabi saw., Bandung: Karisma, 1993. DOI: 10.15408/ref.v18i2.11272 DOI: 10.15408/ref.v18i2.11272
https://openalex.org/W3012285633	https://periodicos.unifor.br/RBPS/article/download/9473/pdf	English	null	Prevalence of osteoporosis in older postmenopausal women	Revista Brasileira em Promoção da Saúde/Revista brasileira em promoção da saúde	2,019	cc-by	5,886	ABSTRACT Objective: To identify the prevalence of osteoporosis in postmenopausal women and its association with risk factors. Methods: Cross-sectional study with 115 older postmenopausal women who participated in the extension project of the Federal District University Center lasting one year, starting in 2017. Bone mineral density was measured using Dual-energy X-ray absorptiometry technique on lumbar spine (L1-L4) and femoral neck. Risk factors for low density were evaluated through interviews. Statistical analysis was performed using the Chi-squared test (p<0.05). Results: The mean age was 67.8 ± 8.4 years and mean time since menopause was 16.7 ± 6.2 years. Mean bone mineral density was -0.96 ± 1.42 in the femoral neck and -1.25 ± 1.75 in the lumbar spine (L1-L4). The prevalence of low density was 66.9% for the lumbar spine and 52.1% for the femoral neck. We found a significant difference in age (50.5% between 51 and 55 years and low density), physical inactivity (82.9%), personal history of fracture in the last 5 years (31.2% with low density) and body mass index - overweight among women with normal bone mineral density (44.7%) when compared with those with low density (p<0.001). Conclusion: Postmenopausal women had a high prevalence of low bone mineral density and associated risk factors. Descriptors: Bone Density; Aging; Health Promotion. Osteoporosis in postmenopausal women DOI: 10.5020/18061230.2019.9473 Original Article Osteoporosis in postmenopausal women DOI: 10.5020/18061230.2019.9473 Original Article PREVALENCE OF OSTEOPOROSIS IN OLDER POSTMENOPAUSAL WOMEN PREVALENCE OF OSTEOPOROSIS IN OLDER POSTMENOPAUSAL WOMEN Prevalência de osteoporose em mulheres idosas na pós-menopausa Prevalencia de osteoporosis en mujeres mayores en la postmenopausia Prevalência de osteoporose em mulheres idosas na pós-menopausa Prevalencia de osteoporosis en mujeres mayores en la postmenopausia Prevalência de osteoporose em mulheres idosas na pós-menopausa Prevalencia de osteoporosis en mujeres mayores en la postmenopausia Milenne da Silva Spinola Federal District University Center (Centro Universitário do Distrito Federal - UDF) - Brasília (DF) - Brazil Maria de Lourdes Alves Carneiro Federal District University Center (Centro Universitário do Distrito Federal - UDF) - Brasília (DF) - Brazil José Maria Thiago Bonardi Ribeirão Preto Medical School - University of São Paulo (Faculdade de Medicina de Ribeirão Preto - Universidade de São Paulo - USP) - Ribeirão Preto (SP) - Brazil Bárbara Katherine Ataide Barros Rodrigues Federal District University Center (Centro Universitário do Distrito Federal - UDF) - Brasília (DF) - Brazil Luciana Zaranza Monteiro Federal District University Center (Centro Universitário do Distrito Federal - UDF) - Brasília (DF) - Brazil Milenne da Silva Spinola Federal District University Center (Centro Universitário do Distrito Federal - UDF) - Brasília (DF) - Brazil Maria de Lourdes Alves Carneiro Federal District University Center (Centro Universitário do Distrito Federal - UDF) - Brasília (DF) - Brazil José Maria Thiago Bonardi Ribeirão Preto Medical School - University of São Paulo (Faculdade de Medicina de Ribeirão Preto - Universidade de São Paulo - USP) - Ribeirão Preto (SP) - Brazil Bárbara Katherine Ataide Barros Rodrigues Federal District University Center (Centro Universitário do Distrito Federal - UDF) - Brasília (DF) - Brazil Luciana Zaranza Monteiro This Open Access article is published under the a Creative Commons license which permits use, distribution and reproduction in any medium without restrictions, provided the work is correctly cited RESUMEN Objetivo: Identificar la prevalencia de osteoporosis en mujeres en la postmenopausia y su asociación con factores de riesgo. Métodos: Estudio transversal realizado con 115 mujeres mayores en la postmenopausia que participaron del proyecto de extensión del Centro Universitario del Distrito Federal con duración de un año e inicio en 2017. Se ha mensurado la densidad mineral ósea por el Dual-energy X-ray absorptiometry en la columna lumbar (primera vértebra lumbar hasta la cuarta) y el cuello femoral. Se evaluaron los factores de riesgo para la densidad baja a través de entrevista. Se utilizó la prueba de chi-cuadrado de Pearson (p<0,05) para el análisis estadístico. Resultados: La media de edad encontrada ha sido de 67,8 ± 8,4 años y el tiempo de menopausia de 16,7 ± 6,2 años. La media de la densidad mineral ósea ha sido de -0,96 ± 1,42 para el cuello femoral y de -1,25 ± 1,75 para la columna lumbar (L1-L4). La prevalencia de densidad baja es del 66,9% para la columna lumbar y del 52,1% para el cuello femoral. Se encontró diferencia significativa para la edad (50,5% tenían entre 51 y 55 años y baja densidad), la inactividad física (82,9%), la historia personal de fractura en los últimos 5 años (31,2% con densidad baja) y se observó el índice de masa corporal – sobrepeso entre mujeres de densidad mineral ósea normal (44,7%) comparadas con aquellas de densidad baja (p<0,001). Conclusión: Las mujeres en la postmenopausia presentaron elevada prevalencia de densidad mineral ósea baja y los factores de riesgo asociados. Descriptores: Densidad Ósea; Envejecimiento; Promoción de la Salud. Descriptores: Densidad Ósea; Envejecimiento; Promoción de la Salud. RESUMO Objetivo: Identificar a prevalência de osteoporose em mulheres na pós-menopausa e sua associação com fatores de risco. Métodos: Estudo transversal realizado com 115 idosas na pós-menopausa que participavam do projeto de extensão do Centro Universitário do Distrito Federal, com duração de um ano, com início em 2017. Mensurou-se a densidade mineral óssea pelo Dual-energy X-ray absorptiometry em coluna lombar (lombar 1 a lombar 4) e colo do fêmur. Por meio de entrevista, avaliaram-se fatores de risco para baixa densidade. Na análise estatística, utilizou-se o teste qui-quadrado de Pearson(p<0,05). Resultados: A média de idade encontrada é de 67,8 ± 8,4 anos e o tempo de menopausa de 16,7 ± 6,2 anos. A média de densidade mineral óssea é de -0,96 ± 1,42 no colo do fêmur e de -1,25 ± 1,75 na coluna lombar (L1-L4). A prevalência de baixa densidade é de 66,9% para coluna lombar e de 52,1% para colo de fêmur. Encontrou-se diferença significativa na idade (50,5% tinham entre 51 a 55 anos e baixa densidade), inatividade física (82,9%), história pessoal de fratura nos últimos 5 anos (31,2% com baixa densidade) e observou-se o índice de massa corpórea - sobrepeso entre mulheres com densidade mineral óssea normal (44,7%) quando comparadas àquelas com baixa densidade (p<0,001). Conclusão: As mulheres na pósmenopausa apresentaram elevada prevalência de baixa densidade mineral óssea e fatores de risco associados. Descritores: Densidade Óssea; Envelhecimento; Promoção da Saúde. Received on: 05/18/2019 Accepted on: 11/21/2019 Received on: 05/18/2019 Accepted on: 11/21/2019 Received on: 05/18/2019 Accepted on: 11/21/2019 This Open Access article is published under the a Creative Commons license which permits use, distribution and reproduction in any medium without restrictions, provided the work is correctly cited 1 Rev Bras Promoç Saúde. 2019;32:9473 Spinola MS, Carneiro MLA, Bonardi JMT, Rodrigues BKAB, Monteiro LZ Descriptores: Densidad Ósea; Envejecimiento; Promoción de la Salud. INTRODUCTION Osteoporosis is a systemic bone disease characterized by loss of bone mass and deterioration of bone microarchitecture and bone quality leading to an increased risk of fragility fractures. Since there are no obvious symptoms of osteoporosis, this condition is often diagnosed after the occurrence of a fragility fracture. Thus, osteoporotic fractures highly burden the healthcare system in terms of increased hospitalizations, surgeries, and prolonged home care and rehabilitation requirements(1). For example, a large study conducted in the United States over a 12-year period (2000–2011) in postmenopausal women ≥55 years of age found that the hospitalization costs for osteoporotic fractures were greater than those for other serious conditions such as stroke, myocardial infarction and breast cancer(2). In the Latin America (LA) region, osteoporosis and osteoporotic fractures continue to be a major healthcare concern(1). The Latin American Vertebral Osteoporosis Study (LAVOS) conducted in five countries across LA with women ≥50 years old found that the standardized prevalence of radiographic vertebral fractures was 11.18 (95% confidence interval [CI] 9.23-13.4)(3). Chronic degenerative diseases appear with aging and there is delayed rehabilitation and signs of disease at advanced stages, which generally compromises older adults’ functionality and quality of life(4). Within this process, there is also a reduction in muscle mass and muscle strength, fatigue, alterations of gait and balance, loss of appetite and a consequent reduction in weight(5). According to the Brazilian Guidelines for the diagnosis and treatment of osteoporosis in postmenopausal women, the most important risk factors related to osteoporosis and postmenopausal fractures are: female gender, White or Asian ethnicity, family history, early menopause, reduced ovarian function before menopause (athlete’s amenorrhea, hyperprolactinemia, anorexia nervosa), poor diet (high caffeine intake, low calcium intake), poor life style (sedentary lifestyle, alcohol abuse, smoking) and fractures(4). Menopause is a period in which women’s health status is significantly fluctuating. Its average onset is at the age of 50. The global average life expectancy for women is 74 years(6), so the time after menopause accounts for nearly one-third of women’s lives. The secretion of estrogen rapidly declines after menopause and leaves postmenopausal women (PMW) at a higher risk of various physical and mental illnesses compared with men(7). Thus, osteoporosis ends up being a common disease in postmenopausal women due to age and hypoestrogenism, with an increased prevalence and incidence of fractures and hence a negative effect on the quality of life of such women(8). METHODS This quantitative cross-sectional study was conducted with 115 older women (out of 155) who participated in the extension project titled “Health Promotion and Healthy Aging” of the Federal District University Center (Centro Universitário do Distrito Federal - UDF) located in the Central region in western Brazil. Inclusion criteria for the present study were: people aged 60 or over with or without hysterectomy who were part of the extension project and whose last menstrual period occurred at least 12 months prior to recruitment. Postmenopause was characterized as absence of the menstrual cycle for the last 12 months(4). Volunteers presenting other conditions or diseases associated with alterations in bone mass were excluded from the study: patients who had undergone oophorectomy, had a history of kidney disease, history of endocrinal disorders or diseases (hyperthyroidism, hypothyroidism, diabetes, Cushing Syndrome, Addison’s Disease), history of heart or lung diseases, Paget’s Disease, auto-immune diseases (Graves’ Disease or Hashimoto’s Disease), history of use of medication associated with alterations in bone metabolism (Hormone Replacement Therapy, corticoids, thyroid hormones, heparin, warfarin, phenobarbital, phenytoin, carbamazepine, lithium or methotrexate, supplementation with calcium or vitamin D). The older women were first approached during meetings of the Health Promotion and Healthy Aging project, which were carried out twice a week for 2 hours/day and included lectures on health promotion and disease prevention. During the meetings, the older women were informed about the objective of the study and invited to participate. Data were collected between September 2017 and February 2018 after signature of the Consent Form. Data were collected through interviews in which the researcher applied a questionnaire with questions addressing the following variables: age, race, gynecological history (ages of menarche and menopause, time since menopause in years, presence of and time since hysterectomy in years), practice of physical activity, current smoking habits, osteoporosis and fractures (history of nontraumatic fractures in the last 5 years, 1st degree relative with osteoporosis). Women that did moderate aerobic exercise for at least 30 minutes five times a week (150 min/week) or did strength training three days a week were considered active(12). After completing the questionnaires, the older women were asked to come to the Physiology Laboratory of the Federal District University Center to carry out BD tests on the proximal femur and lumbar spine (L1-L4) using the Dual-energy X-ray absorptiometry technique (DEXA) and a Hologic QDR-2000 scanner. INTRODUCTION Thus, osteoporosis ends up being a common disease in postmenopausal women due to age and hypoestrogenism, with an increased prevalence and incidence of fractures and hence a negative effect on the quality of life of such women(8). Every postmenopausal woman should be assessed for risk of fractures resulting from osteoporosis as women i thi h f lif ith di i f t i b t ith t h i i d f t h lit Every postmenopausal woman should be assessed for risk of fractures resulting from osteoporosis as women in this phase of life with a diagnosis of osteoporosis – but without having experienced fractures – may have a quality of life similar to postmenopausal women without osteoporosis(9). There is enough evidence to state that bone densitometry (BD) is currently the most effective method to estimate fracture risk in postmenopausal women(10). Preventative measures are especially important given that the available Rev Bras Promoç Saúde. 2019;32:9473 2 Osteoporosis in postmenopausal women treatments may conserve bone mass but cannot restore the osteoporotic bone to normality(11). Therefore, the objective of this study was to identify the prevalence of osteoporosis in postmenopausal women and its association with risk factors. treatments may conserve bone mass but cannot restore the osteoporotic bone to normality(11). Therefore, the objective of this study was to identify the prevalence of osteoporosis in postmenopausal women and its association with risk factors. Rev Bras Promoç Saúde. 2019;32:9473 The project was approved by the Research Ethics Committee of the Federal District University Center (Centro Universitário do Distrito Federal - UDF) under Approval No. 1.931.184. RESULTS The sample consisted of 115 older postmenopausal women who underwent bone densitometry. The mean age was 67.8 ± 8.4 years and the mean time since menopause was 16.7 ± 6.2 years. The mean BMD (in SD of T-score) was -0.96 ± 1.42 for the femoral neck and -1.25 ± 1.75 for the lumbar spine (L1-L4). ( ) Table I shows the distribution of risk factors for osteoporosis. Most of the women in the sample were White (76.5%), sedentary (63.5%) and had normal BMI (50.4%). A few participants reported smoking (27.9%). Table I shows the distribution of risk factors for osteoporosis. Most of the women in the sample were White (76.5%), sedentary (63.5%) and had normal BMI (50.4%). A few participants reported smoking (27.9%). Table I shows the distribution of risk factors for osteoporosis. Most of the women in the sample were White (76.5%), sedentary (63.5%) and had normal BMI (50.4%). A few participants reported smoking (27.9%). Table I - Distribution of risk factors for osteoporosis in older postmenopausal women. Brasília, Federal District, Brazil, 2018. Risk factor n % p value* Age (years) 60 to 65 34 29.5 0.05 66 to 70 57 49.6 71 to 75 24 20.9 Race White 88 76.5 <0.001 Mixed 19 16.5 Black 8 7.0 Body mass index Underweight 11 9.6 <0.001 Normal 58 50.4 Overweight 36 31.3 Obese 10 8.7 Age at Menopause (years) ≤ 45 12 10.4 <0.01 46 a 50 45 39.1 51 a 55 58 50.5 Physical activity Yes 42 36.5 0.03 No 73 63.5 Smoking Yes 32 27.9 0.02 No 83 72.1 Nontraumatic fractures (last 5 years) Yes 18 15.6 <0.001 No 97 84.4 1st degree relative with osteoporosis Yes 24 20.9 <0.001 No 91 79.1 Hysterectomy Yes 28 24.3 <0.001 No 87 75.7 * Chi-squared Test The prevalence rate of low BMD (osteopenia and/or osteoporosis) was 66.9% for the lumbar spine (L1-L4) and 52.1% for the femoral neck (Table II). The classification of the data collected from the T-score was made considering the Brazilian Guidelines for the diagnosis and treatment of osteoporosis in postmenopausal women(4). of risk factors for osteoporosis in older postmenopausal women. Brasília, Federal District, * Chi-squared Test The prevalence rate of low BMD (osteopenia and/or osteoporosis) was 66.9% for the lumbar spine (L1-L4) and 52.1% for the femoral neck (Table II). METHODS The criteria were computed in a single exam (the first exam performed) during the data collection period. The participants were classified according to the T-score and the criteria described in the Brazilian Guidelines for the diagnosis and treatment of osteoporosis in postmenopausal women(4): normal when ≥-1.0 SD, osteopenia when the value was between -1.0 and -2.4 SD, and osteoporosis when ≤-2.5 SD. The participants with total T-score values for spine and/or femoral neck <-1.0 SD (osteopenia and osteoporosis) were considered to have low bone mineral density (BMD). Weight measurement was taken using Filizola scales and height was measured using a stadiometer coupled to the scales. These anthropometric measurements were taken from all the participants – who wore light clothes and were barefoot during measurements – and were used to calculate Body Mass Index (BMI), which is the product of the division of body weight by the square of the height (W(Kg)/H(m)2). The data were classified according to the World Health Organization (WHO)(15) criteria, which classifies adult individuals with BMI < 18.5Kg/m2 as underweight, BMI between 18.5 and 24.9Kg/m2 as normal weight, BMI between 25 and 29.9 Kg/m2 as overweight, and BMI > 29.9Kg/ m2 as obese. The data were analyzed using the Stata software version 12.0 and underwent descriptive statistical analysis using measures of frequency and central tendency in addition to the Chi-squared test. The level of significance adopted was p<0.05. The project was approved by the Research Ethics Committee of the Federal District University Center (Centro Universitário do Distrito Federal - UDF) under Approval No. 1.931.184. 3 Rev Bras Promoç Saúde. 2019;32:9473 3 Spinola MS, Carneiro MLA, Bonardi JMT, Rodrigues BKAB, Monteiro LZ RESULTS The classification of the data collected from the T-score was made considering the Brazilian Guidelines for the diagnosis and treatment of osteoporosis in postmenopausal women(4). Rev Bras Promoç Saúde. 2019;32:9473 4 Osteoporosis in postmenopausal women Table II - Prevalence of low bone mineral density in older postmenopausal women. Brasília, Federal District, Brazil, 2018. DEXA Number of patients (%) Normal BMD Osteopenia Osteoporosis Low BMD* Lumbar spine (L1-L4) 38 57 20 77 (66.9) Femoral neck 55 50 10 60 (52.1) T-score < -1 SD (osteopenia + osteoporosis). DEXA: Dual-energy X-ray Absorptiometry; BMD: Bone Mineral Density Table II - Prevalence of low bone mineral density in older postmenopausal women. Brasília, Fe 2018. The clinical characteristics of the older postmenopausal women classified into normal or low BMD were subjected to statistical comparison and the data are shown in Table III. It There was an association of low BMD with age (p=0.04), White ethnicity (p=0.05), normal BMI (p<0.001), physical inactivity (p<0.001), smoking (p=0.03), nontraumatic fractures in the last five years (p<0.001), and having a first degree relative with osteoporosis (p=0.05). Table III - Association between risk factors and the profile of bone mineral density of older postmenopausal women (n=115). Brasília, Distrito Federal, Brazil, 2018. Table III - Association between risk factors and the profile of bone mineral density of older postmenopausal women (n=115). Brasília, Distrito Federal, Brazil, 2018. ation between risk factors and the profile of bone mineral density of older postmenopausal wome Distrito Federal, Brazil, 2018. Table III Association between risk factors and the profile of bone mineral density of older postmenopausal wome (n=115). Brasília, Distrito Federal, Brazil, 2018. Chi-squared Test. BMD: Bone Mineral Density DISCUSSION Actions concerning the care for older adults taken by the healthcare team are necessary to outline the sociodemographic profile of this population group so as to promote health and prevent and treat diseases in this period of life and facilitate older adults’ access to public services that allow quality treatment and prevention(14). In the present study, there was a high prevalence of osteopenia and osteoporosis among the older postmenopausal women. Densitometric diagnosis of osteopenia and/or osteoporosis in the lumbar spine and/or femoral neck was found in more than half of the women assessed. The prevalence of osteoporosis in postmenopausal women assessed in Indian studies ranged from 12% to 60% and it has been shown to increase with advancing age(15,16). Overall prevalence of osteoporosis in India is found to be higher than that reported in other countries, although studies have not been conducted in rural settings(17). As demonstrated by these studies, the prevalence of osteoporosis in Brazil varies significantly according to the study methodology. Some studies based the diagnosis of osteoporosis on bone densitometry data and others relied on participants self-reporting. In 2010, researchers conducted a cross-sectional study that included 4,332 women aged > 40 years in São Paulo (São Paulo Osteoporosis Study [SAPOS]). The diagnosis of osteoporosis was made by DEXA and the prevalence rate of postmenopausal osteoporosis was 33%(18). Women’s aging entails a decline in the functional capacity of various systems, including the bone system(5). Hormonal alterations in the menopause, especially estrogen deficiency, cause more bone resorption than bone formation, thereby leading to a reduction in bone mass and, consequently, osteoporosis(9). A study of 378 postmenopausal women found that 74% of them presented osteopenia and/or osteoporosis in the lumbar spine and/or femoral neck(19). A Brazilian study conducted in 2012 to evaluate the bone mass of 70 women aged 45-65 years found low BMD in the femoral neck (28.6%) and in the lumbar spine (45.7%)(20). Research shows that with advancing age there is a loss of balance between bone formation and resorption and that although age-related bone loss begins soon after peak bone mass it is more pronounced after the age of 65(21). The study conducted by the Women’s Health Initiative (WHI)(23) evaluated osteoporosis treatment and identified participant characteristics associated with treatment utilization after fracture or diagnosis of osteoporosis in the WHI. DISCUSSION Of the 17,803 women who reported a new diagnosis of osteoporosis or fracture in the interval between enrollment and their final WHI visit, 3,457 reported both fracture and new diagnosis of osteoporosis, 7.926 reported only fracture and 6,420 reported only new diagnosis of osteoporosis(23). A study that assessed the prevalence of osteoporosis in Brazilian women over 50 years of age through bone density measurement found a prevalence rate of 40%(24). In the United States, a study of 600 patients assessed at the Wayne State University in Detroit found a prevalence rate of 52%(2). Recently, a community-based study in Saudi Arabia revealed that 57% of the women presented low BMD (29.8% of them presented osteopenia and 27.2% presented osteoporosis)(25). Another study in South Korea reported a 32.3 prevalence rate of osteoporosis and a 49.9% prevalence rate of osteopenia in the female population(26). A more recent study of postmenopausal women found that 42.5% of them were osteoporotic and 44.9% were osteopenic(27). Several cohort studies have been conducted to assess the family history of osteoporosis and osteoporotic fractures. Researchers have reported that the risk of hip and wrist fracture increased by 50% and 64%, respectively, in the presence of parental history of osteoporotic fracture of the hip or wrist. A family history seems to increase the risk of fracture independently of BMD(28). Other researchers found a negative correlation between a fracture history in a sister and 10-year fracture-free survival in perimenopausal women(29). A cohort study found osteopenia in 51% of the Caucasian women aged 60 to 89 years with a family history of osteoporosis(30). Studies have shown the importance of ethnicity as a factor related to osteoporosis, thus indicating that Black women are at a lower risk of osteoporosis, unlike White and Asian women(31). Researchers have also demonstrated that the prevalence of osteoporosis and osteopenia is higher in White race or White skin individuals(32). In the present study, the individuals with White skin showed greater bone loss (osteopenia and osteoporosis). There is evidence that exercise can prevent some of the complications associated with menopause, such as bone loss, loss of physical fitness and increased risk of osteoporosis(33). Physical exercise effectively decreases risk factors for falling and improves balance(33). Few cross-sectional studies have investigated associations between physical activity and osteoporosis. However, these studies focused on occupational physical activity and other lifestyle factors rather than recreational sports or physical activity(34). RESULTS Low BMD Normal BMD p-value Risk Factor n (%) n (%) Age (years) 60 to 65 10 (15.1) 24 (48.9) 0.04* 66 to 70 39 (59.1) 18 (36.7) 71 to 75 17 (25.8) 7 (14.4) Race White 64 (82.1) 24 (64.9) 0.05* Mixed 11 (14.1) 8 (21.6) Black 3 (3.8) 5 (13.5) Body mass index Underweight 7 (10.3) 4 (8.5) <0.001* Normal 42 (61.8) 16 (34.1) Overweight 15 (22.1) 21 (44.7) Obese 4 (5.8) 6 (12.7) Age at menopause (years) ≤ 45 8 (10.9) 4 (9.5) 0.82 46 to 50 28 (38.3) 17 (40.4) 51 to 55 37 (50.8) 21 (50.1) Physical activity Yes 12 (17.1) 30 (66.7) <0.001* No 58 (82.9) 15 (33.3) Smoking Yes 21 (24.7) 11 (36.7) 0.03* No 64 (75.3) 19 (63.3) Nontraumatic fractures (last 5 years) Yes 10 (31.2) 8 (9.6) <0.001* No 22 (68.8) 75 (90.4) 1st degree relative with osteoporosis Yes 14 (40) 10 (12.5) 0.05* No 21 (60) 70 (87.5) Hysterectomy Yes 16 (37.2) 12 (16.7) 0.06 No 27 (62.8) 60 (83.3) * Chi-squared Test. BMD: Bone Mineral Density 5 Spinola MS, Carneiro MLA, Bonardi JMT, Rodrigues BKAB, Monteiro LZ DISCUSSION A study analyzed women aged ≥50 years and found that women whose work involved heavy physical labor had a lower prevalence of osteoporosis than women who had more sedentary jobs(35). Rev Bras Promoç Saúde. 2019;32:9473 6 Osteoporosis in postmenopausal women Studies have reported that the areas most affected by osteoporosis are the femoral neck, lumbar spine, hip and trochanter and these areas also respond best to the results of the training program(33). Research with women with osteoporosis compared strength training, stretching and control group programs and showed the benefits of strength training interventions in improving posture and balance(36). A study that analyzed the presence of hysterectomy found that women who had undergone hysterectomy presented artificial menopause despite the preservation of ovary functions. In the present study, there was no significant association between hysterectomy and osteoporosis(37). Some limitations of the present study should be mentioned. First, the cross-sectional design of this study made it difficult to establish cause and effect relationships. Moreover, the study suffered losses that could lead to biases in the results. CONCLUSION A high prevalence of low bone mineral density was observed in the lumbar spine and femoral neck among older postmenopausal women. Knowledge of risk factors for low bone mass identified may assist professionals in identifying patients at risk of osteoporosis since evidence demonstrates that clinical history data, physical exams and specific complementary exams combined are key to preventing osteoporotic fractures. CONTRIBUTIONS Milenne da Silva Spinola and Maria de Lourdes Alves Carneiro contributed to the study design and conception; the acquisition, analysis and interpretation of data. José Maria Thiago Bonardi contributed to the writing and/or revision of the manuscript. Bárbara Katherine Ataíde Barros Rodrigues and Luciana Zaranza Monteiro contributed to the study design and conception. CONFLICTS OF INTEREST The authors declare that there are no conflicts of interest regarding this research. The authors declare that there are no conflicts of interest regarding this research. REFERENCES 1. Albergaria BH, Chalem M, Clark P, Messina OD, Pereira RMR, Vidal LF. Consensus statement: osteoporosis prevention and treatment in Latin America-current structure and future directions. Arch Osteoporos. 2018;13:90. 1. Albergaria BH, Chalem M, Clark P, Messina OD, Pereira RMR, Vidal LF. Consensus statement: osteoporosis prevention and treatment in Latin America-current structure and future directions. Arch Osteoporos. 2018;13:90. 2. Singer A, Exuzides A, Spangler L, O’Malley C, Colby C, Johnston K, et al. 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Wright NC, Looker AC, Saag KG, Curtis JR, Delzell ES, Randall S, et al. The recent prevalence of osteoporosis and low bone mass in the United States based on bone mineral density at the femoral neck or lumbar spine. J Bone Miner Res. 2014;29(11):2520-6. 37. Wright NC, Looker AC, Saag KG, Curtis JR, Delzell ES, Randall S, et al. The recent prevalence of osteoporosis and low bone mass in the United States based on bone mineral density at the femoral neck or lumbar spine. J Bone Miner Res. 2014;29(11):2520-6. First author’s address: Milenne da Silva Spinola Centro Universitário do Distrito Federal - UDF - Escola de Saúde SEP/SUL EQ 704/904 Conjunto A. Asa Sul CEP: 70390-045 - Brasília - DF - Brasil E-mail: milennespinola@hotmail.com First author’s address: Milenne da Silva Spinola Centro Universitário do Distrito Federal - UDF - Escola de Saúde SEP/SUL EQ 704/904 Conjunto A. Asa Sul CEP: 70390-045 - Brasília - DF - Brasil E-mail: milennespinola@hotmail.com How to cite: Spinola MS, Carneiro MLA, Bonardi JMT, Rodrigues BKAB, Monteiro LZ. Prevalence of osteoporosis in older postmenopausal women. Rev Bras Promoç Saúde. 2019;32:9473. 9 9
https://openalex.org/W3189519147	https://repositorio.uam.es/bitstream/10486/700454/3/effects_rozas_ACSP_2021.pdf	English	null	Effects of the Linear Polarization of Polariton Condensates in Their Propagation in Codirectional Couplers	ACS photonics	2,021	cc-by	9,685	Article pubs.acs.org/journal/apchd5 Eﬀects of the Linear Polarization of Polariton Condensates in Their Propagation in Codirectional Couplers ABSTRACT: We report on the linear polarization of polariton condensates in a codirectional coupler that allows evanescent coupling between adjacent waveguides. During the condensate’s formation, polaritons usually acquire a randomly oriented polarization, however, our results reveal a preferential orientation of the linear polarization along the waveguide propagation path. Furthermore, we observe polarization-dependent intensity oscillations in the output terminal of the coupler, and we identify the mode beating between the linear- polarized eigenmodes as the origin of these oscillations. Our ﬁndings provide an insight into the control of the polarization of polariton condensates, paving the way for the development of spin-based polaritonic architectures where condensates propagate over macroscopic distances. KEYWORDS: polaritons, condensates, microcavities, optical spectroscopy, polarization, directional couple M i y energy exchange can occur between them.37 The coupled power, limited by the mode’s overlap in the coupler arms, is determined by the separation between the waveguides, the wavelength, the evanescence of the modes, and the interaction length. These devices have proven to be essential for splitting and combining light in photonic systems and have been used widely in the silicon-on-insulator platform.38 Quantum photonic waveguide circuits based on GaAs/Ga1−xAlxAs heterostructures have been demonstrated for the manipulation of quantum states of light.39 These devices have also been exploited for guiding surface plasmon polaritons40,41 and exciton polaritons.42−44 More recently, we have reported on diﬀerent on-chip routing devices: a counter-directional coupler45 and a codirectional coupler for condensates of exciton−polaritons, studying the peculiarities of the polariton propagation46 and how this is aﬀected by the waveguides’ energetic landscape.47 A relevant factor is the spin state of the condensates after polariton’s relaxation processes, leading to their condensation.48−52 Moreover, a spontaneous buildup of the linear polarization of the emitted light above the polariton condensation threshold has been reported both theoretically53−55 and experimentally.4,56−59 The orientation of the polarization plane of the emission is pinned to a crystallographic axis of the microcavity.58,59 This eﬀect has been eﬀusively observed for trapped polaritons using diﬀerent trapping mechanisms, such as photonic disorder,60 stress,5 or M icrocavity exciton polaritons have been, in the latest years, the subject of numerous investigations given their exceptional properties.1,2 These properties, emerging from the strong coupling between their constituents, excitons and photons, allow polaritons to behave as bosonic particles. © 2021 American Chemical Society ■EXPERIMENTAL DETAILS = + ⊥ ∥ ⊥ ∥ m m m m m eff 2 is the reduced eﬀective mass of polaritons, with m⊥and m∥the transverse and longitudinal polariton masses, while Β = − + ⊥ ∥ ⊥ ∥ m m m m deﬁnes the strength of TE− In these equations, R is the coupling parameter between the polaritons and the reservoirs of excitons, and γp is the coordinate dependent losses of the coherent polaritons. We assume that the polariton waveguides are formed by microstructuring, creating a coordinate-dependent eﬀective potential, V, for the polaritons. It is also considered that the microstructuring aﬀects the transparency of the Bragg mirrors, thus, the losses experienced by polaritons become larger outside the waveguides. G (G̃) and GR (ı GR) denote nonlinear corrections, blue shift, to the eﬀective potential due to interactions between polaritons and between polaritons and incoherent excitons of the same (orthogonal) spin, respectively. = + ⊥ ∥ ⊥ ∥ m m m m m eff 2 is the reduced eﬀective mass of polaritons, with m⊥and m∥the transverse and longitudinal polariton masses, while Β = − + ⊥ ∥ ⊥ ∥ m m m m deﬁnes the strength of TE− Figure 1. (a) SEM image of a ﬁeld of directional polariton couplers with diﬀerent coupling region length (L). (b) SEM image of a directional polariton coupler indicating several parameters: coupling length (L = 20 μm), waveguide width (w = 6 μm), and waveguide separation (d = 0.6 μm). Input and output terminals and the coordinates axis are shown, corresponding to the nomenclature used in the text: x (y) parallel (perpendicular) to the main axis of the waveguides in the coupling region and x′ along the axes of the input and output terminals at ∓45° with respect to x and y, respectively. Figure 1. (a) SEM image of a ﬁeld of directional polariton couplers with diﬀerent coupling region length (L). (b) SEM image of a directional polariton coupler indicating several parameters: coupling length (L = 20 μm), waveguide width (w = 6 μm), and waveguide separation (d = 0.6 μm). Input and output terminals and the coordinates axis are shown, corresponding to the nomenclature used in the text: x (y) parallel (perpendicular) to the main axis of the waveguides in the coupling region and x′ along the axes of the input and output terminals at ∓45° with respect to x and y, respectively. ■EXPERIMENTAL DETAILS TM splitting (spin−orbit coupling). Γe accounts for the linear losses in the exciton subsystem, and Pr,l is the intensity of the optical pump creating the exciton baths. In the experiments reported here, an incoherent linearly polarized pump has been used in order to create a bath of excitons that are responsible for setting the polaritons in motion.35 However, polaritons can also be excited resonantly by coherent light. This kind of excitation provides a simpler scenario in numerical calculations to control the properties of the polaritons; we use it in our numerical simulations to clarify some aspects of polariton dynamics when required. In eqs 1 and 2, this pump is accounted for by the driving force Ar,l(x, y, t), the last term on the right-hand side of eq 1 coupling lengths, formed by doubly bent waveguides with input and output terminals rotated ±45° from the longitudinal direction; the geometrical parameters are speciﬁed in Figure 1b. The part of the device where both waveguides remain parallel along the x-direction is dubbed coupling region: a few pairs of mirrors left in the region between the two arms enable the evanescent photonic coupling of polaritons between the guides.46 For the experiments reported here, the dimensions of the selected device are L = 10 μm, w = 2 μm, and d = 0.2 μm. The choice of these parameters allows the coupling of a large fraction of polaritons between the arms of the coupler. In our experiments, we nonresonantly pump the input terminal of the ■PROPAGATION OF POLARITONS ALONG THE WAVEGUIDES ACS Photonics Article Article coupler with linearly polarized 2 ps pulses from a Ti:Al2O3 laser working at 1.664 eV, focusing the beam to a 4.5 μm diameter spot, with a microscope objective (NA = 0.40, f = 4 mm), impinging normally to the sample surface. The photo- luminescence (PL) is collected through the same objective, while the sample is kept at 12 K in a coldﬁnger, He ﬂow cryostat, and detected with a CCD camera attached to a 0.5 m focal length imaging spectrometer. We ensure that the polariton con- densation threshold (12 kW/cm2) has been exceeded and that condensates propagate along the entire device pumping with a power density of 26 kW/cm2. annular optical conﬁnement.61−63 The study and control of the polarization state of polariton condensates has opened new possibilities of designing and improving spin-based devices.64−70 annular optical conﬁnement.61−63 The study and control of the polarization state of polariton condensates has opened new possibilities of designing and improving spin-based devices.64−70 Wire-shaped microcavities are particularly interesting in this respect because, due to their reduced symmetry, each polariton mode shows a polarization splitting into two modes polarized along and perpendicular to the wire axis.71 Here, we theoretically and experimentally study the linear polarization of the emission of propagating polariton con- densates in polaritonic codirectional couplers. Our results demonstrate a coupling between the adjacent waveguides that is not strongly dependent on polarization. However, we encounter striking polarization-dependent emission oscillations at the output terminal of the coupler. For a given set of perpendicular polarizations we ﬁnd a phase shift between the oscillation’s patterns. To better understand our experimental results, a dissipative Gross−Pitaevskii model is used to describe the polarization dynamics in the device. ■EXPERIMENTAL DETAILS The sample used in this work is a λ/2 cavity with top (bottom)- distributed Bragg reﬂectors consisting of 23 (27) pairs of alternating layers of Al0.2Ga0.8As/AlAs. One stack of four GaAs quantum wells of 7 nm of nominal width is placed at the antinode of the electromagnetic ﬁeld inside the cavity, and two additional, identical stacks are embedded in the cavity adjacent mirrors. Low power measurements reveal a Q-factor of ∼5000 and a Rabi splitting of 13.9 meV. The experiments reported here are performed in a region of the sample with a photon-exciton detuning δ ≈−17 meV. The sample has been grown by molecular beam epitaxy and processed by reactive ion etching down to the QWs,46 creating a pattern of adjacent waveguides where length (L), width (w), and separation (d) have been varied. Figure 1a shows a typical ﬁeld of couplers, with diﬀerent γ ℏ∂Ψ = ℏ − Ψ + + \|Ψ \| + ̃\|Ψ \| + + Ψ − ℏ ∇Ψ + Β ∂± ∂ Ψ + ı i i Rn V x y G G G n G n m i A x y t 2 ( ) ( ( , ) ) 2 ( ) ( , , ) t r l r l p r l r l l r R r l R l r r l r l x y l r r l , , , , 2 , 2 , , , 2 eff 2 , 2 , , (1) ∂ = −Γ + \|Ψ \| + n R n P x y t ( ) ( , , ) t r l r l r l r l , e , 2 , , (2) (1) (1) (2) In these equations, R is the coupling parameter between the polaritons and the reservoirs of excitons, and γp is the coordinate dependent losses of the coherent polaritons. We assume that the polariton waveguides are formed by microstructuring, creating a coordinate-dependent eﬀective potential, V, for the polaritons. It is also considered that the microstructuring aﬀects the transparency of the Bragg mirrors, thus, the losses experienced by polaritons become larger outside the waveguides. G (G̃) and GR (ı GR) denote nonlinear corrections, blue shift, to the eﬀective potential due to interactions between polaritons and between polaritons and incoherent excitons of the same (orthogonal) spin, respectively. ■THEORETICAL FRAMEWORK To study the dynamics of exciton-polaritons theoretically, we adopt a well-known model describing the coherent polaritons by two complex order parameter functions Ψr,l for right (r) and left (l) handed circularly polarized polaritons.65,72 The coherent polaritons interact with baths of incoherent excitons having diﬀerent spins. The excitons are characterized by their density nr,l. The whole set of equations can be written as ■EXPERIMENTAL DETAILS Their short lifetime, typically of the order of ps, is comparable or even smaller than their respective thermalization times, so in general these particles do not reach thermal equilibrium. However, a condensation similar to a Bose−Einstein one is observed when the particle density is increased.3−7 Their very low eﬀective mass (∼10−4me, me being the free electron mass) can lead to condensation, even at room temperature. This has indeed been the case in transition metal dichalcogenides, organic semi- conductor materials, and lead halide perovskite structures, where the enhancement of the exciton binding energy, characteristic of these compounds, has enabled the observation of strong light−matter coupling,8,9 polariton lasing and condensation10−18 at room temperature. The ease of use and the ﬂexibility oﬀered by exciton polaritons unwrapped a variety of new proposed devices, such as polariton interferometers,19 logic gates,20−22 or transistors.23−26 Additionally, three-dimen- sional polariton conﬁnement has been achieved in microcavity pillars.27−31 Using two-dimensional lattices of these micro- pillars, it is possible to emulate graphene and its remarkable properties.32−34 All these devices are built using reﬁned lithographic techniques that ensure the polariton conﬁnement along several directions. In the one-dimensional case, only a well-deﬁned longitudinal path along which polariton con- densates can travel remains.21,23,35,36 Received: May 20, 2021 Published: August 3, 2021 https://doi.org/10.1021/acsphotonics.1c00746 ACS Photonics 2021, 8, 2489−2497 Received: May 20, 2021 Published: August 3, 2021 In the present work we will focus on semiconductor microcavity couplers. Such optical directional couplers are formed by parallel optical waveguides, closely spaced, so that © 2021 American Chemical Society © 2021 American Chemical Society https://doi.org/10.1021/acsphotonics.1c00746 ACS Photonics 2021, 8, 2489−2497 https://doi.org/10.1021/acsphotonics.1c00746 ACS Photonics 2021, 8, 2489−2497 2489 pubs.acs.org/journal/apchd5 ACS Photonics ■PROPAGATION OF POLARITONS ALONG THE WAVEGUIDES The propagation distance of these condensates following the input waveguide axis (x′) is displayed in Figure 2. The zero position on the horizontal axis corresponds to the excitation 2490 https://doi.org/10.1021/acsphotonics.1c00746 ACS Photonics 2021, 8, 2489−2497 Figure 2. Polariton propagation along the longitudinal axis of the input arm of a polariton coupler. The x′ origin is placed at the excitation spot. The photoluminescence (PL) intensity (black line) is plotted along the axis of the input terminal, x′, in the region (0 < x < 21, −15 < y < 0) together with the results of the simulation (red line). The inset shows the experimental map of the coupler emission at the condensate energy, 1.583 eV, from which the black curve was extracted; the arrow points to the laser excitation spot. ACS Photonics pubs.acs pubs.acs.org/journal/apchd5 Article ACS Photonics Article particular, we assume that the losses are spatially uniform within the waveguides, although at the excitation spot, the losses can be modiﬁed by the action of the intense pump beam. Other signiﬁcant issues for the polariton condensation, as for example phonon-assisted energy relaxation,73 are not considered in our simple model. All this accounts for the discrepancy at x′ = 0. Away from the excitation area, due to the ﬁnite polariton lifetime, the polariton population decays exponentially as it moves away from the excitation area. The simulations describe well the experimental results up to x′ ∼20 μm, where the intensity droops when reaching the waveguide bend. Now we describe the condensates propagation in the coupling region and in the output terminal. Since the PL intensity in these regions is substantially lower than in the input terminal, we have spatially ﬁltered the emission, so that the PL from the input terminal is removed. The excitation beam is vertically polarized (θi = 90°), that is, parallel to the y axis. We have observed (in contrast with previous works in the literature, which show that the polarization of the nonresonant excitation beam can strongly aﬀect the spinor state of the condensates,51,65 and in agreement with numerous reports) that upon condensation the con- densate’s polarization is independent of the exciting polar- ization, and it is deﬁned by the crystallographic axis of the microcavity4,58,59 and, in our case, by the microstructuring and the presence of the edges of the waveguides. ■PROPAGATION OF POLARITONS ALONG THE WAVEGUIDES The PL is analyzed using a linear polarizer at diﬀerent angles, ranging from θd = 0° (i.e., horizontal polarization) up to 180° in steps of 10°. For simplicity, only a summary of the PL for selected θd is shown in Figure 3. Polariton condensates are generated in the bottom-left input terminal; when they arrive to the coupling region, −5 < x < 5 μm, a large fraction of the population is conveyed from the bottom to the top arm. This oscillation of the polariton population between the two arms has been thoroughly discussed in ref 46, being attributed to the evanescent coupling of two macroscopic wave functions in each arm. It is diﬀerent to those observed in similar wider structures, where polaritons are traveling, without coupling to the neighboring arm, exhibiting a zigzag trajectory,47 that could be interpreted as a manifestation Figure 2. Polariton propagation along the longitudinal axis of the input arm of a polariton coupler. The x′ origin is placed at the excitation spot. The photoluminescence (PL) intensity (black line) is plotted along the axis of the input terminal, x′, in the region (0 < x < 21, −15 < y < 0) together with the results of the simulation (red line). The inset shows the experimental map of the coupler emission at the condensate energy, 1.583 eV, from which the black curve was extracted; the arrow points to the laser excitation spot. spot. Polariton condensates propagate ballistically away from the excitation spot in the lower input terminal after being generated, as readily seen in the inset, which also shows the coupling to the upper arm. The solid black and dot-dashed red lines depict the experimental PL intensity and the numerical simulation (see below), respectively, for polaritons moving toward the coupling region. Close to the origin, the experimental PL is much smaller than that of the simulations due to energy ﬁltering performed by the spectrometer to cutoﬀthe bright background of the pumping light having higher frequency and to some limitations of our numerical simulations around the pump area. The parameters for the linear terms in the equations describing polariton dynamics can be extracted from the experimental data, rendering a satisfactory agreement for the linear polariton propagation outside the pump spot, which is the main focus of our paper. However, we cannot get the nonlinear parameters from our experiment and must use rough estimates in their stead. ■PROPAGATION OF POLARITONS ALONG THE WAVEGUIDES The density of polaritons is depicted in a normalized logarithmic false-color scale. Figure 5. (a) Solid/dash-dotted line depicts the experimental normalized PL intensity vs y, the transverse to the axis of the coupling region coordinate, at a position close to the entrance/exit of the coupling region, −3.2 μm/+3.2 μm, for θd = 0°. (b) Corresponding simulated polariton densities at the same positions of the coupler as those shown in panel (a). Figure 5. (a) Solid/dash-dotted line depicts the experimental normalized PL intensity vs y, the transverse to the axis of the coupling region coordinate, at a position close to the entrance/exit of the coupling region, −3.2 μm/+3.2 μm, for θd = 0°. (b) Corresponding simulated polariton densities at the same positions of the coupler as those shown in panel (a). erasing of polariton’s spin memory during the relaxation processes (see also Figure S2). of polaritons experiencing zitterbewegung.74 After the coupling, polaritons continue propagating throughout the top arm until the edge of the waveguide at the output terminal, while only a minor fraction of the population remains in the bottom arm. By increasing either the length (L) or the spacing (d) between the arms, the fraction of coupled polaritons can be controlled.46 Drastic intensity variations along the device are observed when the polarization of the emission is analyzed. We ﬁnd a considerably large intensity when the polarization is analyzed at 0°. By contrast, a remarkable intensity reduction is observed around and above 30°. A further increase of θd results in a slow PL recovery for θd ≳90°. p ( g ) Let us now discuss how the theoretical model introduced above describes the eﬀects observed in the experiment. We performed numerical simulations of the waveguides with the experimental geometrical parameters and with a depth and width of the eﬀective polariton potential providing the width of the fundamental mode (full width at half-maximum of the intensity) to be very close to 1 μm: the width of the mode in the modeled waveguide is equal to the width of the fundamental mode of an inﬁnitely deep rectangular potential of 2 μm wide. In the numerical simulations, we use a random noise of low intensity as initial conditions and excite the system by an incoherent pump creating a bath of excitons, which eventually create the polariton condensates. ■PROPAGATION OF POLARITONS ALONG THE WAVEGUIDES In Figure 3. PL of polariton condensates propagating along a coupler. The emission is analyzed at diﬀerent linear polarizations (black arrows) ranging from θd = 0° (a) to 180° (h). The excitation at the input terminal (not shown) is performed with a vertically polarized laser beam [denoted by the red arrow in (a)]. The output terminal in which polarization-dependent oscillations are visible for some angles θd’s is indicated by a black dashed rectangle in (c); the axis along this terminal is deﬁned as x′. The positions labeled as Pi (where i = 1 and 3) mark the maximum amplitude of the aforementioned oscillations for θd = 0°. The PL emissions are ﬁltered at an energy of 1.583 eV and depicted in a normalized logarithmic false-color scale. A power density of 26 kW/cm2 has been used for the measurements. Figure 3. PL of polariton condensates propagating along a coupler. The emission is analyzed at diﬀerent linear polarizations (black arrows) ranging from θd = 0° (a) to 180° (h). The excitation at the input terminal (not shown) is performed with a vertically polarized laser beam [denoted by the red arrow in (a)]. The output terminal in which polarization-dependent oscillations are visible for some angles θd’s is indicated by a black dashed rectangle in (c); the axis along this terminal is deﬁned as x′. The positions labeled as Pi (where i = 1 and 3) mark the maximum amplitude of the aforementioned oscillations for θd = 0°. The PL emissions are ﬁltered at an energy of 1.583 eV and depicted in a normalized logarithmic false-color scale. A power density of 26 kW/cm2 has been used for the measurements. https://doi.org/10.1021/acsphotonics.1c00746 ACS Photonics 2021, 8, 2489−2497 2491 pubs.acs.org/journal/apchd5 ACS Photonics Figure 4. Simulations of the polariton distribution along a coupler. The emission is analyzed at diﬀerent linear polarizations (black arrows) ranging from θd = 0° (a) to 160° (g). The excitation is performed with a linearly polarized laser beam at the input terminal (not shown). The density of polaritons is depicted in a normalized logarithmic false-color scale. p g j p Figure 4. Simulations of the polariton distribution along a coupler. The emission is analyzed at diﬀerent linear polarizations (black arrows) ranging from θd = 0° (a) to 160° (g). The excitation is performed with a linearly polarized laser beam at the input terminal (not shown). https://doi.org/10.1021/acsphotonics.1c00746 ACS Photonics 2021, 8, 2489−2497 ■POLARITON’S POLARIZATION DYNAMICS ■POLARITON’S POLARIZATION DYNAMICS To quantitatively describe the polarized PL, we focus now on two regions of the coupler: the coupling region and the output terminal. In the coupling region, the PL intensity is found experimentally to be maximum for θd = 0° (=180°), that is, when the polarization is horizontal, parallel to the longitudinal axis in this region. On the contrary, the intensity drops with increasing θd reaching its minimum at ∼45°. A further increase of θd leads to a partial recovery of the emission, resulting in a PL for θd = 90° (vertical polarization), ∼50% lower than that of θd = 0°. These results reveal a preferential orientation of the polarization along the longitudinal axis of the waveguide. For larger θd, being the polarized emission direction closer to the orientation of the pumped (input) terminal, the PL intensity in the coupled region becomes high again as a consequence of a larger population with this polarization in the input terminal. This is readily seen in the polarization maps compiled in Figure 6. Panel (a)/(c) shows the Additionally, the numerical modeling replicates qualitatively the experiments bearing out the tunneling of polaritons from the lower to the upper waveguide in the coupling region. Figure 5 presents a comparison between the experimental results [panel (a)] and the calculations [panel (b)]: the normalized PL intensity is plotted as a function of y, the transverse to the axis of the coupling region coordinate, for a position close to the entrance/exit (−3.2/+3.2 μm) of that region with a solid/dash- dotted line (for θd = 0). Note that y = 0 marks the center of the gap between the arms, therefore, the signal from y < 0 and y > 0 arises from the pumped and coupled arm, respectively. It is apparent that, at the entrance, a larger polariton population is present in the pumped arm of the coupler (y < 0) than that in the coupled arm (y > 0), both in the experiments and the simulations. This situation is reversed toward the exit, where the population becomes larger in the coupled arm. Figure 6. (a) Spatial maps of the polariton polarization degree, P = − + I I I I H V H V, for θd = 0° (H) and θd = 90° (V) polarizations of the emission in the coupler. ACS Photonics Article the pseudospin of the polaritons excited by an optical pulse. This precession takes place because of the TE−TM splitting in the dispersion relation. This splitting also aﬀects the spatial evolution of the polaritons polarization when they propagate under cw-excitation conditions. The conﬁnement of the polaritons inside the microstructured waveguide aﬀects both the eigenmodes and the dispersion relations. For our narrow structures only the eﬀects in the fundamental mode need to be considered. The details of the theory necessary to understand the linear polariton propagation are given in the Supporting Information (SI). waveguide is unmistakably obtained in the simulations. This eﬀect originates from the TE−TM splitting of the polaritons: it is theoretically discussed in detail in the next section and in the SI. ■POLARITON’S POLARIZATION DYNAMICS (a) Spatial maps of the polariton polarization degree, P = − + I I I I H V H V, for θd = 0° (H) and θd = 90° (V) polarizations of the emission in the coupler. (b) Corresponding maps for θd = 50° (D) and θd = 130° (A) rendering P = − + I I I I D A D A. (c, d) Analogous degrees of polarization Figure 6. (a) Spatial maps of the polariton polarization degree, P = − + I I I I H V H V, for θd = 0° (H) and θd = 90° (V) polarizations of the emission in the coupler. (b) Corresponding maps for θd = 50° (D) and θd = 130° (A) rendering P = − + I I I I D A D A. (c, d) Analogous degrees of polarization obtained from the simulated polariton densities for the same analyzer angles as those presented in (a) and (b), respectively. The degree of polarization is plotted in a false color scale with red (blue) corresponding to positive (negative) values. obtained from the simulated polariton densities for the same analyzer angles as those presented in (a) and (b), respectively. The degree of polarization is plotted in a false color scale with red (blue) corresponding to positive (negative) values. experimental/simulated degree of polarization, deﬁned as P = − + I I I I H V H V, where IH (IV) is the PL intensity for θd = 0° (90°), and demonstrates the preferential polarization orientation in the coupling region, where a positive degree of polarization (red coded values) is obtained both in the experiments and the simulations. A positive degree of polarization in this region is also obtained in the diagonal basis, as depicted in panel (b)/(d) for P = − + I I I I D A D A obtained in the experiments/simulations; in this case I (I ) is the PL intensity for θ = 50° (130°) experimental/simulated degree of polarization, deﬁned as P = − + I I I I H V H V, where IH (IV) is the PL intensity for θd = 0° (90°), and demonstrates the preferential polarization orientation in the coupling region, where a positive degree of polarization (red coded values) is obtained both in the experiments and the simulations. ■POLARITON’S POLARIZATION DYNAMICS (b) Corresponding maps for θd = 50° (D) and θd = 130° (A) rendering P = − + I I I I D A D A. (c, d) Analogous degrees of polarization obtained from the simulated polariton densities for the same analyzer angles as those presented in (a) and (b), respectively. The degree of polarization is plotted in a false color scale with red (blue) corresponding to positive (negative) values. The amount of polaritons transferred to the upper arm depends on the height and the width of the potential separating the upper and the lower arms as well as on the dissipation rate of the propagating polaritons, which experimentally is greatly inﬂuenced by sample inhomogeneities. The lack of a full quantitative agreement between the theoretical and the experimental polariton transfer can be attributed to the not perfect ﬁtting of the experimental polariton dissipation rate and the height of the eﬀective conﬁnement potential used in the simulations. It should also be stated that, both in the experiments and theory, the population transfer is not strongly polarization dependent and 65(±4)% of the polaritons are transferred in the coupling region, regardless of their polarization, for our speciﬁc sample and experimental conditions. The coupling region where the waveguides go parallel to each other is short (10 μm) and the distance between the waveguides is much shorter; therefore, the tunneling length is less than 10 μm and most of the polaritons are transferred from the lower to the upper waveguide. Furthermore, the interaction between the diﬀerent polarizations is relatively weak, so considerably longer propagation distances would be required to observe polarization eﬀects in the tunneling of polaritons. From a theoretical point of view, the eﬀect of the coupling between the polarizations and the waveguides produces four eigenmodes having diﬀerent prop- agation constants for the same frequency. One can anticipate that this should result in a complex behavior of the polaritons bouncing between the waveguide and changing the polarization state. However, for our structures and experimental conditions, the eﬀects originating from the coupling between the wave- guides are much stronger than those caused by the spin−orbit interaction. Thus, the consequences of the latter on the propagation within the relatively short coupling area can be neglected. Figure 6. https://doi.org/10.1021/acsphotonics.1c00746 ACS Photonics 2021, 8, 2489−2497 ■PROPAGATION OF POLARITONS ALONG THE WAVEGUIDES The results of the numerical simulations shown in Figure 4 attest to a good qualitative agreement with the experiments: they reproduce the observa- tion that the polariton condensate is preferentially polarized along the direction of the waveguide. Furthermore, a conspicuous additional eﬀect is observed at the output terminal. The PL on this terminal displays two local maxima at P1 = (13, 7) and P3 = (30, 22) μm when the polarization is ﬁltered at 0°. This is in stark contrast with the emission observed at θd = 90°, in which local minima appear at the same set of coordinates. These intensity oscillations in the PL do not exist for intermediate polarizations (see for instance 50° and 130°): the emission shows just an exponential decay with propagation distance along the output terminal. This behavior is independent of the linear polarization of the excitation laser as borne out by our experiments, since the nonresonant excitation conditions in our case guarantee the Let us mention that, for our experimental conditions, the polaritons propagation is linear away from the excitation spot. The polarization dynamics observed in the experiment and in the numerical simulations is directly related to the optical spin Hall eﬀect appearing because of the TE−TM splitting.65,75 Glazov et al. pioneering work75 reports a temporal precession of 2492 https://doi.org/10.1021/acsphotonics.1c00746 ACS Photonics 2021, 8, 2489−2497 pubs.acs.org/journal/apchd5 ACS Photonics https://doi.org/10.1021/acsphotonics.1c00746 ACS Photonics 2021, 8, 2489−2497 Corresponding Author M. D. Martín −Departamento de Física de Materiales and Instituto Nicolás Cabrera, Universidad Autónoma de Madrid, 28049 Madrid, Spain; orcid.org/0000-0003-2256-1418; Email: dolores.martin@uam.es where k∥and k⊥are the wavevectors of the TE and TM modes, respectively. This is the case along the output terminal for θd = 0° and 90°, which are linear combinations of the TM (45°) and TE (135°) modes in this part of the coupler, where clear beatings are observed both in the experiments and simulations. However, the beats are absent for θd ∼45° and ∼135°. * sı Supporting Information * sı Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsphotonics.1c00746. SEM image of the microcavity structure together with refractive index and electromagnetic ﬁeld proﬁles. Addi- tional experiments on polarization beatings at the output terminal. Simulations of the polariton dynamics. Ex- citation by either a coherent or incoherent pump (PDF) Movie of polarization degree of propagating polaritons (MP4) We can explain these polarization beatings by considering the TE−TM splitting in the waveguide and therefore taking into account the diﬀerent group velocities for each polarization. In the framework of the mathematical model introduced above, the splitting lifts the degeneracy between the eigenmode polarized along (TE) and that polarized perpendicularly (TM) to the waveguide axis.76 At a given frequency, these two modes have diﬀerent wavevectors, and therefore, if both modes are excited, interference fringes are obtained when the polarization of the propagating polaritons is a linear combination of those of the eigenmodes. The beating period is given by = π \| − \| ⊥ L k k beating 2 , ■POLARITON’S POLARIZATION DYNAMICS A positive degree of polarization in this region is also obtained in the diagonal basis, as depicted in panel (b)/(d) for P = − + I I I I D A D A obtained in the experiments/simulations; in this ( ) ( ) case, ID (IA) is the PL intensity for θd = 50° (130°). case, ID (IA) is the PL intensity for θd = 50° (130°). We focus now on the polarization-dependent oscillations at the output terminal. These oscillations are clearly seen as a sign change of P (going alternatively to red- and blue-coded values), Finally, and most importantly for the present work, the polarization beating seen in the output terminal of the upper 2493 https://doi.org/10.1021/acsphotonics.1c00746 ACS Photonics 2021, 8, 2489−2497 pubs.acs.org/journal/apchd5 ACS Photonics Article Article both in the experiments and in the simulations, in panels (a) and (c) of Figure 6, that show the polarization degree in the H/V basis. However, they vanish when the polarization is analyzed in the D/A polarization basis [see panels (b) and (d)]. For a better understanding of this eﬀect, PL proﬁles have been extracted along the x′ direction of the terminal, in the region marked with a dashed line in Figure 3c. Figure 7 summarizes the experimental propagating polariton waves that interfere with the incoming ones.41 Finally, let us stress that the theory presented here does not aim to give a comprehensive description of the quite large variety of polarization eﬀects that can be observed in exciton polariton systems, but to back our experimental results. A systematic analysis of diﬀerent linear and nonlinear regimes of polarization beats is a challenge of great interest, but is out of the scope of the present paper. Figure 7. (a) PL intensity, integrated along the transverse coordinate in the output terminal of the coupler, vs distance, measured along the longitudinal axis at this terminal, for diﬀerent analyzer angles, ranging from θd = 0° (top) to 180° (bottom) in steps of 10°. (b) Corresponding simulated polariton densities for the same θd’s. ■CONCLUSIONS In summary, we have evidenced the rich phenomenology of polariton propagation in codirectional couplers when the population is analyzed into its linearly polarized components. A detailed analysis of the PL has been accomplished by mapping the polarized-dependent emission at the condensate’s energy. Two diﬀerent sections of the coupler have been studied in detail, the coupling region and the output terminal. The former region shows a transfer of polariton population from the pumped- to the coupled-arm of the device that is not strongly polarization dependent. In the latter region, on the contrary, polarization- dependent oscillations emerging from the TE−TM splitting of the fundamental modes of the waveguide have been found. In these codirectional couplers, based on waveguide microcavities, which provide a polarization-dependent mode spectrum, the polariton spin gains crucial importance. Our work paves the way for the use of polariton codirectional couplers, taking advantage of the changes of the polariton’s polarization state during their propagation. Further eﬀects of the geometry of the couplers on the spin of the polariton condensates, such as the inﬂuence of the bends on the circular polarization, are currently under investigation. Figure 7. (a) PL intensity, integrated along the transverse coordinate in the output terminal of the coupler, vs distance, measured along the longitudinal axis at this terminal, for diﬀerent analyzer angles, ranging from θd = 0° (top) to 180° (bottom) in steps of 10°. (b) Corresponding simulated polariton densities for the same θd’s. (a) and simulated (b) emission proﬁles for diﬀerent polar- izations between θd = 0° and 180° in steps of 10°. The zero position denotes the beginning of the output terminal, while the end of the structure is located at x′ ∼45 μm. A transition between diﬀerent patterns can be distinguished. For θd close to 0° and 180°, beatings separated by ∼24 μm are observed, with the maxima at x1′ ∼4 μm and x3′ ∼28 μm. In contrast, for θd close to 90°, a weak signal is obtained at x1′ being the maximum now at x2′ ∼16 μm, that is, out of phase from x1′ by half the beating distance (12 μm). ■ASSOCIATED CONTENT ■ASSOCIATED CONTENT * sı Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsphotonics.1c00746. ACS Photonics pubs.acs.org/journal/apchd5 Article Article Savona, V.; Littlewood, P. B.; Deveaud, B.; Dang, L. S. Bose−Einstein condensation of exciton polaritons. Nature 2006, 443, 409−414. Sebastian Klembt −Technische Physik, Wilhelm-Conrad- Röntgen-Research Center for Complex Material Systems, and Würzburg-Dresden Cluster of Excellence ct.qmat, Universität Würzburg, D-97074 Würzburg, Germany (5) Balili, R.; Hartwell, V.; Snoke, D.; Pfeiffer, L.; West, K. 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Oleg Egorov −Institute of Condensed Matter Theory and Optics, Friedrich-Schiller-University Jena, D-07743 Jena, Germany (9) Ardizzone, V.; de Marco, L.; de Giorgi, M.; Dominici, L.; Ballarini, D.; Sanvitto, D. Emerging 2D materials for room-temperature polaritonics. Nanophotonics 2019, 8, 1547−1558. Ulf Peschel −Institute of Condensed Matter Theory and Optics, Friedrich-Schiller-University Jena, D-07743 Jena, Germany (10) Kéna-Cohen, S.; Forrest, S. R. Room-temperature polariton lasing in an organic single-crystal microcavity. Nat. Photonics 2010, 4, 371−375. I. A. Shelykh −Faculty of Physics and Engineering, ITMO University, St. Petersburg 197101, Russia; Science Institute, University of Iceland, Reykjavik IS-107, Iceland (11) Daskalakis, K. S.; Maier, S. A.; Murray, R.; Kéna-Cohen, S. Nonlinear interactions in an organic polariton condensate. Nat. Mater. 2014, 13, 271−278. Manuel Gundin −Departamento de Física de Materiales, Universidad Autónoma de Madrid, 28049 Madrid, Spain (12) Plumhof, J. D.; Stöferle, T.; Mai, L.; Scherf, U.; Mahrt, R. F. Room-temperature Bose−Einstein condensation of cavity exciton- polaritons in a polymer. Nat. Mater. 2014, 13, 247−252. Ignacio Robles-López −Departamento de Física de Materiales and Instituto Nicolás Cabrera, Universidad Autónoma de Madrid, 28049 Madrid, Spain (13) Su, R.; Diederichs, C.; Wang, J.; Liew, T. C. H.; Zhao, J.; Liu, S.; Xu, W.; Chen, Z.; Xiong, Q. Room-temperature polariton lasing in all- inorganic perovskite nanoplatelets. Nano Lett. 2017, 17, 3982−3988. L. Notes The authors declare no competing ﬁnancial interest. ■ACKNOWLEDGMENTS (20) Espinosa-Ortega, T.; Liew, T. C. H. A complete architecture of integrated photonic circuits based on AND and NOT logic gates of exciton-polaritons in semiconductor microcavities. Phys. Rev. B: Condens. Matter Mater. Phys. 2013, 87, 195305. We thank C. Schneider and H. Suchomel for sample growth and useful discussions. (21) Antón, C.; Liew, T. C. H.; Cuadra, J.; Martín, M. D.; Eldridge, P. S.; Hatzopoulos, Z.; Stavrinidis, G.; Savvidis, P. G.; Viña, L. Quantum reflections and shunting of polariton condensate wave trains: Implementation of a logic AND gate. Phys. Rev. B: Condens. Matter Mater. Phys. 2013, 88, 245307. ACS Photonics Viña −Departamento de Física de Materiales, Instituto Nicolás Cabrera, and Instituto de Física de la Materia Condensada, Universidad Autónoma de Madrid, 28049 Madrid, Spain; orcid.org/0000-0002-6376-6703 (14) Su, R.; Wang, J.; Zhao, J.; Xing, J.; Zhao, W.; Diederichs, C.; Liew, T. C. H.; Xiong, Q. Room temperature long-range coherent exciton polariton condensate flow in lead halide perovskites. Sci. Adv. 2018, 4, No. eaau0244. Complete contact information is available at: https://pubs.acs.org/10.1021/acsphotonics.1c00746 (15) Dusel, M.; Betzold, S.; Egorov, O. A.; Klembt, S.; Ohmer, J.; Fischer, U.; Höfling, S.; Schneider, C. Room temperature organic exciton-polariton condensate in a lattice. Nat. Commun. 2020, 11, 2863. Funding (16) Su, R.; Ghosh, S.; Wang, J.; Liu, S.; Diederichs, C.; Liew, T. C. H.; Xiong, Q. Observation of exciton polariton condensation in a perovskite lattice at room temperature. Nat. Phys. 2020, 16, 301−306. ( ) This work has been partly supported by the Spanish MINECO Grant Nos. MAT2017-83722-R and PID2020-113445GB-I00. A.Y. and I.A.S. were ﬁnancially supported by the Ministry of Science and Higher Education of the Russian Federation through Megagrant Number 14.Y26.31.0015 and Goszadanie No. 2019-1246. I.A.S. acknowledges also the support from the Icelandic research fund, Grant No. 163082-051. The Würzburg and Jena group acknowledges ﬁnancial support within the DFG Project Nos. PE 523/18-1 and KL3124/2-1. 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S.; Flayac, H.; Galopin, E.; Lemaître, A.; Sagnes, I.; Solnyshkov, D.; Amo, A.; Malpuech, G.; Bloch, J. All-optical phase modulation in a cavity-polariton Mach-Zehnder interferometer. Nat. Commun. 2014, 5, 3278. Authors Elena Rozas −Departamento de Física de Materiales and Instituto Nicolás Cabrera, Universidad Autónoma de Madrid, 28049 Madrid, Spain We would like to mention here that PL intensity fringes having a short spatial period are also clearly seen at the ends of the waveguides. These short-period oscillations are visible since polariton coherence is preserved during their propagation.77,78 They originate from the reﬂection of the polaritons at the waveguide end, resulting in the formation of counter- We would like to mention here that PL intensity fringes having a short spatial period are also clearly seen at the ends of the waveguides. 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https://openalex.org/W2117229500	https://zenodo.org/records/2111957/files/article.pdf	English	null	V.— THE MEANINGS AND SYNONYMS OF PLUMBAGO.	Transactions of the Philological Society	1,908	public-domain	23,016	Kd $ pohhB8alva plyVVp&$ $8RTi, 8 K d &d,a, <[ dhfyov r € nohbv 6yKov sol€; KRl $6 b y P o 5 UTl$pbV KCd <K /L6‘hRVOS hEVKdY. * MOhhB8RlYR 6€ b p h q h h ’ $ h l ~ R p y V p O $ R V ? S , [We, baourfA/3ovua KR1 Kl#h Jv rp; A E t o r p r B t i d a i , i$vxd r e J A ~ Q jsarorib?s ry1 X p B p a r i y f v t r a r . $ 6; ~ C ~ ~ ~ O V U R 8 ~ O A V B ~ ~ X ~ O U S $ a h h ~ yeuv~rar 6; $6 d r p y d p o v K a l x p u u o i . tori 66‘ 5 UKU~LOEIBJIE, p$l hleB6qs, &ve? 8; ltal UT~ABOVUU. ~ ~ V R ~ L V 6i txel spoiuy T1S K a l d p U K 7 4 KR7R &BRUT$V K d K B p v ~ o v CbpIUKOp&‘? K d rudrqs f U d B E h T h Y A r O a p y d p y Kai a K w p f q pohbfi6ov. ”Evior 6k T ~ O U C ~ ~ C ~ A O V I T I 7 0 % plviupaulv hA”yqv pohd~8arvu~. TRANSACTTONS OF THE PHILOLOGICAL SOCIETY, V. -THE MEANINGS AND SYNONYMS OF PLUMBAGO. By JOHN W. EVANS, LL.B., D.Sc., Adviser in Geology and Mineralogy to the Imperial Institute. [Read at the Jfedtiizg of the Philological Society oia January 11, 1907.1 [Read at the Jfedtiizg of the Philological Society oia January 11, 1907.1 HAYING been consulted by Dr. Murray on the history of the word plumbago’ in connection with the Oxford Dictionary of the English Language on Historical Principles, I gave some attention to the subject, and my interest carried me further than I had originally intended. The main conclusions at which I arrived will be found in the article on that word in the dictionary, but the limits within which it was necessarily compressed did not permit of the presentation of many of the facts disclosed, and at Dr. Murray’s kind suggestion I have communicated the present paper to this Society. I have endeavoured, as far as possible, to exclude matters of purely mineralogical or metallurgical interest, except so far as they have a direct bearing on the subject of the paper, but it will be found that the variations in the use of the words with which I deal reflect in a remarkable manner the changing fortunes of the arts and sciences in the centuries covered by the survey. I have only attempted to follow the history of the word plumbago ’ and its synonyms so far as they relate to minerals or metallurgical products. The botanical aspects of the subject lie beyond my province. I had not completed my investigations when the portion of the dictionary which included the word ‘plumbago’ went to press, and in some cases I have since found earlier instances of the use of words than those which will be found in its pages. p g The subject being most conveniently presented chronologically, I begin with the word ‘molybdaena,’ whose history was for long intimately connected with that of plumbago, but goes back to a somewhat earlier date. Molybdana is the Latinized form of the Greek poXd/3Earva, and is Phil. Trans. 1908. Phil. Trans. 1908. 10 THE MEANINGS AND SYXSONYMY O F PLUMBAGO. 134 derived from po’Xv/380~, lead. Mox4/3aa~va was applied to various things connected with the metal, such as a plummet used to obtain a vertical line, a leaden sinker attached to a net, a bdlet for a catapult, and a plant associated in some way with lead. TRANSACTTONS OF THE PHILOLOGICAL SOCIETY, In the mineral sense it appears to hare been applied to lead oxide obtained as a bye product in smelting gold and silver ores, as well as to a natural substance with similar properties and of similar composition. p Aristotle (obiit B.C. 321) tells us (‘I De generationc animalium,” ii, 2) that molybdaena mixed with water and olive-oil makes a large mass from a small one, solid from liquid, and pale from dark.‘ q p Four hundred years later Dioscorides gave a more systematic account of the same substance (“Materia Medica,” v, 100):- The best molybdsena is similar in appearance to litharge, yellow in colour and somewhat lustrous. When ground to an impalpable powder it is pale yellow, and on boiling with olive-oil becomes liver-like in hue. Such as is bluish or lead-like in colour is bad. It is also found as a mineral in the neighbourhood of Sebaste and Korykos [both on the coast of Cilicia], and of this [substance] the better is that which has the appearance neither of slag nor of stone, but is yellow and shining. It has a [medicinal] power similar to that of litharge and lead slag.” In the same treatise (v, 95) he states that some use molybdana instead of lead filings in preparing lead 10tion.~ It is formed from silver and gold. There can be little doubt that poX6/3Gaiva, U K W P ~ U poX;/3Bov, and X~~Lppryvpor are all varieties of lead oxide produced by the smelting of lead and silver ore and the cupelling of nrgentiferous lead, but it is not apparent what was the exact difference in the application of these terms. The normal lead oxide (Pb 0) is yellow or yellowish red, but lead slag is often bluish or greyish black from the presence of a lower oxide of lead or of impurities. It has been employed from time immemorial in the manufacture of lead plaster by boiling T H E MEANINGS AND SYNONYMY OF PLUMBAGO. 135 with olive-oil and watcr, olcate of lead and glycerine being formed. The thickening and increase in volume in the process might give some justification for Aristotle’s statement. Apparently he applied the term ,uoXt$Xalva to an impure variety of the oxide which produced a dark mixture. 1 It has been contended that this was graphite (post, pp. 149-54), but that it was a lead ore is probable from the statement that its therapeutic properties were the same as those of iitharge. It has also been suggested (by Gimma, “ Fisica Sotteranea,” Naples, 1730, ii, p. 131) that the rrrpdywvor of Hippocrates (“De internalibus affectionihus,” cap. 45 and 49, ed. LittrC, 1851, vol. vii, pp. 278, 290) was galena, but there is no evidence in favour of this view. ’Avrl pohvj36abqs AlOapyvpov 1 It has been contended that this was graphite (post, pp. 149-54), but that it was a lead ore is probable from the statement that its therapeutic properties were the same as those of iitharge. It has also been suggested (by Gimma, “ Fisica Sotteranea,” Naples, 1730, ii, p. 131) that the rrrpdywvor of Hippocrates (“De internalibus affectionihus,” cap. 45 and 49, ed. LittrC, 1851, vol. vii, pp. 278, 290) was galena, but there is no evidence in favour of this view. ’Avrl pohvj36abqs AlOapyvpov. ’ Mohb,%arva h1OapyhpQ 7rapaRAqUiav gX€L 8hVClplV, . . . h 7 1 6’ &p$W 7h Ka6pfh K d $ $‘dppOS &r?lKrR. qbdppUKa 7 q K 7 & Kai O b x &UR€p 0; h h K d 7axLurq 6’ a h & 4 rfi(cs ylvrrai rrpouhabdvror i;[ovs 70; ihaiou. 775~~raJ yt pAv K R ~ E i 68wp pitar RA&TOY 2$$uair. . . . poh&?6arvav, @pPipp&qv Z ~ ~ ~ A A O L S gfis 7 0 ; s &AAO~S hieorr d&audp?)v ~ a r h rhv €is ‘Epyaurfpa +ipouuav d6bv &b Ilrpydpou. Kahfirac G’Epyaurfipra Kdpq TLP, ( V 8 K a l pkakAd &Srf, pera(b nrpydpv Kal & I ( ~ K O U , r d i o v s brlxouua tkpydpov rtspa~ouiovc 7fucapdKoy7a. Mohb,%arva h1OapyhpQ 7rapaRAqUiav gX€L 8hVClplV, . . . h 7 1 6’ &p$W 7h Ka6pfh K d $ $‘dppOS &r?lKrR. qbdppUKa 7 q K 7 & Kai O b x &UR€p 0; h h K d 7axLurq 6’ a h & 4 rfi(cs ylvrrai rrpouhabdvror i;[ovs 70; ihaiou. 775~~raJ yt pAv K R ~ E i 68wp pitar RA&TOY 2$$uair. . . . poh&?6arvav, @pPipp&qv Z ~ ~ ~ A A O L S gfis 7 0 ; s &AAO~S hieorr d&audp?)v ~ a r h rhv €is ‘Epyaurfpa +ipouuav d6bv &b Ilrpydpou. Kahfirac G’Epyaurfipra Kdpq TLP, ( V 8 K a l pkakAd &Srf, pera(b nrpydpv Kal & I ( ~ K O U , r d i o v s brlxouua tkpydpov rtspa~ouiovc 7fucapdKoy7a. lldvra yhp aua 8th Ar8affhpov uuwkvrar xal 8rh poAvBsa[aqr 86varar uKfud(m8at. . . . rpbs 82 7hs Xpdas r6v +appdfcwr, ilua 81’ a h & uK€Ud~EraI Cm@opd 7 1 5 ah+ brl,.Kakbov 6n: rb rpatdrfpov lyar rhs xpdar $ pohhB6arva. This y$ &~SEA?TIS seems to have been a kind of bituminous earth a lied to vine8 attacked by injurious insects (Dioscorides, op. cit., v, 180, an8’liny, “Hist. Nat.,” XXXY (16), 56). The French have applied the term ‘ am elite ’ to a soft carbonaceous or bituminous shale, which was sometimes emplo ei’fbr drawin and known as ‘ crayon des char entiem.’ See P. Pomet, “ dist. Gen. des kooues,” 1694, iii, p. 87 ; Dezatier D’Argenville, li L’Histoire Naturelle,” Pat%, 1742, p. 70, modem French dictionaries, andpost, pp. 150, 152, 155, 166. 2 ‘Avd -$s &raAijs .r) h p r ~ h h 8 o s pohhB8aiva. p pp , , , 3 Zephyrium is in Cilicia close to the localities mentioned by Dioscorides. TRANSACTTONS OF THE PHILOLOGICAL SOCIETY, This would become lighter as the reaction proceeded, for the oleate is, at least at first, light yellow in colour, and by its increase in bulk would mask any impurities which were present. ’ The ‘ natural ’ ~ o X ; / ~ S ~ C V Q probably comprised various ‘ oxidized ’ ores of lead, such as the oxide, carbonate, sulphate, phosphate, and the light-yellow molybdate known as wulfenite. Even the carbonate and sulphate, which are properly white, are often yellowish from the presence of hydrate of iron. The inferior rarieties may have included more or less un- decomposed lead sulphide, the modern galena, but pure specimens of that mineral were probably represented by the p 0 h p S o ~ t 8 j r X;eos of Dioscorides.‘ Galen, who lked a century later (obiit A.D. 200) than Dioscorides, appears to have used the word p~X$?Gatvn in a similar sense. In hia ‘(De succedaneis” he gives litharge as a substitute for p o X . ; P h l ~ ~ , ~ and in his “ De simplicium medicamentorum temperamentis,” ix, 3. 22, he states that poX&,B:PGaivrc has an effect similar to that of litharge, that both are soluble, not insoluble like stones, calamine, and sand, but that the solution is most rapid when vinegar is associated with oil, though it 2150 takes place on long boiling with water [and presumably oil]. He saw ph’p8utvu scattered about with numerous other stones on the road that leads from Pergamon to Ergasteria, a village, where there were mines, between Kyzikos and Pergamon, 410 stadia from the latter.3 ’ Mohb,%arva h1OapyhpQ 7rapaRAqUiav gX€L 8hVClplV, . . . h 7 1 6’ &p$W 7h Ka6pfh K d $ $‘dppOS &r?lKrR. qbdppUKa 7 q K 7 & Kai O b x &UR€p 0; h h K d 7axLurq 6’ a h & 4 rfi(cs ylvrrai rrpouhabdvror i;[ovs 70; ihaiou. 775~~raJ yt pAv K R ~ E i 68wp pitar RA&TOY 2$$uair. . . . poh&?6arvav, @pPipp&qv Z ~ ~ ~ A A O L S gfis 7 0 ; s &AAO~S hieorr d&audp?)v ~ a r h rhv €is ‘Epyaurfpa +ipouuav d6bv &b Ilrpydpou. Kahfirac G’Epyaurfipra Kdpq TLP, ( V 8 K a l pkakAd &Srf, pera(b nrpydpv Kal & I ( ~ K O U , r d i o v s brlxouua tkpydpov rtspa~ouiovc 7fucapdKoy7a. TRANSACTTONS OF THE PHILOLOGICAL SOCIETY, 136 THE MEANINGS AND SYNONYMS OF PLUMBAGO. In his ‘‘ De compositione medicamentorum per genera ” (i, 11) he states that ,uoXdPGuiva can always be substitut,ed for litharge, but it makes the colour of the product darker.’ It is therefore probable that it was a darker, less pure variety. It cannot have been the hard metallic sulphide, for in his “ De succedaneis ” he says that it may be used as a substitute for ‘ soft ’ or ‘ vine ’ earth.* Paulus Bgineta (vii, 3), who probably lived in the sixth century A.D., appears to employ poXd@Gaivu in the same sense aR Galen and his predecessors. y y Paulus Bgineta (vii, 3), who probably lived in the sixth century A.D., appears to employ poXd@Gaivu in the same sense aR Galen and his predecessors. I n the “Historia Naturalis” of the younger Pliny we find passages closely similar to those which I have extracted from the writings of Dioscorides, who was a contemporary, if the date usually assigned to him be correct. It is probable that Pliny took them from the Greek author, though, it may be, they both borrowed from the same source. As a general rule we find the word poX~$Gucva, when used for a mineral or a metallurgical product, simply transliterated into the Latin form ‘molybdaena’: “Est et molybdaena, quam alio loco galenam appellavimua, Fena argenti plumbique communis. melior haec quauto magis aurei coloris, quantoque minus plumbosa, friabilis et modice gravis. cocta cum oleo iocineris colorem trahit. adhaerescit et auri argent.ique fornacibus. hanc metallicam vocant. laudatissima quae Zephyrio fiat. probantur minime terrens minimeque lapidosa” (xxxiv (ls), 53). Elsewhere Pliny describes molybdsena as one variety of spuma argenti, viz., litharge obtained in the smelting of silver : “ Quidam duo genera faciunt spume qua vocant scircrytida et peumenen, tertium molybdanam in plumbo dicendam ” (xxxiii (6), 35). However, in the passage (xxxiv (IS), 50) corresponding to the other quotation from Dioscorides (v, 95) Pliny uses the word plumbago instead of molybdsna : “ quidam limatum plumbum sic terunt, quidam et plumbaginem admiscent.” 131 THE MEANINGS AND STNOXYMS OF PLUMBAGO. Elsewhere Pliny uses a third word, galena, both for the native mineral and the furnace product : “ Plumbi nigri origo duplex est, aut enim sua prooenit vena nec quicquam aliud ex sese parit, aut cum argent0 nascitur, mixtisque venis conflatur. ’ This is substantially the explanation miyen by Johann Beckmsnn, (‘ Beitrage zur Geschichte der Erfindungen,” vol. %, Leipzig, 1797, p. 331 ; ‘( History of Inventions and Discoveries,” vol. iv, London, 1514, p. 11. The colophon reads : “ Explic” dyascorides quG etrus paduanFsis legendo eorexit et exponendo utiliora s‘ut T lucem deduxit. &I ressus Colle p- magis- tnim joh’m allemanum de medemblick. anno xpi miiesimo. cccco.lxxviii0. mense iulii.” TRANSACTTONS OF THE PHILOLOGICAL SOCIETY, hujus qui primus fluit in fornacibus liquor stagnum appellatur, qui secundus argentum, quod remansit in fornacibns galena, quze fit tertia portio addits yen=. h s c rursus conflata dat nigrum plumbum, deductis partibus non [probably a mistake for ‘nonis’] duabus” (xxxiv (16), 47). ’ ‘ Plumbum nigrum ’ is lead as opposed to ‘ plumbum candidum,’ or tin. ‘ Stagnum’ or ‘ stannum’ appears t o be the alloy of lead and silver first obtained. The lead of this alloy was then conyerted int,o slag or ‘ galena,’ also known as ‘ molybdena ’ or ‘ plumbago.’ This was again smelted, and the third product, pure lead, equal in amount to seven-ninths of the slag, was obtained.’ Elsewhere, speaking of silver ore, Pliny tells us : “ excoqui non potest, nisi cum plumbo nigro aut cum vena plumbi, galenam vocant, qus juxta argenti venis plerumque reperitur ’’ (xxxiii (61, 31). I n modern times lead is mainly obtained from the sulphide, but in the shallower mines of ancient times the oxidized ores which are found near the surface must have been largely worked, as they are now in uncivilized countries, and the word ‘galena,’ when used for the mineral, appears, like molybdsna and plumbago, to have primarily signified these oxidized products, though it may have included the sulphide as an inferior variety. ‘ ’ ‘‘ ‘ Plnmbago ’ is only once used in the ‘‘ Historia Naturalis ” in the sense of a mineral product, but it is elsewhere employed in other senses, twice as the Latin equivalent of the plant ~ O X ; / ~ ~ ~ U L V ( L (xxv (13), 97; xxix (4), ZS), and twice for a lead-like hue in the zmaragdus, which diminished its value (xxxvii ( 5 ) , 18). The words niolybdtena and plumbago do not occur in any other l i l L ti th g The words niolybdtena and plumbago do not occur in any other classical Latin author, 10 spite of the fact that plumbago is only once used in the sense of a kind of litharge also denoted by the words molybdaena and galena, I am inclined t o believe that this was its earliest meaning. Just as ferrugo means the rust of iron and srugo that of copper, THE MEANINGS AND SYNONYMS OF PLUMBAGO. TRANSACTTONS OF THE PHILOLOGICAL SOCIETY, 138 so plumbago originally meant the product obtained by the corrosion of lead when heated. The derivation of the word ‘ galena,’ which is also confined to the pages of Pliny among ancient authors, is very obscure. The writers of the renascence (post, pp. 140-2, 144, 146) believed that it was of Spanish origin, and the fact that Pliny, who used it, obtained much of his information on metallurgical matters in connection with lead and silver from Spain (see for instance xxxiii (6), 31), lends some countenance to the suggestion. The derivations which have been proposed from the Greek yak-jq, a calm, and yeXE‘w or +Lw, I laugh or shine, do not seem very probable. If it be Latin or Greek, or derived from an allied Aryan idiom, it may be akin to the English ‘ cloam ’ and the old Sclavonic glina (see the Oxford Dictionary, under ‘clay’ and ‘cloam’). It would then have originally meant any yellowish earthy material, and only secondarily that from which lead was obtained. T have been unable to find a reference to any of the words plumbago, molybdsena, or galena in the writings of the Middle Ages earlier than the “ Bibliotheca Mundi ” of Vincentius Bellovacensis (A.D. 1190 to 1260), who repeats tho statement of Pliny that molybdsena is a third genus of Spuma argenti or Iitharge. y g p g g A fuller reference to the same word is found in the “ Pandects,” or Dictionary of Medicine, of Matthsus Silvaticus (obiit 1340), Physician to Robert, King of Sicily, first printed in 1474. Here (cccccliiii) we read “ Molibdena .i. plumbum ustiim et stercus plumbi sed Dyas. cap. molibdena dicit quod est quasi stercus auri vel argenti,” etc. The quotation is, however, not from Dioscorides himself, but from an alphabetical compilation founded on his writings, which appears to have been current in Italy during the later Middle Ages. This was printed in 1478, in black letter with marginal comments, apparently of later date than the text, at Colle (probably the town of that name near Florence). 3 Another edition of the translation by Ruellius appeared at Strasburg in the same year. It contains the commentaries of Narcellus Virgilius as well as the corollaries of Hermolaus Barbarus. I had concluded from internal evidence that this book (which is usually described as a translation of Dioscorides) was printed from a manuscript version - of much earlier date, before I discovered that it was quoted by Sitvaticus, who appears to have been under the impression that it was the actual work of Dioscorides, though it includes materials from other sources. It must have been compiled long before Silvaticus wrote, and is quite distinct from a brief abstract of Dioscorides in Greek by Stephanus, arranged alphabetically by diseases, a Latin translation of which was published in 1581. 3 The paragraphs in the “ Corollarii ” do not exactly correspond with those in the text. 3 Another edition of the translation by Ruellius appeared at Strasburg in the same year. It contains the commentaries of Narcellus Virgilius as well as the corollaries of Hermolaus Barbarus. p 3 The paragraphs in the “ Corollarii ” do not exactly correspond with those in the text. I had concluded from internal evidence that this book (which is usually described as a translation of Dioscorides) was printed from a manuscript version ) p p - of much earlier date, before I discovered that it was quoted by Sitvaticus, who appears to have been under the impression that it was the actual work of Dioscorides, though it includes materials from other sources. It must have been compiled long before Silvaticus wrote, and is quite distinct from a brief abstract of Dioscorides in Greek by Stephanus, arranged alphabetically by diseases, a Latin translation of which was published in 1581. TRANSACTTONS OF THE PHILOLOGICAL SOCIETY, It purports to have been edited and corrected by Petrus of Padua, but whether from the carelessness of the editor or the defective state of the manuscript there are numerous blunders.’ It reads “ Molipdina est quasi stercus auri et argenti,” and continues in the same words (except for evident mistakes) as the quotation THE MEANINGS AND SYNONPMS O F PLUMBAGO. 139 by Silvaticus. I n the margin we read “molebdina planta est latine dicta plumbago.” ’ p g The first Latin translation of Dioscorides to appear in print was probably that of Herrnolaus Barbarus, well known for his Latin abridgement of Aristotle. The earliest edition in the library of the British Museum is believed to have been printed at Venice in 1516. It is accompanied by “Annotamcnta” by Joannes Baptista Egnatius, 6‘ in usum etiani mcdiocriter eruditorum,” and followed by ‘‘ Corollarii,” by Hermolaus Barbarus, “ libri quinque non ante impressi.” In the principal paragraph already cited (v, 100 ; paragraph 935 of this edition) ‘molibdana’ appears both in the heading and the translation, while in that (v, 95; 930) corresponding to the passage in which Pliny uses the word plumbago (xxxiv (18), 50) me find “rnolybdaenam hoc est plum- baginem.” In the ’‘ Corollarii” (paragraph 936) ’ we read with reference to the former paragraph of the text: “Xolybdaena hoc est plumbago, quam et Galenam vocmius: communis est plumbi, et argenti vena. . . . Sunt q u i molibdanam inter spumas argenti collocent. Fossilis Molibdaena est et ad vicum Ergasteriam. . . . Nominatur, et Nolibdana, id est Plumbago, herba Plinio,” etc. I n the same year a translation by Joannes Ruellius appeared at Paris. I n paragraph v, 91 (= v, 100 suprfi), the heading is “De molibdaena seu plumbagine,” while in thc text only ‘molybdaena’ is used. In paragraph v, 86 (= v, 95 s u p a ) , ‘ plumbago ’ is employed instead, exactly as in the parallel passage in Plioy. L4 later translation and commentaries by Marcellus Virgilius, ‘‘ Secretarius Florentinus,” were printed with the Greek original at Cologne in 1529.3 Here in paragraph v, 54 (= v, loo), we have the heading : “ De plumbagine metallica ” followed by “ hfoiybdanam (Romani plumbaginem dicunt).” The translation THE MEANINGS AND SYNONYMS OF PLUMBAGO. F. L. Becher (“ Die Mineralogen,” Freiberg, 1819), however, states that it w89 issued in 1518 (p. 15) or 1528 (p. 21). p y 2 Bleischweif mas granular or fibrous galena. See post, pp. 144, 160, 163, 168. I t appears also to have been applied to some sterile mineral of a lead-like appearance, possibly graphite. “ Bleyschweiff ist eine leere Berg-art, so das dnsehen hat, als ware es gediegen Bley ” (Christianus Berwardus, ‘‘ Interpres Phraseologis Metallurgics,” Franckfurt am Mayn, 1684 ; also “ Erklarung derer Bergmanuischen Worter und Red-Arten,” which forms an appendix to the Institutiones Metallice of G. C. Rirschmaier, Wittenberg, 1 687, and other vocabularies ; see also p. 29). 1 In a small treatise published in 1566, entitled “ De metallicis rebus ac nominibus observationes varie et erudita, ex schedis Georgii Fabricii : quibus ea potissimum explicautur, qua Georgius Agricola prsteriit,” we read (p. 19b) : ‘‘ Dfierunt lapis plumbarius, et plumbago. hzc enim pura est, quam Hispani galeuam nominarunt” ; and (p. 21.h) “ Plumbago. Glantz, oder gedigen Bley.” ‘‘ Lapis Plumbarius. Bleyertz.” TRANSACTTONS OF THE PHILOLOGICAL SOCIETY, 140 follows, iu which plumbago is used throughout, as it is also in paragraph v, 50 (= v, 95). In the commentary we are told: “Galenam bis terve . . . Graecorum metallicam molgbdreuam Plinius vocavit : non tam ut a molybdsna plumbagineque sui generis herba, . . . appellatione secerneret, quam quia scribente eo warn historiam, haec erat privatim metallicae plumbaginis appellatio, ab Hispanis Galecia u t credimus facta, in qua cele- berrims quondam metallorum fere omoium fodim fuerunt. &use quoniam nostra &.ate nulla est, nec habet ea vox aliquam nunc significationis aestimationem, impune et sine inviiiia relicta a nobis fuit, prssertim in re q u s ex substantia sua certius indicsnda erat. Plumbago ob eam causam molibdaena hac dicta: et ne cum herba misceretur, ex metallo nota illi adjecta est.” part of the sixteenth century as the Latin equivalent of poXz$36atva, not only in the botanical but also in the mineral sense, although Pliny had used by preference molybdaena or galena. We therefore find the word ‘ plumbago ’ recognized in the early. y y p y g The use of these words were discussed at length in the Bermannus sive de re metallica” of Georgius Agricola, first published at Basle in 1530.’ A preface by Erasmus (missing in the British Museum copy) was at first undated, but in the edition of 1546 bears the date of 1529. This short work forms an introduction to the study of mineralogy and metallurgy, and is cast in the form of a dialogue. It is remarkable for shrewd good sense and an incisive style. The author argues (pp. 41-4) at some length that the natural mineral substance referred to by Pliny as galena, molybdaena, and plumbago was the mineral principally worked for lead at the time he (Agricola) wrote, viz., the lustrous black sulphide of lead, the galena of modern mineralogy. ‘‘ ‘‘ Bermannus :-In his omnibus argenti materia eat, atque hic primum videa Galenam sive plumbaginem. Nsvius :-Estne haec plumbago quam Plinius poX;flGat~~p [sic] etiam vocat ? Ber- mannus:-Ita sentio. . . . 1 J. H. Yott, ‘ I Dissert. Chimiques,” vol. ii, 1759, p. 560; Dana’s “Mineralogy,” 6th ed., 1892, p. 59 ; and Hintze, “Handbuch der Mineralogie,” vol. i, 1905, pp. 466, 557, but see post, pp. 146, 155, 156. See alsopost, pp. 156, lay, 161. TRANSACTTONS OF THE PHILOLOGICAL SOCIETY, Colore plumbi, ut videtis, est, atque ob id Grscis poX;/?Batua,w, Latinis plumbaginem dictam arbitror, nisi quis iccerco potius, quod ipsum etiam pro me facit, sic dictam velit, quod ex ea plumbum fiat.’’ He thought, as I believe correctly, that this lead - coloured mineral was the X1‘Bor wuoXvflGoer8~~ of Dioscorides (v, 98), which 141 THE MEANINGS AND SYNONYYS OF PLUMBAGO. he translates by ‘lapis plumbarius,’ and states that it differs from ‘‘ Molybdena nativa ipsius Dioscorides magis colore quam materia ” (p. 45). (p ) The ‘ molybdzna ’ found in the furnace had, on the other hand, Agricola admits, different physical characters (p. 43). g p y (p ) He discusses the derivation of the word ‘ galena,’ remarking : “ Galena sive Hispanicum, sire alterius gentis vocabulum sit, nihil moror: nam nostrum non esse hinc perspicuum puto, quod serius metalla fodi cepisse in Germania constet: id certe nostri imitati, eandem rem similiter, nltimis tantum mod0 literis mutatis, appellarunt,” referring apparently t o the word ‘ Glantz.’ It is possible, of course, that German miners may have converted a foreign word into one with a similar sound that mas already familiar to them. He strongly condemns the suggestion that galena itself was derived from Galicia in Spain, pointing out that Pliny states that only tin, ‘plumbum candidum,’ is found there. As a matter of fact lead ore does occur in Galicia, but there seems no real evidence in favour of the derivation suggested. He also speaks (p. 42) of a sterile rariety of galena-“Aliud preterea genus Galen% est, huic, quod jam \-obis ostendi, colore nihil dissimile, sed prorsus sterile et ita subtile ut totum violentia ignis consumatur, ac per fumum evaporet.” g p p The book concludes with an appendix, ‘( Rernm metallicarum appellationes . . . autore Plateano,” where we read ‘‘ Galena sive Molibdsna-glantz und blyertz, auch blyschnueiff ’.” g y y In his treatise “De natura fossilium,” ed. 1546, p. 366. he adheres to the views expressed in his earlier work, remarking on the question of the derivation of galena, “Qiiod vocabulum utrum Hispaniense sit, an alterius gentis et lingue, si Hispani nesciunt, nemo, ut opinor, poterit scire.” 142 THE MEANINGS AND SYNONYMS OF PLUMBAGO. TRANSACTTONS OF THE PHILOLOGICAL SOCIETY, absimilis frugiferae, sed prorsus est sterilis, et totam vis ignium consumit .” The endeavour to follow the steps by which these words or their derivative forms became introduced into the modern languages of Europe and the variations in their meaning presents some difficulty on account of the widely extended use of Latin in writings on scientific and even technical subjects. Latin was in fact a practical means of expression, independent of nationality, and lucidity and accuracy were considered of more importance than a Ciceronian style. The earliest vernacular publication which is of interest for our present purpose is a translation of Dioscorides into Italian, printed in Venice in 1542. Here (17, 95 = v, 100) we have the heading ‘‘ De la piombaggine ” followed by “ Molibdena (Romani pliim- bagine) ” ; piombaggine (in one place spelt biombaggine) is used in both passages. In another translation by Xarcantonio Montigiano, printed at Florence in 1547, the corresponding heading (v, 54 = v, 100) is ‘(Della Tena di Piombo cio 2 Piombaggine,” and both terms are employed in the text. I n a third translation into Italian by Pier Andrea Mattioli of Siena, with a commentary by the same author, printed at Venice in the following year, the paragraph describing molybdaena (v, 59 = v, 100) is headed “ Della Molibdena, over0 Piombaggine,” but molibdena alone is used in the text, though in the earlier passage (v, 54 = v, 95) piombaggine is substituted, as plumbago is in Pliny. This translation and commentary proved very popular, and were translated into Latin and French. The later editions owe much to the writings of Georgius Agricola. The first French edition was an abbreviation published in 1553, under the title ‘‘ Les six livres de Pedacion Dioscoride d’hnazarbe de la niatiere medicinale, translatez de latin en Francois ” ; y adioustant,” we are told later, “ quelques petites annotations (sachant tres bien le nature1 de la nation Francoise, s’estudier et oomplaire brevet@) extraict du battu B tout marteau l’entier commentaire du S. AndrO Pierre Yatthioli Medecin Senois.” Here under the heading ‘( De la Plombagine que les Grecs appellent niolyfdena : les Latins Plombago, les Italiens : Piomba- gine” (v, 50 = v, 100) we read: “Et par ainsi la Plombagine n’est autre chose, que la Litharge remassee depuis le coder dee minieres comme un lict en la fournaise. TRANSACTTONS OF THE PHILOLOGICAL SOCIETY, I n his “ Rerum metallicarum interpretatio ’’ (1546) we find the following definitions : “ Galena- Glantz unnd pleiertz ” ; ‘L Galena iuanis-Blende ’’ ; “ Molibdaena, idem quod plumbago ” ; (‘ Plumbago metallica - Pleiertz, pleischweis ” (a misprint for ‘ pleischweif ’) ; “ Plumbago fornacum-Herdplei ” ; ‘‘ Plumbarius lapis-Glantz.” It is not clear whether galena sterilis and galena inanis are the same. Blende meant originally any mineral with a deceptive appearance, but at the present time, when used without quali- fication, it means zinc-blende and galena inanis has therefore been identified with that mineral.’ Agricola was, however, too careful an observer to assert that the colour of zinc-blende was “nihil dissimilis” to that of galena. There are several minerals which might be converted into volatile products in the furnace, and bear a closer resemblance to galena than zinc-blende. Some of these, such as stibnite (sulphide of antimony) and bismuthine (sulphide of bismuth), were well known to Agricola as distinct minerals. There only remain graphite, the black lead of our pencils, a crystalline form of carbon, and molybdenite, a sulphide of molybdenum, which present great resemblance to each other, and certain rare minerals containing tellurium. There can be little doubt that graphite was the substance mainly referred to as galena sterilis.2 Christophorus Encelius Salueldensis (Entzelt of Saalfeld), in his “De re metallica,” Frankfort, 1551, i, 34, pp. 66-9, also contends that plumbago, galena, and molybdaena are identical with Glantz, the modern galena. “ Ergo nostra plumbago unser glantz, est galena, et molybdsena.” ‘“am quod Plinius . . . aut Hispani galenam vocant, nos jam glantz dicimus.” H e too condemns the derivation from Galicia. He suggests as a distinction between molybdaeua and galena, that the latter should be employed for lead ore containing silver, and the former for ore free from that metal. After referring to the different colours of lead ores, he says : “ A Germanis omnes hse species dicuntur generali nomine, bleiertz, glantz a splendore, bleischweyff etc.” His frequent use of the term ‘ nostra plumbago ’ seems to imply that it was in his time the ordinary Latin expression in Germany for lead ore. He characterizes the ‘ sterile ’ variety as “ nullius momenti, colore non 143 THE MEANINGS AND SYNONYMS OF PLUMHAGO. “ Glantz, welches die Lateiner Galenam nennen, is ein glauch oder gliiu Metall, bricht gern auff silberoengen, helt offt hlei’ unnd silher . . . Bleischweiff oder lumbago i t ern gelblicht metall, voller schwebels, darumb es von bley und scfwefel den namen haben 8011, oder das es des gauges schweiff ist, Disz helt offt ble7 und silber ” (“Die neundte Predigt,” p. 1016). “Etlich pley venincket in den luckern herd, oder trencket sich drein, disz nennen die gelerten Molybdenam ” (“ Die dreyzehende Predigt,” p. 1496). Cotgrave’s explanation is reproduced in the Glossographia of Thomas Blount (1656 and 1674) under ‘ plumbagin.’ The definition by E. Phillips in his 6 ‘ New World of English Words,” 1658 and later editions, “silver mingled with lead stone, or oar,” and that of N. Bailey in his “Universal Etymological English Dictionary,” 1721 and later editions, “ Lead naturally mingled with Silver,” are evidently derived from this source. I am indebted for these and a number of other references to Dr. Murray. TRANSACTTONS OF THE PHILOLOGICAL SOCIETY, La Plombagine minerale, n’est autre chose que la vene, qui tient I’Argent, et le Plomb par THE MEANINGS AND SYNONYMS OF PLUMBAGO. 144 ensemble.” This is, so far as I know, the first recorded use of plombagine in French, though the word may no doubt be older. A complete French edition appeared in 1572. p pp There is a rather earlier use of molybdena in French in the “Epitome des trois premiers livres de Galien” (1549), by M. Gregoire, where we find in book i (p. 167 of the edition of 1552), under the heading “Des Nedicamens qui se font de Molybdaena,” “ La Litarge et Molybdana sont presque appliquez it rnesme usage.” g A Spanish translation of Dioscorides and a commentary by Doctor Andres de Laguna were printed at Salamanca in 1566. Under the heading (‘ De la Molibdena ” we find: “ Griego MoX@a~va. Lat. Molybdaena, et Galena. Molibdos en Griego significa el Plomo de do tom0 el nombre la molibdena, . . . la qua1 no diffiere de la Galena tan celebrada” (book v, paragraph 59). This reference to the celebrity of galena is not easy to understand, but it indicates that the word was well known in Spain in spite of the fact that it appears in a list of ‘nombres latinos’ at the end of the book, while molibdena is found in a similar list of ‘ nombres castellanos.’ Dioscorides was not translated into German, but we find the same words explained in the ‘< Sarepta” of Johann Mathesins, the friend and biographer of Luther. This curious blend of mining, metallurgy, and theology appears to have been originally delivered in the form of sermons to the mining population of Joachimsthal. The preface is dated in 1562, and the book is stated to have been issued in that year, but the earliest edition which I have seen was printed at Nuremberg in 1571, after the author’s death in 1565. Here Glantz and galena are employed for argentiferous galena, and Rleischweiff and plumbago for an ore of sulphur and lead. Molybdena, on the other hand, is used in the sense of litharge.’ I n the “ Meisznische Bergk Chronica,” by Petrus Albinus, Dresden, 1590, we find (p. 133) the form plumbagine: ‘( Item es henget auch offt an der plumbagine, ein viride, welches vie1 Bley THE MEANINGS AKD SYXONYMS OF PLUMBAGO. 1 On p. 612, however, it is also rendered by ‘‘ congealed specks resembling . . . spots of lead.” C ’ l i i d di h Gl hi Th Bl TRANSACTTONS OF THE PHILOLOGICAL SOCIETY, 145 gibt.” ‘ Viride ’ may be pyromorphite and mimetite (phosphate and arseniate of Iead with some chloride or fluoride), which are often greenish in colour. It was not till the publication in 1601 of the translation by Philemon Holland of the “ Historia Naturalis ” of Pliny that we find any of the words we have been considering used in English. I n this translation the words galena and molybdsna are retained throughout. Thus in vol. ii, p. 472, we read : “ This minerall or mettall they call Galena,” with a marginal note “ o r Molyb- dsna ” ; on p. 474, ‘( As for the third, named Molybdsna, they reckon as a thing by itselfe; to be treated of in the discourse or chapter of Lead”; on p. 517, “As for the third part of the reine which remaineth behind iu the furnace, it is Galana, that is to say, the verie mettall it selfe of lead.” The heading of chapter 18, p. 518, includes “of the veine of Lead called Nolybdzena or Galena.” See also p. 520. When plumbago is used in the Latin original for the plant (pp. 236, 359) or for a lead-coloured tinge in the zmaragdus (p. 612), it is translated by the same word.’ On the other hand, when it is employed as a synonym for the mineral molybdsna (p. 519), it is merely rendered by “ some lead ore.” ‘Plombagine,’ however, is used both for the natural and artificial mineral product in Cotgrave’s French and English Dictionary published in 1611. “Plombagine: f. Pure lead turned almost into ashes by the vehemence of the fire: This is the artificial1 Plombagine, and comes of lead put into a furnace with gold, or silver oare, to make them melt the sooner; (by which imployment it gaines some part in the worth of those metalls;) there is also a naturall, or minerall Plombagine, which (as Mathiolus thinketh), is no other then silver mingled with lead- stone, or oare.” “ Yolibdene as plombagine; also the herb leadw ort. ” Six years later, in the “Surgion’s Mate,” by John Woodall, printed in London in 1617, we find (p. 113) under the heading 146 THE MEANINGS AND SYNOSYMIY OF PLUMHAC~O. See also post, p. 168. 1 Pencils of the modern type do not appear to hare been introduced till about a century later. 2 See G. Agricola, ‘‘ De natura fossilium,” rol. ii, ed. 1546, p. 197 ; J. II. G. von Justi, 6 ‘ Gruudriss des gesoniten Mineralreiches,” 1757, p. 113 ; Beckmaun, op. eit., vol. v, 1903, p. 245; vol. iv, 1814, p. 353. Beckmann, op. cit., vol. v, 1803, pp. 237, 260; vol. iv, 1814, pp. 347, 356 ; 1‘ Anthologia Grwca Palatina,” vi, 67 and 68 ; l’liny, “ Hist. Nat.,” xxxiii (3), 19, and (6), 31. Liddell 6- Scott, on the other hand, under fidhu@os, suggeet that in one of the passages from the “ Anthologia ” that word is used in the sense of graphite (black lead). They also quote authority for the use of ‘ fikul88or ’ as a test for gold, and believe that here a150 graphite is referred to. See note, p. 156. There seems, however, no evidence of this. This is, however, most improbable. TRANSACTTONS OF THE PHILOLOGICAL SOCIETY, ‘ g Ninium” : L L Plumbago, or red leade, hath the force of binding, mollifying, filling up hollow ulcers with flesh.” This is the only instance, so far as I know, in which ‘plumbago’ is used for minium, the red oxide of lead (PbO), which was in ancient and medizval times sometimes confounded with cinnabar, the red sulphide of mercury, but nerer with litharge. The mistake may have arisen from the statement by Johannes Gorrzus (Jean des Gorris) the elder in his “Definitionum Medicorum Libri 21,” first published in 1564 at Paris, that )ILOXL/~:IJSULZ~, or plumbago, was made from boiling lead-“ Est medicamenturn metallicum ex plumho fervescente factum ” (p. 300, ed. 1578). According to the manner in which the operation is carried out either litharge or minium may be obtained.’ I n 1565 appeared a volume of tracts on minerals, rocks, and cognate matters, mostly by Conrad Gesner of Zurich. One of these, entitled “De omni rerum fossilium genere,” appears to be a catalogue of a collection of minerals, metallurgical products, and fossils, which must have been very complete for the time. The Latin and Gcrman names for the specimens are given, and appear in some cases at least to be derived from Entzelt. Among the entries are the following (p. 74 and following pages) :-‘‘ Molybdaena vel plumbago metallica. Hartblei under ofenbruch” . . . Undcr the heading “ Plumbago” :-“Plumbago metallica vel nativa, verb0 Hispanic0 Galena, id est, vena plumbi et argenti communis. Glantz.” “ Plumbago simplex qu3e nihil nisi plumbum in se continet. Reiner glantz, der nichts dann blep helt.” “ Tessellata in lapide calcareo. Wurfflichter glantz in weissem kalchstein.” This is crystallized galena in the modern sense. Under ‘‘ Plumbago sterilis” are a number of entries, which show that the term was very loosely employed, at any rate by this author; they include the following :- 6‘ Plumbago sterilis pici sirnilis. Bech blende.” This is pitch- blende, at present the principal source of uranium and radium. It is the earliest known mention of the mineral. Pitchblende would not be volatilized in an ordinary furnace, so it cannot be the galena sterilis of Agricola. “ Flava nitens, Scharfenbergia prope Misenam. Licht gelbe blende.” Probably a variety of zinc-blende. ‘‘ Sterilis galena sirnilis. Glantz blende.” Probably THE MEANINGS AND SIINOXYMS OF PLUMBAGO. 147 graphite or molpbdenite. “ Sterilis Ten% cupri sirnilis. TRANSACTTONS OF THE PHILOLOGICAL SOCIETY, Kupffer blende.” Perhaps kupfernickel, an arsenide of nickel. In the “De rerum fossilium, lapidurn et gemmarum maxime, figuris et sirnilitudiuibus Liber,” published in the same volume, we find (p. 10.1) one of the earliest references t o graphite and its use for pencils : ‘( Stylus inferius depictus, ad seribendum factus est, pluinbi cujusdam (factitii puto, quod aliquos fitimmi [antimony] Anglicuni vocare audio) genere, in mucronem deraoi, in manubrium ligneum inserto.” A drawing shows the black lead fixed into one end of the handle.’ The flaky graphite of Bavaria had been worked from prehistoric times for mixing with clay to form pottery, and the Passau or Ips crucibles, in which this material was employed, were widely used, but, it does not seem to have been employed for writing so early a8 that from the Borrowdale mine, near Keswick, in Cumberland, which mas for some three centuries the principal source of supply of the mineral for this purpose. Metallic lead and silver were used both in ancient and mediaeval times for drawing lines. Subsequently in the early renascence an alloy of two parts of lead nnd one of tin, known as ( ( lo stile del piombo ” or ‘( lapis piombino,” was employed for drawing3 These terms were probably transferred to graphite when it came into use. Gesner appears to have had this alloy in mind when he wrote, but the name ‘( stimmi anglicum ” shows that it was in fact Borrowdale graphite. g p In the “Sarepta” of Nathesius, from which I have already quoted, we read (Predigt ix, p, 1036) : “ Wie man hernach mit silbern stefften auff die hultzern weissen plancketen ocler tefelein, oder mit bleyeneo auff die gefirnsten pergamenenen und mit dinten auff die Eselsheute, und jetzt auff schiferne tnfeln mit schifersteiu oder auffs papir mit einem newen unnd sclbwachssenen metal1 zu- schreiben pfleget.” 148 THE MEANINGS ASD SYNONYMS OF PLUMBAGO. we ead, Ot e s w t b ac e eade t ust a qu . 4 J. Otley, ‘‘ Account of the Black lead Mine in Borrowdale.:: : Memoirs of the Literary and Philosophical Society of Manchester, ser. 11, vol. 111, 1819, p. 168. 6 G . Jars: “Voyages Netallurgiques,” vol. ii, 1780, p. 554. I ‘ Mine de plomb pour les crayons nommes Black-lead ou Wad-Lead.” 1 If, as is no doubt the ewe, these words occur in the first eaition issued in 1562, this is the earliest unmistakable reference to graphite. , g p 3 The terms in which Dioscorides refers to these minerals precludes the possibility of either of them being graphite. p y g g p 3 The use of black lead is again referred to in the “ Ludus Literarius, or the Grammar Schoole,” by John Brinsley, pFblished in 1612, where (p. 47) the reader is told to “ note them with a pensil of black lead” ; and in the margin we read, I‘ Others with blacke leade thrust into a quill.” ‘‘ 1 If, as is no doubt the ewe, these words occur in the first eaition issued in 1562, this is the earliest unmistakable reference to graphite. 3 The terms in which Dioscorides refers to these minerals precludes the possibility of either of them being graphite. 3 The use of black lead is again referred to in the “ Ludus Literarius, or the Grammar Schoole,” by John Brinsley, pFblished in 1612, where (p. 47) the reader is told to “ note them with a pensil of black lead” ; and in the margin we read, I‘ Others with blacke leade thrust into a quill.” 4 J. Otley, ‘‘ Account of the Black lead Mine in Borrowdale.:: : Memoirs of the Literary and Philosophical Society of Manchester, ser. 11, vol. 111, 1819, p. 168. 6 G . Jars: “Voyages Netallurgiques,” vol. ii, 1780, p. 554. I ‘ Mine de plomb pour les crayons nommes Black-lead ou Wad-Lead.” TRANSACTTONS OF THE PHILOLOGICAL SOCIETY, This new and self-grown metal that was used for writing on paper can scarcely have been anything else than graphite.’ This new and self-grown metal that was used for writing on paper can scarcely have been anything else than graphite.’ p p y y g g p A few years later we find mention of graphite in English books under the name of ‘ black lead.’ In the “Jewel1 House of Art and Nature, containing divers rare and profitable Inventions,” by Eugh Platte, 1583, we read: “upon the which you may trick, either with a fine pointed Cole, blacke lead or pen ” ; ‘‘ and drawe thereon with blacke lead” (p. 39, ed. 1594). (p , ) In the second edition, published in 1587 (but not the first, published in 1586), of Camden’s ‘‘ Britannia,” there is (p. 523) the following reference to the Borrowdale mine : ‘‘ Hic etiam passim reperitur terra illa rnetallica, sire saxum induratum et micans, nobis Black Leade dictum, quo ad ducendas lineas, et mono- chromata pictores utuntur. Quod an sit Dioscoridis Pnigitis, vel Melanteria,a vel ochra terra calore in nigrum adusta, aut veteribus incognitum, non facile dixerim, et perquirant alii.” This passage is literally translated in the first English edition of Camden, published in 1610 (p. 767). “ Heere also is commonly found that mineral1 kind of earth or hardned glittering stone (we call it Black-lead),s with which painters use to draw their lins and make pictures of one colour in their first draughts,” etc. p g The mines of Borrowdale were included in the manor of the same name, which, having belonged to the Abbey of Furness, passed to the Crown in the reign of Henry VIII. It was granted by James I, before the end of the year 1614, to William Whitmore and Jonas Verdon, with ‘‘t,he Wad Holes and Wad, commonly called black Cawke, of the yearly rent or value of fifteen shillings and fourpence.”4 The word ‘cawke’ is a form of ‘cauk’ or chalk, but is usually applied to the mineral barytes, sulphate of barium. is now applied to the soft hydrous binoxide of manganese. Its Wad,’ also written ‘ wadt ’ or ‘ wadd ’ or ‘ wad-lead,’ THE MEANINGS AND SYNONYMS OF PLUMBAGO. 149 use in the sense of graphite appears to be earlier. It appears from the statements of Camden (loc. 1 Apparently ,uohuSSosrS+p h[Oos, as understood by Falloppius, included (ilzter aIia) gTaphite, for it was ‘‘lapis nihil plumbi in se continens,” which mas employed by the potters ( ‘ I De Metallis seu Fossilibus,” cap. xxv, ed. 1606, p. 327). Others (e.g., Entzelt, loc. cit. ; L. Fuchs, ‘I Oper. Didact.,” Pars 11, De Compos. &led., ed. 1604, p. 65 ; A. Libavius, “Comment. Alchym.,” ii, ed. 1606, p. 116) followed Agricola in considering it to mean our modern galena, as well as in other respects. Falloppius used molybdma and plumbago for the furnace product, lac. cit., cap. xxyi. Th iffi l i d h d b i i f h i h Several subsequent writers took the same view. p y 2 The oriffinal matita was red, the word being a corruption of hpmatites, the red oxide ofairon. TRANSACTTONS OF THE PHILOLOGICAL SOCIETY, Cssalpinus seems to have thought that both were varieties of lead ore : ‘‘ Hi lapides si urantur, in Lythargyrum vertuntur, ut vena PIumbi.” I n the “ Historia Naturale ” of Ferrante Imperato, published at Naples in 1599, me read in book iv, chapter 43, p. 122 : ‘( I1 grafio piombino si preferisce a tutte le materie, che preparino il disegno alla penna e l’inchiostro : percioche facilmente, usandovi industria, si cancella: e no volendo cancellurlo si conserva. Non da im- pediment0 a1 maneggio della penna, il che fa it piombo per un modo, e il carbone per un’ altro . . . i! ontuoso a1 tatto: e a1 fuoco sommamente indurisce. Puosi rngionevolmente locare nel geno de talchi.” ‘ Grafio‘ is an Italianized form of the Latin graphiuna, a stile, corresponding to the Greek ypa@Ls, also used in Latin with the Same meaning. The form ‘ graffio’ is employed by Gimma (op. cit., vol. ii, 296, 291), who includes in it ‘ lapis bianco,’ ‘ lapis nero ’ or ‘ ampelite,’ ‘ lapis romo ’ or ‘ ematite,’ and ‘ graffio piombino.’ ‘ Ampelite ’ is also described as ‘ terra nera,’ and comprises both the French ampelite and graphite. p g p In book xxv, chapter 6, we find a further description of graphite, and also, it may be, molybdenite, under the heading “Gleba Piombina e congeneri ” : “La Gleba piombina B di color bigio, e di piombo, lubrica nell’esser maneggiata, e eh‘imbratta le mani, . . . ritrovasi parte fogliosa che si rissolve tutta in scame: parte con- sistente in forma soda, qua1 si taglia in fette lunghe, e se ne fa il grafio detto piombino ” (p. 678). Its use for crucibles is referred to. In book xxvi, chaptor 2, p. 694, ‘ moludena’ is employed in the old sense of litharge, obtained in refining silver and gold by means of lead. In book xxv, chapter 6, we find a further description of graphite, and also, it may be, molybdenite, under the heading “Gleba Piombina e congeneri ” : “La Gleba piombina B di color bigio, e di piombo, lubrica nell’esser maneggiata, e eh‘imbratta le mani, . . . In book xxvi, chaptor 2, p. 694, ‘ moludena’ is employed in the old sense of litharge, obtained in refining silver and gold by means of lead. TRANSACTTONS OF THE PHILOLOGICAL SOCIETY, cit.) and others that German miners were employed in Cumberland in the reign of Elizabeth, and it is possible that the first part of Wasscr-blei,’ the old German name for graphite, may be a corruption of the Cumberland term, or, on the other hand, the latter may have been introduced from Germany. y The term black lead was also employed in England in the sense of metallic lead, as a transIation of the Latin ‘plumbum nigrum.’ It is thus used in Trevisa’s translation of the ‘{De Proprietatibus Rerum ” of Bartliolomsus Anglicus, made towards the end of the fourteenth century, and first printed about 1495. “But of blacke leed is dowble kynde. For blacke leed comith alone of a veyne : other is gendred wyth splver in medled veynes” (xvi, 80). Black lead was used in the same sense in ‘ L A greene Forest, or a Natural1 Historie,” by John Marplet, published in 1567 (p. 13). There can, however, be but little doubt that at this period the term usually signified graphite from the Borrowdale mine. Andreas Caesalpinus of Arezzo, in his ‘‘ De Metallis libri tres,” first pubIished at Rome in 1596 (book iii, cap. 22, p. all), employs ‘ molybdaena,’ ‘ plumbago,’ and ‘ galena ’ in the same manner as Pliny and early commentators on Dioscorides. He refers elsewhere (book iii, cap. 8, p. 186) to graphite in the following terms : “ Puto autem molybdoidem [viz. the ‘ p o X v ~ G o e ~ G j s XLBor’ of Dioscorides ’1 esse lapidem quendam in nigro splendeiitem colore Plumbeo, tactu adeo lubrico, ut perunctus videatur, manusque tangentium inficit colore cinereo, non sine aliquo splendore Plumbeo: utuntur eo pictores coticulis in cuspidem excisis, ad figuras designandas : appellant autem lapidem Flandriae, quia ex BeIgia affertur.” This is evidently Borrowdale graphite, which entered the Continent by way of Belgium. He, however, confounds it with bismuth, and says it was used in casting type. He also refers to another ‘ genus ’-“ nigrum ut carbo et crustosum, quem pictores Matitam Phil. Trans. 1908. Phil. Trans. 1908. 11 11 150 THE MEANINGS AND SYNONYMS O F PLUMH.4GO. nigram vocant.” This may have been B graphitic or carbonaceous shale, the French ampelite (see ante, p. 136). 1 Many later ivriters also referred to graphite as a variety of black talc. Talc, it must be remembered, included (as it still does to some extent in popular use) not only the talc but the mica of modern mineralogists (see also pp. 161-73). TRANSACTTONS OF THE PHILOLOGICAL SOCIETY, Graphite, including probably molybdenite, is also identified with the pokvP6o~d$s XtOop of Dioscorides by Francisco Imperato in hi8 “De fossilibus opusculum,” published at Naples in 1610: ‘ I Molubdoides, seu plumbaris lapis a plumbo est longe diversus, licet in plumbi venis reperiatur, cuius succum tantum emittit ; et propterea pictores ill0 ad designandum utuntur ; nonnulli stimmi anglicum illum appellitant, propter similitudinem ; verum D molgbdsna Plinii differt, quam Galenam nuncupant, ut THE MEANINGS AND SYNONYMS OF PLUMBAGO. 151 infra ; at ex dicto plumbari qusedam construuntur wsa ad aurum, ac argentum purgandum destinata, necnon alterum ab altero separandum ; nam d d e ignis repugnat potentise ” (p. 58). On the other hand, it is distinguished from galena, molybdsena, and plumbago as used by Pliny : ‘( Interdum quoque aurum, cuprum, et argentum, quod lapidum genus, plumbum, et argentum continens, communiter Galena nuncupatur, seu molybdena apud Plinium ; quo nomine appellat etiam illud, quod in fornacis parietibus inharet, durn plumbum cliyuatur, admisto auro, vel argento, quod vere molybdense, seu blumbaginis’ nomen retinet; Bed ambs a puro plumbario lapide distant, qui ejus colorem tantnm refert, non autem plumbum, nec ejus pondus habet, et molybdoides nominatur ut supra” (p. 92). ’’ The ‘( Lexicon Alchimiq ’’ of Martinus Rulandus, published at Frankfort in 16 12, distinguishes between molipdina and molybdenn. The former being described as “ Gold haat, oder silber haat oder Triisen.” The explanations of molybdena, plumbago, and galena are extracted from previous authors. An English translation appeared in 1892 bearing the date of 1612 and printed in imitation of the style of that time. Most unwarrantably the words referring to plumbago “ factitia,” “ a Germanis dicitur Thest, oder Herd- klegen,” * are translated ‘‘ it is called by the Germans Graphite, or Compressed Galena,” giving the incorrect impression that the word ‘graphite’ was in use in its present sense as early as the beginning of the seventeenth century. I n another place the words “ Plumbago, galena et molybdena, m u m et idem sunt” are rendered “Plumbago, Galena, and Black Lead are one and the same,” quite misrepresenting the meaning. A little later we find galena in use in the sense of graphite, as the following extracts from the ‘‘ De Pictura plastice statuaria libri duo ” of Julius Csesar Bulengerus (Boulenger), a Jesuit father, published in 1627 at Leyden, will show (pp. p “ Thest oder Herdtbley.” * This is apparently taken from L. Fuchs, loc. cit., where, however, we read See p. 143 ante. * TRANSACTTONS OF THE PHILOLOGICAL SOCIETY, 103-5): “Ante omnia sciendum est, aliud esse levi manu sine coloribus adumbrare creta, sive rubrica, carbone, terra sanguinea, vel galena, seu ~ ~ ~ G u L ’ v ; : I , quam Plinius lib. 34. cap 18. venam plumbi, et argenti communem esse ait; aliud, coloribus adhibitis pingere . . . Pictoris stylus, seu cretacea graphis, est frustum oblongum rubrice aut terrae sanguine%, aut carbo oblongus, aut plumbea graphis, seu THE MEANINGS AND SYNONYMS O F PLUMBAGO. 152 designatrix galena pictoris, vulgo dicitur, crayon, charbon, crayon de mine de plomb de mer, marquant de ,oris. Opus graphide adumbratum, rubrica, aut plumbo designata pictura. Rudis, et informis designatio totius operis, carbone, plumbo, vel rubrica impolite designatum opus.” Again, “ pastquam rudi Minerra carbone, rubric6 vel galena sine coloribus adumbraverimus,” and “ I n rudi illa designatiane, quze carbone fit, aut creta, utimur cretacea graphide, stylo rubricato, vel frusto oblongo rubrics, carbone, plumbo.” ‘ p Here “ plumbea graphis,” ‘< designatrix galena pictoris,” and similar expressions appear to be synonyms, represented in French by “crayon de mine de plomb de mer,” and can only refer to graphite. ‘ Plomb de mer ’ was often used for ‘ graphite ’ in the seventeenth and eighteenth centuries. It is probably a trans- lation of the German ‘ Wssserblei.’ Another Jesuit, Bernardus Caesius, quotes Bulengerus almost verbatim in his (‘ De Mineralibus,” published at Leyden in 1636. On the other hand, he describes plumbago as (1) ‘( communem venam plumbi,” (2) “ purissimum plumbum, quod ignis ustione, cineris speciem induit ” (pp. 257, 613, and 625). ‘‘ p pp In the ‘‘ Museum Metallicurn ” of Ulgsee Aldrovandi of Bologna (Parma and Placentia, 1648), we read, p. 167: “Plumbago aliquibus dicitur erugo plumbi : attamen Plumbago proprie est materia nuncupata Galena, quie, post fusionem plumbi in fundo fornacis remanet. . , . Terum, apud Plinium, Plumbago triplex constituitur : prima species est Galena vocata, quae argentum, et plumbum participat, secunda est plumbago, seu Molybdena arti- ficialis turn Plinii, tum I)ioscoridis,2 tertia est substantia qusdam fossilis lapidosa, que Plumbago vel Molybdena naturalis vocatur.” He afterwards quotes the passage in Csesalpinus which refers to graphite under the name of ‘molibdoiiles,’ and continues: “ Hujus lapidis aliud genus crustosum, et instar carbonis, nigrum invenitur, quo similiter Pictores utuntur. Ad nostros pervenerunt manus duo lapides crustosi, seu potius escharam zemulantes cum aliquo livoTe plumbi . . . , , y p g See also op. cit., p. 182 : “Et quod in fundo catimi remanebat ad mentem Plinii Galena nempe Plumbago vocabatur.” He writes, however, ‘ molybdena ’ for pohuB6aivg. He writes, however, ‘ molybdena ’ for pohuB6aivg. He writes, however, ‘ molybdena ’ for pohuB6aivg. See also op cit p 182 : “Et quod in fundo catimi remanebat ad mentem y p y ( pp , , ) 2 I n the Notitia R e 4 Mineralis” of Jobannes Jonstonns (Leipzig, 1661), however, galena is used ?or an ore of lead and silver, ‘‘ melior quo magis aurei coloris ” ; galena factitia and molybdaena for the artificial oxide, and plumbago, ‘‘ si solum sterilis ab igne consumitur, coloris magis lurido ” (p. 59). 1 Blyerts became the ordinary Swedish term for graphite, hut the German Bleiertz was only exceptionally used in the same sense (see pp. 165, 167, 172). I “Galena (forte a yahsiv splendere ; quod instar Argenti vens spiendeat) ex qua Metalla excoquuntur, vel Plunibum solum (et turn Plumbago proprie dwitur, Anglice, Lead Ore) vel et Plumbum et Ar entum (et turn Galena audit),” p. 298. “ Plumbago Graecis Moh@Baiva a &umb [sic] appellatione sumpta. Fit enim ex Plumbo,” etc., p. 307. 2 I have been uneble to see the original from wbicb this was taken, but the I‘ Pharmaco ceia Schrodero-Hoffmanniana,” published at Cologne in 1684, reads (p. 307) L‘ holybdsna seu plumbago . . . nativa et factitia,” and continues in the same sense as the English translation. . 253 there is a note by Hoffmanu :-“ De Plumbo Scriptorio oder Wasserbyey sciendum, vocari illud Molybditem Lapidem, de quo erudite scripsit Cesalpiniis, et primam facit speciem lapidis plumbarii; . . . Multo lsvior ac friahilior minera Plumbi nigri parurn aut nihil fere Argenti in Be habens. Exteri et in primia ltali hoc crudum a Nobis petunt, illudque erepolientea denuo iterum ad Nos remittunt pro usu smiptorio.” This is the first mention of Wasserblei I hare found, but the word probably came into use much earlier. On TRANSACTTONS OF THE PHILOLOGICAL SOCIETY, Primus vocabitur Escharites niger molybdoides, secundus dicetur Escharites cum aliquo rubore molybdoides alter.” These specimens apparently consisted either of molybdenite, graphite, or graphitic or carbonaceous shale. 153 THE MEANINGS AND STKONTMS OF PLUhIBBGO. Later on, p. 177, he adds--“ Insuper Pictores, ut nonnulli asseverant, ad imagines designandas, stylo ex materia plumbea parato utuntur. Hodie hujusmodi stylus ex lapide plumbario fabricatur . . . Aliqui ad signandas chartas modico plumbi acuminati utuntur, idque Piombino nominant. Reprssentamus hie iconem styli ex cujusdam plumbi genere facti in mucronem derasi, et manubrio ligneo inserti. Putat tamen Gesnerus esse genus plumbi factitii, quod apud aliquos Stimmi Anglicum appellatur.” He gives a copy of Gesner’s drawing. pp g py g Ole Worm, a Norwegian, in his “ Museum Worniianum,” published at Leyden in 1655, foIIows Agricola and Entzelt in identifying galena, plumbago, and molybdaena (spelt molybdena) with our modern galena, while he distinguishes plumbago, instead of molybdiena, as containing lead alone, from galena, with lead and silver. He admits, however, that some believe galena, plumbago, and molybdsna to be “tria diversa corpora” (p. 127). At the same time he treats graphite as a variety of galena. ‘‘ Ex nostris officinis Norvagicis, frugiferse Galens tria genera accepi, unum quod Plumbum refert, et manus plumbeo colore inficit, quo etiam ad lineas ducendas utimur, vulgo plumbago Bley-ertz (nobis Bleyas) vulgaris pictoribus usus ” (p. 128). The second genus was a liver- coloured ore associated with native silver ; the third, perhaps, granular galena. His sterile galena “ coloris magis lurido ” may possibly be zinc-blende. He also refers (pp. 128, 135-6) to the use of the words molybdaena, plumbago, and galena for furnace products of the nature of litharge. The two former words continued to be empIoyed in this way for some time after galena had become restricted to the sulphide of lead, with or without silver.2 p I n the latter part of the seventeenth century increased interest was taken in mineralogy in this country. ‘‘ ‘‘ I n the ‘‘ ~ T C L U O ~ V K T O X O ~ ~ ~ , sive Panmineralogkon,” or ‘‘ An Universal History of Minerals,” by Robert Lovell, published at Oxford in 1661, we find (p. 38) the following: “Plumbage, PZum- hago [Latin]. P[Iace]. It sticks to the furnace in the purifying 154 THE MEANINGS AND SYSONYMS OF PLUMBAGO. of SiIver or Gold. M[atter]. TRANSACTTONS OF THE PHILOLOGICAL SOCIETY, of Silver or Gold purified, with lead. N[ame]. MoX+?;PGULV~, Molybdsena.” ; , y Apparently none of the words galena, plumbago, or molybdens were yet associated in this country with graphite, for in the Pinax Rerum Naturalium Britannicarum ” of Christopher Merrett, 1666, p. 218, we read : l L Nigrica fabrilis, Black ledd, Peculiaris haec terra Angliie Europaez et American= et bactenus nomine caruit, ideoque faveute Analogia hoc ipsi imposui ad Keswick, in Cumbria. . . . Lapis cseruleus Killow dictus ducendis lineis idoneus in Agro Lancast.” Walter Charleton, in ‘ I De Yariis Fossilium Generibus,” appended to his Onomasticon Zoicon ” (1668), follows Yerrett in the use of “nigiica fabrilis” for graphite (p. 219), and the “Xuseum Wormianum” with regard to the meaning of the words galena and plumbago,’ exoept that there is no hint of the use of galena for graphite. “The Compleat Chymical Dispensatory, in Five Books . . . Written in Latin by Dr. John Schroder . . . and Englished by William Rowland ” (1669), contains the following (p. 246) : “ Molybdaena, or Plumbago. It is Natural, or Artificial ; the first is Lead Oar, or that mixed with Silver. The Artificial is a kind of Litharge.” This is chiefly of interest as being one of the very few instances in which plumbago has been used in English for compounds of lead.z Of more importance is the ‘‘ Metallogaphia, or An History of Metals,” by John Webster (1671), who refers (p. 20) to Camden’s mention of I‘ a Mine of Black Lead, for which we yet want a Latine name, but that of late Dr. Merrett hath given it the title of Nigrica.” On p. 280 he returns to the subject : ‘‘ Here it cannot THE MEANISGS AND SYSONPMS OF PLUMHAGO. 155 be amiss to say something of that which we commonly call Black- Lead, because it discoloureth the hands far more then common Lead, and is that whereof Pencils are made for Painters and Scriveners, and many other such like uses. In the North we usually callit Rellom, and some call it Wadt.” Kellow or killow was applied not only to graphite, but to a soft, black, earthy mineral, possibly a carbonaceous or graphitic shale. The word is usually derived from the North Country Collow or Colley soot,’ but Dr. Xfurray believes that the change in the first rowel is improbable. 1 Beckmann (op. cit., \-ol. v, 1503, p. 246; vol. iv, 1814, p. 354, states that See both wad and killow or collow meant ‘ black’ in the Cumberland dialect. ante, pp. 148, 154, andpost, pp. 158-9, 163. 8 The Crown is entitled to all silver-mines. See also p. 205 ; on p. 221 we read ‘‘ in Galena inani, Fhich the Germans call Blend, and our miners in the North, Blue Blindake ” ; and me p. 250. 4 “There is also a mineral Lead, which we call Black Lead, something like Antimony, but not so shining or sollid; . . . of late, it is curiously formed into cases of Deal or Cedar, and so sold as dry Pencils.” Laws, part ii, under “ lead” ; Fodina, pp. 5, 7. 1 Beckmann (op. cit., \-ol. v, 1503, p. 246; vol. iv, 1814, p. 354, states that See both wad and killow or collow meant ‘ black’ in the Cumberland dialect. ante, pp. 148, 154, andpost, pp. 158-9, 163. h ll TRANSACTTONS OF THE PHILOLOGICAL SOCIETY, On pp. 344-5 me read : “ All that we shall say here concerning Galena, Plumbago, Lapis Plumbarius, and Molybdma . . . is that there is much said to little purpose, and that in some respects they may be taken for all one ; . . . I hold that the main difference lieth in this, that it is to be accounted Galena when it holdeth a sensible quantity of Silver, or however when it holdeth as much Silver as may make it a ;\line Royal: but if it hold no sensible quantity of Silver, then it may be called Plumbago; and this I wish every Test-master and every Miner seriously to mind and consider of.” The “Fodina? Regales” (1670) and “Laws of Art and Xature,” 1683, by Sir John Pettus, contain references to black lead,’ but no use of the words galena, plumbago, or molybdsena. The uncertainty that prevailed in the use of these words is well illustrated in the (‘ Mineralia ” of Joachim Junge, published at Hamburg in 1689. It appears to have been compiled from the author’s notebooks after his death, and consists largely of extracts from previous works, with notes, queries, and suggestions. Molyb- daena (with plumbago as a synonym) appears to be employed in the sense of litharge (p. 163). The artificial form is distinguished as molybclsena fornacum,” and the mineral as “molybdsna 156 THE MEANINGS AND SYNONYMS OF PLUMHAGO. metallica, fossilis,” while the sulphide is referred to as ‘‘ galena non flava.” Then follow two queries :-“ Ob Tetting wisse was und woher das schreibblei sei ? Obs aus Engelland”; “ bei Lann- giessern‘ zu fragen. Ob sie wissen wasz bleyertz (damit wir schreiben) sei: und woher es komme. etliche nennens wasser- bley.” Under L( Observanda” we read, “ Plumbago scriptoria nee lapis plumbarius est G. Agric. quia hic durior stibio, nec plumbago G. Agric. quia haec flava est.” In a note (p. 166), apparently made at a subsequent date, under the title “ Plumbago Anglica sive Galena inanis,” he refers to “Terra illa metallica et micans Anglis blaclre-leade dicta,” and after quoting further from Camden, continues : ‘ I Anglica hsc plumbago nee lapis plumbarius sive plumbi speciem gerens, nec plumbago est secundum Q. Agricolam quia lapis ita durus est ut f a d e teri non possit, et plumbum continet, interdum etiam una argentum. Ex eo [viz. p y Galena inanis or sterilis, blende, and pseudo-galena continued to be used in a very general sense. In the ‘ Mineralogia” of J. G. Wallerius (1747), p. 249, for instance, Beckbliinde is defined as ‘‘ Pseudo galena picea.” See also ‘‘ De Matricibus Metallorum,” by J. G. Hoffmannand J. B. Bcehmer (1738), pp. 68-9, and A. G. Monnet, “Syst&me de MinBralogie” (1779), p. 180. Kevertheless, as we have seen (p, 142 ante), the most usual signification was zinc-blende or sulphide of zinc. 1 Smelters who cast pewter or other metals or alloys into various vessels. TRANSACTTONS OF THE PHILOLOGICAL SOCIETY, lapis plum- barius} ad rubedinem exusto fit minium secundarinm menninge.” He says that the galena inanis or blende of Agricola seems to differ from the galena ‘‘ simpliciter ” or Glantz of the same author, “non u t inane a fertili sed specie.” The former he renames pseudo-galena, a name employed by several subsequent writers. At the same time he sags that plumbum scriptorium appears to differ from galena sterilis.’ g As Junge died in 1657, his reference to plumbago scriptoria and plumbago anglica constitutes the earliest known definite use of plumbago in the sense of graphite, though, as I have shown (pp. 151-31, galena and even molybdBna had been so used. In the ‘‘ Teutsche Material Kammer ” by J. J. Marx, published at Nuremberg in 1 6 8 i , graphite is referred to (p. 7 8 ) as “Cerussa nigra. schwartz Bleyweiss,” while in the “Vollkommenes Lexicon,” which forms an appendix, plumbago fossilis appears as synonymous with Bley Aertz and Bley Sohweisz, plumbago with Bley Glantz, and plumbago Plinii with Molybden and Molybdaena. As Junge died in 1657, his reference to plumbago scriptoria and plumbago anglica constitutes the earliest known definite use of plumbago in the sense of graphite, though, as I have shown (pp. 151-31, galena and even molybdBna had been so used. ‘‘ In the ‘‘ Teutsche Material Kammer ” by J. J. Marx, published at Nuremberg in 1 6 8 i , graphite is referred to (p. 7 8 ) as “Cerussa nigra. schwartz Bleyweiss,” while in the “Vollkommenes Lexicon,” which forms an appendix, plumbago fossilis appears as synonymous with Bley Aertz and Bley Sohweisz, plumbago with Bley Glantz, and plumbago Plinii with Molybden and Molybdaena. p g y y In the “Histoire Generale des Drogues,” by Pierre Pomet, published at Paris in 1694, we read :-(part iii, p. 42), L L Le 157 THE MEANINGS AND SYNONYMS OF PLUMBAGO. Troisidme Plomb mineral est tout au contraire fort usit&, et est ce que nous appellons Mine de Plomb noire, Plomb de Nine, ou crayon, parce quc le plus parfait sert 2~ designer. Les Anciens luy ont donne le norn de Plombagine et de Plomb de mer, en ce qu’ils ont pretendu qu’il se tiroit du fonde de la mer ; les Etrangers [the Dutch] le nomment Potelot ” (viz. pot-lead).’ A translation into English, with additions from other sources, appeared in 1712. Here (vol. In the ‘ Museum Museorum ” of M. B. Valentini, published at Frankfort in 1704, graphite is referred to as ‘‘ Wasserbley welches sonsten plumbago, cerussa nigra . . . genennet wird.” See, however, Gimrna, op. eit. (1730), ii, p. 141, who, however, only quote9 previous authors. TRANSACTTONS OF THE PHILOLOGICAL SOCIETY, ii, p. 351) we find: “The third sort of Lead Oar is very much us’d, and ’tis that we call Black Lead, or Crayon, . . . The Ancients gaye it the Name of Plumbago, and of Sea Lead.” This statement, which represented for a long time the only use of the word plumbago for graphite in English, is repeated in the subsequent editions of 1737 and 1748. Sir’ John Hill, who was actor, playwright, physician, botanist, zoologist, and mineralogist, and in more than one capacity called forth the satire of Garrick and the discriminating condemnation of Dr. Johnson. He supplied a note:-“The Nolybdana or Plumbago is a substance of the Litharge kind, . . . Black Lead is the Nigrica Fabrilis, Charlt. Foss. 2. Massa Nigra ad Pnigitem referenda, Worm. 5, . , . It is rather an earth than a metal.” The latter was edited by the versatile As a matter of fact molybdana and plumbago had long since ceased to be used in the sense of litharge. Plumbago led the way in this respect, for in Blancard‘s ‘( Physical Dictionary ” (second edition, quoted in the Oxford Dictionary), published in 1693, we find an explanation of the word molybdena which is evidently taken from SchrGder, but the word plumbago is dropped. It is significant too that in the Latin edition, published at Leipzig in 1695, of the “ Historia Naturale ” of Ferrante Imperato, we find (p. 133) ( plumbago ’ substituted for ‘ grafio piombino ’ in the sense of graphite, while ‘ molibdtena ’ is still used like moludena’ in the sense of litharge (p. 787). This use of molybdana did not, however, extend, except for Hill, beyond the first quarter of the eighteenth century.? On the other hand, there is, as I have said, no evidence outside the translation of Pomet of the use of plumbago for graphite in England. For instance, in ‘( Some Observations concerning the THE MEANINGS AND SYNONYMS OF PLUMBAGO. 158 substance commonly called Black Lead,” by the “late Dr. Rob. Plot, F.R.S.” (Phil. Trans., 1698, p. 183), we are told that “The mineral substance, called, Black Lead (our common Lead being the true Black Lead, and so called, in Opposition to Tin, which is the White Lead) found only at Keswick, in Cumberland, and there called, Wadt, or Kellom ; by Dr. Merrett, Nigrica Fabrilis, . . . * After discussing its various appiications (except, curiously enou h, its manufacture into pencils) he concludes : “ for these and other Uses, it’s %oughh up at great Prices by the Hollanders, and others,” * p g y , , * A French translation of this wa3 published at Paris and Amsterdam in 173.5, an Italian at Venice in 1139, and a German at Erfurt in 1746. See J. E. J. Walch, s’ Dns Steinreicfi ” (1169), i, p. 11. * After discussing its various appiications (except, curiously enou h, its manufacture into pencils) he concludes : “ for these and other Uses, it’s %oughh up at great Prices by the Hollanders, and others,” * A French translation of this wa3 published at Paris and Amsterdam in 173.5, an Italian at Venice in 1139, and a German at Erfurt in 1746. See J. E. J. Walch, s’ Dns Steinreicfi ” (1169), i, p. 11. TRANSACTTONS OF THE PHILOLOGICAL SOCIETY, Among the lead ores he refers to L i the sparkling or Steel-grain’d ; this commonly yields more or less Silver and is what Dioscorides, and the Naturalists after him, call Molybdaena : Pliny, Galena.” Among “Mock ores ” he mentions “ Blind,” “ Blend,” and “ Black Talk, or as the Germans call it, Ste5ile-Nigrum ” ; the last may be molybdenite or a variety of graphite.’ Among the lead ores he refers to L i the sparkling or Steel-grain’d ; this commonly yields more or less Silver and is what Dioscorides, and the Naturalists after him, call Molybdaena : Pliny, Galena.” Among “Mock ores ” he mentions “ Blind,” “ Blend,” and “ Black Talk, or as the Germans call it, Ste5ile-Nigrum ” ; the last may be molybdenite or a variety of graphite.’ y y g p I n the “Attempt towards a Natural History of the Fossils of England” black lead and galena are referred to in similar terms.2 I n the portion entitled 6 L A Catalogue of the Foreign Fossils in the Collection of J. Woodward, M.D.,” we find (p. 29), under the heading l‘Nigrica fabrilis, Black Lead or Wad,” a specimen described as ‘( Lapis Plumbarius sterilis, cum quo Scribi potest. Altenbergae in Saxonia. Wasserbley Ertz ; i.e. Black-Lead Ore. N. de Schonberg. (’Tis the Nigrica fabrilis or Black Lead.) ” The locality leaves, however, little doubt that this was not graphite, but molybdenite. The following labels (pp. 37-40) are also of interest : ‘ 6 Plumbago super Pyritem aureo colore. Fribergs in Sasonia. Silberhaltiger Blcyschweiff nff Kupffer Eies.” This is argenti- ferous galena associated with copper pyrites. “ Plumbago in Talco cinereo. Rnebergae in Saxonia. Bley-glantz in grauen talc, i.e. Lead-Glitter in grey Tale. N. de Schonberg.” Plumbag? is explained in similar terms on two other labels. The expression ‘lead glitter ’ is a very fair translation of ‘ Bley-glantz,’ but it was not adopted by other authors. Plumbago tesselata. Fribergs in Saxoniq. Wurfflicht Glantz-Ertz, i.e. Diced Glitter Ore. M. de Schonberg.” There are several references to galena. “An addition t o the Catalogue of the Foreign Native Fossils in the collection of J. Woodward, N.D.” (1725), includes the following entries (p. 16): Plumbago ad Altenburgia [ ? Altenberg], ex Minis Stanni. Dr. Henckell. This is Wad, or Black-Lead, with White-Spar.” The association with tin ore makes it probable it was molybdenite. pp p y y g p 3 Dr. A. Hutchinson, of Pembroke College, Cambridge, has kindly examined the specimens referred to, whjch are still preserved in the Woodwardian Museum, with their original labels, and has enabled me to verify my surmises as to their real nature. 1 See p. 8 of the ‘‘ Methodica Distributio,” and pp. v, 2, 3, 43, 55, and 56 of the “Fossils of all kinds.” ’ 2 pp. 185, 211. I n some eases the ‘black lead ’ is stated to be connected with copper ore ; it mould therefore probably be molybdenite, not graphite. TRANSACTTONS OF THE PHILOLOGICAL SOCIETY, whence the most Proper Name that can be given it, perhaps, may be Ochra Nigra, or Black Ochre.” Nowhere is there any mention of plumbago. p g The same is the case with the “Natural History of Westmoreland and CumberIand,” by Thomas Robinson, published in 1109, where “ Wadd, or Black-Lead” is described as a “ black pinguid and shining Earth.” g In the early years of the eighteenth century there was a com- parative dearth of textbooks on mining and mineralogy. Our chief information is obtained from the writings of Dr. John Woodward : his “ Methodica Fossilium in Classes Distributio,” an appendix to his ‘‘ Naturalis Historia Telluris ” (1 714) ; his ‘ I Fossils of all kinds Digested into a Method suitable to their mutual Relation and Affinity” (1728); and “ An Attempt towards a Natural History of the Fossils of England,” an explanatory catalogue of his collection, which was afterwards presented to the University of Cambridge. Bound up with the latter are subsidiary catalogues of additions and of the portions of his collection obtained from abroad. The details in the latter are, he tells us, copied from the labels on the specimens, and accordingly furnish us with information of the contemporary use of mineralogical terms on the continent of Europo. This catalogue bears as a whole the date 1729, but portions appear to have been issued earlier. Both in the (‘ Methodica Distributio ” and in the “ Fossils of all kinds ” he identifies nigrica fabrilis with wadd and black lead. I n the latter publication he also distinguishes between the softer killow (Killoia molliuscula) and the harder killow (Killoia duriuscula). The former is described as ‘‘ of a blackish or deep blue Colour, and, doubtless, had its name from Kollow, by which name in the North, the Smut, or Grime on the 3aeks of Chimneys, is call’d.” 159 THE MEANIKGS A N D SYXONYhZS OF PLUIBAGO. TRANSACTTONS OF THE PHILOLOGICAL SOCIETY, ‘‘ Molybdena grossior grober bleyglantz, i.e. Coarse Lead, shining, hic ubivis obvia, continens 60 Libras Plumbi, et 1, 2, 3. Lotos Argenti. Saxoniae. Dr. Henckell. ” “ Xolybdena, Granis minutioribus Saxoniae. Dr. Henckell.” 160 THE MEANIKGS AND SYNONYMS OF PLUMBAGO. These and other labels illustrate the variations in the use of these words at this time. Plumbago is used by Dr. Schonberg for the mineral galena, and by J. F. Henckel, the author of the “ PyritoIogie ” and other mineral works, for molybdenite and probably graphite. Molybdgna is employed both by Woodward and Henckel in the sense of lead sulphide. GaIena is used by Scheuchzer with the same meaning, but Woodward, De Schonberg, and Leopold apparently. employ it to include other sulphides. I have been unable to find any of these words in Henckel’s German publications, but in a note in Latin on Zinc, Observatio Ixxx, ‘‘ Acta Phyeico-Medica . . . Nature Curioeorum [Dresden] Nurimbergae,” vol. iv, 1737, pp. 308-11, we find molybdena’ employed for zinc-blende, sulphide of zinc, while galena is used by Henckel for the common ore of lead, the sulphide. I n the years that intervene between these catalogues and the birth of modern chemistry towards the end of the eighteenth century, the most striking feature is the predominant position taken by the Swedish men’ of science, whose industry and enterprise laid the foundations for the marvellous advances that followed. This was especially the case with mineralogy, where the. volume of their work exceeded that of the rest of Europe. We have seen that during the seventeenth century ‘lapis plum- barius,’ ‘ plumbago,’ ‘ galena,’ and, exceptionally, molybdena were at one time or another employed in the sense of graphite, including probably molybdenite, with which it was confounded. In the period now under consideration it was ‘ molybdeena ’ that was usually employed in this Bense ; ‘ galena ’ became identified, as we have seen, with the mineral that now bears that name, and it will not be necessary to follow ite history further in much detail. This is translated in the French edition of Henckel’s works, ubliahed in 1760, by ‘prombaghe,’ vol. ii, p. 494-6. ‘ Plumbagof on the o&er hand, is employed (vol. i, p. 35) to transkte ‘ Bleyschweiff,’ ‘ Arsenicalisches Bley-Ertz ’ in the ‘* Pyritologia,” published in 1725 (p. 91). * hi fi i d i b h b li d i i y g , p (p ) * This fine-gained variety was by some authors believed to contain arsenic au well as sulphur and lead (see preceding note and pp. 163, 165, 167). This is translated in the French edition of Henckel’s works, ubliahed in 1760, by ‘prombaghe,’ vol. ii, p. 494-6. ‘ Plumbagof on the o&er hand, is employed (vol. i, p. 35) to transkte ‘ Bleyschweiff,’ ‘ Arsenicalisches Bley-Ertz ’ in the ‘* Pyritologia,” published in 1725 (p. 91). * This fine-gained variety was by some authors believed to contain arsenic au well as sulphur and lead (see preceding note and pp. 163, 165, 167). p ( p g pp , , ) well as sulphur and lead (see preceding note and pp. 163, 165, 167). 1 In the German edition of 1740 (Halle), Sterile nigrnm is translated Schwartze Blende ; and Molybdsna by Wasser-Bley (pp. 8 and 14). TRANSACTTONS OF THE PHILOLOGICAL SOCIETY, Plumbago, on the other hand, was used i n three distinct senses :- (1) following Worm, for galena free, or nearly free, from silver, in which case the word galena was restricted to the argentiferous varieties ; (2) for the fine-grained and occasionally fibrous varieties of galena, which were known in German as Bleyschweiff ;z and (3) for graphite or molybdenite. 161 THE MBlNINGS AND SYNONYMS OF PLUMBAGO. THE MBlNINGS AND SYNONYMS OF PLUMBAGO. It was soon disco>-ered that the supposed mineral which corre- sponded to both our graphite and molybdenite did not contain lead, as the earlier writers had supposed; but there was much difference of opinion as to its real composition. Those that experimented with molybdenite came to the conclusion that it was a compound, probably a sulphide, of zinc, and, as we have seen, Henckel confused it with zinc-blende. Others thought it might contain tin. Those who had graphite to deal with believed it to be a kind of ‘talc,’ either steatite or mica, combined with some combustible material, or, what was the same thing, some material containing the principle of combustibility, ‘ phlogiston.’ Iron was known to be present, and by some it was thought to be the substance that was ‘ phlogisticated ’ or combustible. Gradually these ideas, which contained distinct elements of truth, became more definite, till the results of Scheele’s work only required the magic of Laroisier’s theory to transform them into the views that we now hold. Graphite (including molybdenite) is deaIt with kt some length in vol. iv of the I L Universal Lexicon” of J. H. Zedler, Halle and Leipzig, 1733. Among the names with which it is credited are Schreibe-Bley, Bley-Schweiff, Test, Zwitter, and others, such as Plumbago and Xolybdsena, that have already been mentioned. The Italian Narchesita di plombo and Spanish Marquesita del plomo are also said to have the same meaning. The article appears to be largely founded on Pomet. I n the first edition of the ‘ I Systema Ratura ” of Linnaus, published at Leyden in 1735, we read, “Mica . . . particulis impalpabilibus. Sterile nigrum,” and “ Zincurn . . . sterile micaceum ? an hujus loci ? Molybdzna, Blyerts.” The former was probably molybdenite, the latter iz confusion between graphite and molybdenite.‘ I n the revised edition of 1740, published at Stockholm, these species are merged into one (p. 4) :--“Mica particulis squamosis, inquinantibus Molgbdana Blyack.” I n the edition of 1744 (Paris) there are again (p. 11) two species, “Zincurn micaceum atrum. Sterile nigrum ” and “ Zincum cinereum fusco-inquinans. Molybdma, le Plomb de Mer ou Plomb de Mine.” The former appears to be molybdenite, the latter graphite. 162 THE MEANINGS AND SYNONYMS OF PLUMBAGO. In the “ Tabuls Metallurgico-docimastica ” of A. G. 1 Neither graphite nor molybdenite is volatile, when heated out of contact with the air. THE MBlNINGS AND SYNONYMS OF PLUMBAGO. Berlichius, which forms an appendix to the edition of the “Schediasma de Tinctura ” of Gabriel Clauderus, published at Nuremberg in 1736, galena, Blende, and Bley Glantz are apparently synonymous, in the sense of lead sulphide. ‘Plumbago metallica’ or ‘Bley- schweiff,’ which is ‘‘ Splendens instar Plumbi nigri ” and heavy, and is said to contain more or less silver, is, I imagine, granular galena. ‘ Galena inanis ’ is evidently zinc - blende. Finally, ‘ 6 Molybdena, Wasser-Bley,” ‘‘ Ex atro paulo splendescens instar plumbi,” and more or less heavy, probably includes graphite and molybdenite (see tables K to N). y ( ) In 1737 appeared at Leyden a ‘‘ Dissertatio Academica sistens Nihil,” by Isaac Lawson, a Scotch medical student, who afterwards became a medical officer in the British Army. ‘ Nihil ’ or ‘pompholyx’ is sublimated oxide of zinc, and in the course of the dissertation (p. 13) attention is incidentally drawn to a very slight sublimate obtained from the mineral known as molybdsena when heated in a retort.‘ “Datur minera, quse dicitur Molybdsena; sub quo nomine mineram plumbi quidam intelligunt ; nos autem hic intelligimus mineram plumbei colons, micaceam, haud duram, ponderosam satis, ad tactum admodum saponaceam, pinguem, corpora solida lubri- cantem, ex cujus frustis purioribus et longionbus hodie fiunt styli sci-iptorii.” ‘‘ Pondus hujus miner= specificurn insigne docere videtur metallici quid inesse, quamvis nullo experiment0 hactenus mihi noto constitit, quodnam metallum in ea reperiatur.” After describing the results of .his experiments, he continues : “ Unde probabile ridetur semimetallum quoddam contineri in Molybdsena, ipmm forte Zincum, quamquam nulla arte adhuc nota potuit extrahi.” There can be no doubt that the mineral with which he experi- mented was molybdenite, which, however, the author believed to be identical with graphite. The mineral described by J. G. Hoffmann and J. B. Bcehmer, in their “De Matricibus Metallorum,” published at Leipzig in 1738, as associated with tin, was also probably molybdenite, though they too identify it with graphite used in lead pencils. “ Hi ipsi lapides stanni divites, aliam insuper satis sterilem THE MEAKIXGS AND SYKONPXS OF PLCYHAGO. 163 secum ducunt Mineram, quce Plumbago ab aliis Molybdsna nuncupatur” (p. 69). THE MBlNINGS AND SYNONYMS OF PLUMBAGO. Plumbago seu Molybdsna mihi est illud minerarum sterilium genus, quod calore [&I cum Galena convenit, seil leve, molle atque friabile est, plumbi nihil continet, digitos, chartam aliaque corpora livido nigricante colore pingit, proptereaque ad scripturas atque picturam pleruruque aclhibetur ; nostratibus Reiss-Bley, Bleystifft-Ertz dicitur. Equidem me non latet Agri- colam. . . . Plumbaginem atque Xolybdenam veram Plumbi Mineram nuncnpare, ast hodie prsdictus significatus magis obtinet. Interim nonnulli aliam Molybdaenam inter atque Plumba,’ einem faciunt differentiam et [Plumbaginem] Plumbi venam radiatam Antimonio similem, scil. Bley-Schweif, salutant. Molybdsnam vero Wasser-Bley nuncupant ” (note, p. 70). The word ‘ plumba- ginem ’ appears to have been omitted. I n the I ‘ Elementa Artis Docimastics,” a treatise on metallurgy by J. A. Cramer, published at Leyden in the succeeding year, vol. i, p. 262, we read: LLInter Nineralia nondum examinata imprimis considerationem meretur Molybdsna, alias quoque vocata Cerussa Nigra, Plumbum marinum Germ. (Wasser Bley.), non confundenda cum Galena Plumbi quae, licet eodem nomine quando- que designetur, prorsus tamen ab illa discrepat. Est Molybdsna Minerale eoloris Plumbei, ex squammulis micaceis contextum, mollius, nt cultro facile corradi queat, pondere Lapides Micaceos simplices, quos fere quoad texturam refert, longe superans, ad tactum valde saponaceum, corpora solida affrictu suo lubrica reddens : . . . Stylis itidem Scriptoriis usu pervulgato inscrvit.” There can be little doubt that the author included both graphite and molybdenite in his molybdtena. His ‘ I Galena Tesselata, Germ. Bley Glantz ” (p. 214), is evidently galena in the modern sense of the word. Plumbago ’ is not mentioned, but ‘ Bleyschweiff ’ is described as an arsenical sulphide of lead (p. 215). Two editions of an English translation by Cromwell Mortimer were publivhed in 1711 and 1764. They do not differ in any important respect from the Latin original. Both read (‘ Molyb- diena . . . in English, Wad or Black Lead,” arid refer to the mine at Borrowdale, p. 181 (1741). They are chiefly of interest as representing one of the few instances in which molybdsena ha3 been used in English in the sense of graphite. ‘‘ A similar use of the word is found in the ‘‘ Natural History of Cornwall,” by William Borlase, published in 1758 (p. 130) : “ Nolybdaena, or the pencil lead . . . some small gravels of this 164 THE MEANINGS AND SYNONYMS OF PLUMBAGO. 2 A French translation is included in “ Dissertations chpmiques de M. Pott,” p w y . vol. iv, p. 1. Pans, 1759. 1 The occurrence of the Cornish mineral in a metalliferous mine renders it probable that it was molybdenite. 3 I have not been able to identify this reference, but a later publication of vo . v, p. . Bohn is referred to on p. 169. , 2 “cum 2. p. Nitri mixtum . . . demum levissime detonat, cujus ratio forte in involutione parci principii inflammabilis sita est” ; . . . ‘4 cum p. Remis Nitri mixtum itidem transpellit spiritum nitrosum sub ruhris vaporibus.” 3 Other authors who class these minerals among the micas are A. F. C . Hempel (“ Terrarum atque lapidum partitio,” Gcittingen, 1762, p. 18) and J. E. I. Walch (“ Das Steinreich,” Halle, 1769, vol. ii, p. 37). See also p. 150. 1 See the third edition of an “ Allgemeines (Economisches Lexicon,” Leipzig, 1753, edited by G . H. Zincken, p. 362. Pott refers, of course, to an earlier edition which I have not seen. THE MBlNINGS AND SYNONYMS OF PLUMBAGO. will mark paper as free as the molybdaena from Cumberland . . . They came from a work in Camborn, called Huelcrafty” [Wheel Crafty].' ‘‘ y In 1739 the ‘‘ Mineralogie ” of Magnus von Bromell was published in Swedish at Stockholm. A German translation appeared in the following year, under the title “Mineralogia et Lithograpia Suecana ” (Stockholm and Leipzig). Here we find (p. 106, corresponding to p. 59 of the original), “Plumbago, Molybdzena, oder Bley-Ertz, ist eine andere weiche, leichte, glantzende und gar zu reiffe Bley-Malms-Art, welche die Hande farbt, wann man sie bearbeitet, und dienet dam, dasz man damit auf Knochen, Pappier und Pergament mahlen iind schreiben kan.” The Bley-Ertz of some localities was apparently a bituminous or oil-bearing shale. He also mentions its use for ‘ Bley-Federn,’ also referred to as “ Reisz- und Schreib-Federn.” In 1740 an interesting paper by J. H. Pott appeared in FOI. vi, p. 29, of the “ Miscellanea Berolinensia ” of the Societas Regia Scientiarum of Prussia,* under the title ‘‘ Examen chymicurn plumbi scriptorii vulgo plumbaginis,” in other words graphite. He commences with a long list of synonyms which had been employed at different times and in different languages. These include, besides ‘ plumbago,’ ‘ plumbago scriptoria,’ and ‘ plumbum marinum ’ in Latin ; ‘ molybdites,’ ‘ molybdoides,’ and ‘ molybdzena ’ in Greek ; ‘ plomb de mere ’ [sic], ’ plombagine,’ ‘ mine de plomb noire,’ ‘ plomb de mine,’ and ‘ plomb minerale ’ in French ; ‘ Black Lead ’ in English ; and ‘ Wasser-Bley,’ ‘ Reisz-Bley,’ ‘ Schreibe- Bley,’ and ‘ schwartz Bley-Weisz ’ in German. The mineral was, he says, called by the old workers in France ‘Pott Loot’ or ‘ Poteloot ’ (“ quasi Topfer-Bley ”), and it was also known as ‘ Crayon ’ (“ quasi Creta nigra”) and ‘ Cerussa nigra.’ Inferior varieties were referred to as ‘ Eisen-Farbe ’ and ‘ Eisen-Schwiirtze.’ Other names quoted from various authors are ‘ Eisendach,’ ‘ argilla ferri,’ ‘ Ochra nigra,’ nigrica fabrilis,’ ‘ Cadmia ferruginea,’ and ‘galena sterilis.’ It is doubtful whether some of these really referred to either graphite or molybdenite. He cites (p. 33) as an error the statement in Bohn’s Kauffmann3 (ii, p. 61) that ‘Reisz 165 THE MEANIEGS AND SYNONYMS OF PLUMBAGO. 2 It is also closely followed in the “Lithophylacium Bornianum, Index Fossilium quae collegit et in Classes &c Ordines hsposuit Ignatius S.R.I. Eques I\ Born I’ (lgnaz von Born), publishes at Prague in 1772. THE MBlNINGS AND SYNONYMS OF PLUMBAGO. Bley ’ is prepared by the Italians from Bley Ertz, and also that of the author of a ‘L Lexicon Economicon ” (p. 326),’ who thought that plumbum scriptorium was a pure mineral of lead found in mines, but that coinmon plumbum scriptorium was prepared from lead, especially in Saxony. Bley ’ is prepared by the Italians from Bley Ertz, and also that of the author of a ‘L Lexicon Economicon ” (p. 326),’ who thought that plumbum scriptorium was a pure mineral of lead found in mines, but that coinmon plumbum scriptorium was prepared from lead, especially in Saxony. He then describes his own experiments, admitting, however, that he does not know if the plumbago he treated was the same as that employed by Lawson. He declares that he obtained no evidence of the presence of sulphur or zinc. He notes (p. 36) that the mineral decomposes potassium nitrate [a characteristic reaction of gra~hite].~ Finally, he comes to the conclusion that he was dealing with a “ terra talcosa, igni et menstruis indomita, pauco martiali [iron] et pauciore acido Vitriolico.” It was probably Borrowdale graphite with a little iron pyrites, which is nearly always present. y p Seven years later appeared the first edition of the “Mineralogia” of J. G. Wallerius (Stockholm, 1747). Here, under “Lapides bpyri,” we find (p. 131) : “Blyertz Spec. 126. Mica pictoria nigra, manus inquinans. Molybdzena.” He describes it a8 consisting of small scales arranged without order, grey-black in colour, with feeble lustre, and communicating to the hands, paper, and linen a grey colour like that of lead. It preserves its colour and consistency in the fire. (1) “ Ren Blyertz, Molybdsena pura ” ; (2) ‘‘ Oren Blyertz, Molyb- dzena impura” ; (3) “ Blyertz Tarningar, Nolybdana tessularis.” He refers to Lawson’s experiments, and alludes to the possible presence of zinc. The two former varieties no doubt included both graphite and molybdenite; the third would seem to be the modern galena. This, however, appears (p. 292) as a separate species : “Bly glants Tarninge &Calm-Plumbum, sulphure et argento mineralisatum, minera, tessulis minoribns vel majoribus, vel granulis, micante. Galena. Plumbago metallica.” Plumbago is, aIso, used as a synonym of Bleischweiff, which i R supposed to contain lead, sulphur, and arsenic : “ Blyschweif - Plumbum, sulphure et He enumerates three varieties : Phil. Trans. 1908. 12 166 THE MEANINGS AXD SYKONTMS OF PLUMRAGO. 1 Graphite is also classed under the ores of iron as “ Ferri Minera pictoria : Molybdaena” by M. T. Briinnich in his Mineralogy, published in 1777 in Danish (p. 247) and iu 1781 in German (p. 255). See also post, pp. 168, 172, 174. 177. THE MBlNINGS AND SYNONYMS OF PLUMBAGO. arsenic0 mineralisaturn, minera pinguiori fere malleabili, Plum- bago.” On p. 136 me read “Klitbarg. Ollaris mollior, pinguis, niger, micaceo - lamellosus, yix coherens, pictorius. Talcum nigrum.” This may include amp6lite (ante, p. 136) or graphite. Translations were subsequently published in French and German. q y p In the Latin edition of the “ Systema Naturz ” of Linnsus, published both at Stockholm and Leipzig in 1748 and Leyden in 1756, graphite is not referred to, and neither ‘molybdaena’ nor ‘ plumbago is recognized in the nomenclature. However, in the Catalogue of the Museum Tessinianum, published at Stockholm in 1753 and believed to be the work of Linnsus, the following entry appears (p. 54) : ‘‘ Molybdaena. Zincum fusco inquinans. Mica pictoria nigra, manus inquinans (Wall. Min., 131). Hue Refertur usquedum certiora innotescant.” In the “ Systerua Minerale ” of Johann Lucas Woltersdorff, published at Berlin, also in 1748, we find graphite described under “Metalla ignobilia” as “Ferrum . . . Nigricans, splendens, unctuosum, inquinans. Nolybdsna. Wasser - Bley. Nigrics Fabrilis, Reiss-Bley.” In 1754 there appeared at Stockholm, in the ‘‘Kongl. Svenska Vetenskaps Academiens Handlingar ” (Proceedings of the Royal Swedish Academy of Science), vol. xv, p. 189, a paper entitled “R6n om Bly-Erts” (the usual Swedish term for graphite), by B. Qrist, describing experiments on a mineral occurring in flexible plates, which must have been molybdenite. He heated it in a current of air and obtained a white sublimate. I n the “ Elementa Mineralogie ” of F. A. Cartheuser, publislicd at Frankfort in 1755, there is no undoubted reference to graphite, but fine-grained galena is separated from the cubical variety under the name of ‘ Plumbago ’ or ‘ Bleischweiff (p. 66). g p The year 1758 was marked by the appearance at Stockholm of yet another Swedish Mineralogy, the ‘< Forsijls ti1 Mineralogie ” of Axel von Cronstedt. This proved very popular, and was translated into French, German, English, and Italian, in Borne cases more than one edition being published.* On p. 139 of the original edition, which was issued anonymously, we read : “ IIrn THE MEANINGS AND SYNOPFYMS OF PLUMBAGO. 167 och Tenn, Sulphur ferro et stanno saturatum. Blyerz. Wasser- bley. Yolybdena.” It is divided into three varieties. See ante, p. 160, for the we of phmbagine and plumbago in the French edition of Henckel’s works, published in 1760. 1 Born (op. cit., i, p. 61) gives “ICeswig Anglie ” as the locality for * S ‘‘ Molybdtena textura chalybea.” * See note, p. 150 am^ y THE MBlNINGS AND SYNONYMS OF PLUMBAGO. 168 He ip the earliest, so far as I am aware, to assert the existence in graphite of phlogiston, the first step to the recognition of the mineral as a form of carbon. Pott, however, recognised the possibility of the occurrence of a “parcum principium idammabile” (ante, p, 165). Vogel distinguishes three varieties of galena: (1) “Galena tessulata”; (2) “Galena granulata, punctata”; (3) “Bleischweif, Plumbago,” the last being “in einer derben und fast streifichten Gestalt” (p. 456). p I n the fourth edition, published in 1762, of the dictionary of the Academie Franqaise, plombagine is explained as “ Substance minerale de la nature du talc. O’est la m6me qui est plus connue sous le nom de Crayon, ou de Mine de plomb.” In the edition of 1718 the word is not included, while in the “Dictionnaire des Arts et des Sciences,” part of the first edition, printed in 1694, it is explained in the terms employed by Mattioli. y y Some entries in the “ Dictionnaire Universe1 des fossiles propres et des fossiles accidentels” of E. Bertrand, published in 1763, illustrate the confusion in the nomenclature at this time. “Le Crayon des Peintres, appelle mine de plomb, est aussi un Mica. C’est le Molybdsena de Pline, le Molybdoi’des de Dioscoride, . . . On appelle aussi en Franqois ce crayon fossile, plombagine et plombacine, du Latin plumbago. . . . I1 y a une matiere qu’on appelle aussi mine de plomb, qui est rouge. Quelques Droguistes le nomment tout - aussi ma1 - B - propos minium ” (vol. ii, p. 43). ‘( Plombagine. Plombago. On s’accorde peu sur la vraye application de ce nom. 1’. Les uns entendent par I$. les glebes de plomb mineral cubiques qu’on appellent galhes. 2’. D’autres designent par 1& une autre sorte de mine de plomb qui est arshicale et sulphureuse. 3’. Henckel’ appelle de ce nom une sorte de crayon, plumbago scriptoria. . . . C’est le mica des peintres . . . Ce dernier fossile ne contient point de plomb. Henckel croit qu’il est plut6t ferrugineux. . , . C’est que les Anglois nomment Black Lead . . . Nous croyons qu’il feroit plus exact d’appeller galkne la premihre espbce de rninhral, plombagine la seconde, molybdkne la troisihme. I1 feroit souhaiter que les Naturalistes s’accordassent m e fois dans leur nomenclature” (vol. ii, p. 1333. On the other hand, we find (~01. THE MBlNINGS AND SYNONYMS OF PLUMBAGO. The first is “ Molybdena membranacea nitens,” which is described as platy and shining, and of the same colour as ‘ Blgglants.’ Bispbergs Xlack, Bastniis wid Riddarhytta, and Altenberg are given as localities, and it is stated that a specimen from Bispberg was that employed by Qvist in his experiments. This, therefore, is molybdenite. The second, with “ Textura Chalybea,” is apparently graphite j and the third, with ‘‘ Textura rnieacea et granulata, Grof Blyertz,” described as consisting of small flakes or granules, appears to represent flaky graphite. I n 1762 J. C. Valmont de Bomare published a Mineralon at Paris, which was largely founded on Wallerius. Here species 87 (POI. i, p. 124) is “ Molybdhe, Mica des Peintres, Crayon ou Mine de plomb, etc. (Molybdena Mica pictoria. . . . Pseudo-Galena Wolt. Plumbanus, etc.).” He remarks : “ Le crayon se trouve commun6ment arec les mines d‘Qtain ; il en contient aussi quelque- fois abondamment.” He believed it, however, to be essentially a kind of talc.2 Other species are ‘< G a h e ou Mine de plomb en cubes . . . plumbago metallica . . . ,” and “Mine de plomb sulphureuse et arsenicale, . . . Bleyschweiff Germanorum, . . . Plumbago nonnullorum ” (vol. ii, pp. 98, 103). g pp I n the same year appeared at Leipzig the “ Practisches Mineral System ’’ of R. A. Vogel. Here, under the heading “ Wasserbley, Reissblep. Nolybd~na, Plumbum scriptorium ” (p. 66), we read: ‘( Das Wasserbley ist ein leichter, schwarzgrauer zerreiblicher und abfarbender Glimmer; aus dem man lange nicht gewusst hat, was man machen soll, und es daher fiir eine Art eines Ifleyerzes gehalten hat. Es ist abcr nicht ein Gran Bley daTinn, sondern vielmehr etwas, obwohl sehr weniges eisenhaftes j hierniichst aber ein wenig Phlogiston : das iibrige uud meiste ist eine talkichte, dem Feuer widerstehende Erde.” He describes how the Germans made the ‘ leads’ of pencils by cementing the powdered graphite, and continues : ‘‘ Es ist aber noch ein Geheimniss, mit was fur einer Materie die Englinder ihr Wasserbley schmelzen.” As a matter of fact the English graphite was cut directly from the mineral. a THE MEAEilNGS AND SYNORYMS OF PLUMBAGO. I cannot find any evidence of the use of the word in this sense by Henekel, exceptid the labels given by Dr. John Woodward. 1 See also C . F. G . Westfeld, ‘‘ Mineralogiiche Abhandhngen,” GiittinGeu and Gotha, 1767, p. 51, and the “ Catalogue Systkmatique,” by P. F. Davila, assisted by J. B. L. de Rom6 de Lisle, published in the same year, where Molybdene DU Crayon ” is classed under tale, and “ Molvbdene ou Mica des Peintres,” from Bisphergs-Klack and other Swedish locaiities, and therefore presumably molybdenite, under zinc (pp. 120, 372). THE MBlNINGS AND SYNONYMS OF PLUMBAGO. ii, p. 63) ‘‘ Molybdsene. Molybdsena. En Allemand Bleiertz. THE IvIEAKIKGS AHD SYNONYMS OF PLUMBAGO. 169 Mine de plomb. Ce mineral contient toujours du plomb.” See also vol. i, p. 166. Mine de plomb. Ce mineral contient toujours du plomb.” See also vol. i, p. 166. p The first definite recognition of the fact that molybdsna and its numerous synonyms comprised two eptirely different substances is in the “ Naturgeschichte des Nineralreichs ” of J. W. Bnumer (Gotha, vol. i, 1763 ; vol. ii, 1764). Here in part 4, devoted to the earths, chapter ir dealing with the clay-earths (thonerde), we find (vol. i, p. 151) : ‘( Das Wasserbley, molybdzena, kan am fiiglichsten unter die glimmrigen, etwas Eisen, Zinn und Schwefel haltigen Erden gerechnet werden. Nan findet dasselbe zu Bispberg, BastnLs und Gran in Schweden, und Altenberg in Sachsen.” Both the supposed composition and localities point t o molybdenite (see also ii, p. 105). On the other hand, in the fifth part, Stones, chapter iv, clay-like (thonartig) stones, we are told (i, 1). 217) : ‘( Das Wasserbley, Reissbley, Molybdana, Plumbum scriptorium, bestehet aus kleinen dunnen unordentlich zusammengef ugten Schuppen, und ist ein leichter schwaregrauer abfarbender Glimmer. Es bestehet aus einem brennbaren und eisenhaften Wesen, nebst einer talckigen Erde.” He refers to its occurrence at Kesmick, and its use for pencils and crucibles (see also ii, p. 139). There can be no doubt that the combustible substance containing iron was graphite. It is curious that Baumer should have retained the same names for two substances which he evidently thought were unrelated the one to the other.’ Galena is described as the commonest lead ore, and as containing lead and silver, while bleischweiff or galena punctata is stated to contain arsenic in addition. The term ‘plumbago’ is not used by Baumer, but in the 4‘ Neueroffnetes Waarenlaager,” by G. C. Bohn, published in the same year at Hamburg, we have (col. 134) the entry : “Bbyweiss, das schwame, oder Wasserbley, sonst auch Reiszbley, Schreibbley, Ylumbago, cerussa nigra, und von den Franzosen Crayon genannt.” On the other hand, in an English book of a somewhat similar character, the ‘‘ Commercium Philosophico Technicum,” by W. Lewis, M.B., F.R.S., also published in 1763, we find neither molybdsna nor plumbago employed, only black lead (p. 325). The term ‘plumbago’ is not used by Baumer, but in the 4‘ Neueroffnetes Waarenlaager,” by G. C. THE MBlNINGS AND SYNONYMS OF PLUMBAGO. Bohn, published in the same year at Hamburg, we have (col. 134) the entry : “Bbyweiss, das schwame, oder Wasserbley, sonst auch Reiszbley, Schreibbley, Ylumbago, cerussa nigra, und von den Franzosen Crayon genannt.” On the other hand, in an English book of a somewhat similar character, the ‘‘ Commercium Philosophico Technicum,” by W. Lewis, M.B., F.R.S., also published in 1763, we find neither molybdsna nor plumbago employed, only black lead (p. 325). THE MEANIXGS AND SYNONYMS OF PLUMBAGO. 170 The author cites both Qvist and Cronstedt, and describes experiments in which he himself demonstrated the almost entire dissipation of graphite by heat. y In the edition of the “Systema Naturae” published in 1768, which was, as usual, more detailed than those that preceded it, Linnsus dealt at some length with these minerals, but he had no suspicion of the wide difference between graphite and molybdenite. He makes (p. 121) a genus of ‘Molybdenum,’ of which Plumbago or Bleyertz is one species. ‘‘ Molybdenum tritura cerulescente,” or, as we should now say, molybdenum with a bluish streak. He identifies it with the mineral investigated by Qrist, the ‘‘ Sulphur ferro et stanno saturatum ” of Cronstedt, and the “Mica pictoria nigra manus inquinans” of Wallerius. He is rather oracular as to its composition : “ Metallurn proprium inde inducere nulla ars chemica etiamnum didicit. An metallum oppositum Hydrargyro, quod nunquam fusile, ut illud semper 2 Non introduco ideam novi metalli sed colloco obscuras species metallicas in loco gratis expetito, usquedum Regulus coronetur.” This appears to point to moljbdenite, but he refers to its use for pencils, crucibles, and other purposes for which graphite is employed. The second and third species of the genus molybdsenum included manganese ore and wolfram (tungstate of iron and manganese). The different forms of galena appear as species of the genus Plumbum (p. 312). In the first English edition of Cronstedt, published in 1770, under the title “An Essay towards a System of Mineralogy,” we find (p. 156) the heading ‘< Iron and Tin, Sulphur ferro et stanno saturatcm, Black Lead, or Wadd, Molgbdsna.” The term plumbago is nowhere used. In the following year appeared ‘( Fossils arranged according to their obvious characters,” by J. Hill, M.D., to whom reference has already been made. He followed Linnseus in employing molybdenum as a generic name with a number of species, some of which corresponded to graphite or molpbdenite. 1 Giovanni Antonio Scopnli, in his “Principia Mineralogize,” Prague, 1772, tells UB I‘ Yeterum Molybdrena ad genus plumbi pertinebat. Recentinres nimia licentia nomen hoc dedere Mieae particulis minimis, mquinantibus, atro-plumbeis ” (PP. 38, 40). THE MBlNINGS AND SYNONYMS OF PLUMBAGO. Another reference to graphite is met with in “ A Political Survey of Britain,” by John Campbell, LL.D., published in 1774 (vol. ii, p. 37). “Black Lead is what some have supposed, with very little Reason, to be the Molybdena or Galena of Pliny ; THE MEANINGS AND SYNONYMS O F PLUMBAGO. 171 others stile it Plumbago.” I n a note he says : “Foreign authors call by that Name [molybdena] a Substance found in Prussia, which serves for making Pencils, and comes from thence to be confounded with ours, vhich it in no other Circumstance rcsembles.” This is the first instance outside the pages of the translation of Pomet in which plumbago is employed in English in the sense of graphite. Even ou the Continent the use of the various forms of the word in that sense had been comparatively rare, but it was gradually becoming more common. For instance, in the I‘ filQmens de Xin6ralogie Docimastiquc,” by B. G. Sage, published at Paris in 1772, the third species of tin is “Nolybdhe, plombagine, crayon noir” (p. 241). Here both graphite and molybdenite appear to be included and considered to form one mineral species.’ p In the first edition of Valmont de Bomare’s Mineralogy, published in 1762. plumbago was, as we have seen, only employed in the sense of galena. In the second edition, published twelve years later, we find a list of synonyms (rol. i, p. 193), including, amongst others, molgbdsne, mica des peintres, mine de plomb noire des peintres, crayon ; molybdena, sterile nigrum, plumbago scriptoria, mica nigrica aut colore vario fabrilis. A list of vernacular names follows, including potelot, mine de plomb noire ou savonneuse, plomb de mer, plombagine, plomb de mine, ceruee noire, talc noir friable, blende and fausse gal&ne. H e supposes it to contain zinc, and possibly lead. H e still use8 (vol. ii, pp. 176, 186) plumbago metallica and plumbago nonnullorum in the same sense as in the first edition. In his ‘‘ Dictionnaire raisonn6 Universel,” published in the succeeding year, he gives a similar list of scientific and popular synonyms of graphite or molybdenite, including molybdhe, molyb- d e w , nigrica fabrilis, plumbago scriptoria, and plombagine. He expressea his belief that the mineral is a steatite, viz. massive talc in the modern sense, formed of iron, sulphur, and zinc, and similar in nature to zinc-blende (vol. ‘F, p. 468). I He believed it to he an altered form of tin. See note to the German edition On the other hand, in the 2;: edition, (vol. i, by N. G. Leske, Leipzig, 1775, p. 248. published in 1777, he describes it as “ un mica martial et alumineux p. 194), and as Mica gris, onctueux, color6 par le fer ” (vol. ii, p. 206). THE MBlNINGS AND SYNONYMS OF PLUMBAGO. A t the same time he abandons the use of plumbago in the sense of galena. I n 1775 the second volume of a Latin revision of the Mineralogy of Wallerius was published a t Stockholm under the title ‘ I Systema THE MEANINGS AND SYNONYMS OF PLUMBAGO. 172 Mineralogicum,” in which (vol. ii, p. 249) we find: “Ferrum corrosum, Volatile, mineralieatum, minera nigrescente, squamosa, pictoria, magneti refractaria. Molybdaena.” ‘( Plumbago. Non- nullorurn.” A number of synonyms are given, and we are told that it is “ in igne aperto ad maximam partem Volatilis, ad 70 vel 80 pro-Centenario avolans; in igne rero clauso fortissime persistens sine aliqua mutatione.” The first variety, “ Molybdaena pura, membranacea, nitens,” is evidently the molybdana membranacea nitens ” of Cronstedt, and therefore the m i n e d molybdenite. The other varieties, M. micacea, arenaria ” and “ M. textura chalybea,” are apparently both graphite. y pp y g p The references to galena and bleischweif (1-01. ii, pp. 302-6) are practically the same as in the original work. There is an interesting note on plumbago : “ Plumbaginis rox diversirnodo sumitur a Mineralogis; alii hoc nomine Galenam plumbi . . . indicarunt, quam distinctionis gratia, vocarunt Plumbaginem Metallicam ; alii Molybdsnam hoc nomine compellarunt, quam inter Mineras ferri descripsimus, eandemque rocarunt Plumbaginem scriptoriam. Alii ham, quam heic descripsimus, mineram plumbaginem simpliciter vocarunt, quos, ad evitandam confusionem secuti sumus.” q I n 1778 a German edition of the mineral portion of the “Systema Naturae ” of Linnaeus, translated and enlarged by J. F. Gmelin, was published at Nuremberg under the title ‘‘ Vollstandiges Natursystem des Mineralreichs.” Here (rol. iii, p. 66) the genus Molybdsnum is used in the same manner as in the Latin edition of 1768 already quoted. The first species is M. plumbago, with the synonyms Wasserbley, Loschbley, Reissbley, Schreibbley, Topferbley, Schwarzbleiweisse, Rleyerz, schwarz Bleyerz, Eisep- farbe, Eisenschwarze, schwarze Kreide, schwarzer Ocker, Blende, Blpertz (&‘wed.), black lead ore, blacklead, plomb de mer, mine de plomb noire, crayon npir, plombagine, molybddne, ,~oXZIP&T< s, plumbago, plumbum nigrum, mica pictoria, and molybdsna. The translator seems to have had no suspicion that there were two entirely different substawes tQ which these different expressions were applied. The two other species of Molybdsnum are the same as in the Latin edition. In the genus Plumbum there are several species, including P. galena, Bleyglanz, galena ; P. THE MBlNINGS AND SYNONYMS OF PLUMBAGO. compactum, Bley- schweif, plumbago (pp. 212, 222). Another genus, ‘ Galena,’ included a number of sulphides of different metals (pp. 85, 96). The obscurity that had so long prerailed with regard to the 173 THE idEANINCiS AND SYKONYMS OF PLUMRAGO. true nature of graphite and molybdenite, then known alike as molybdaxa or plumbago, was at length dissipated by the work of C. W. Scheele, which was published in the Proceedings of the Royal Society of Sweden in 1778 and 1773. y y The. first paper, which appeared in 1778 (Kongl. St-en. Vet. Acad. Hand., ~ o l . xxxix, p. 247), and was entitled “Fiirsiik rned Blyerts, Molybdznn,” commences by the statement that the author is not treating of the common blyerts of the shops, but with what Cronstedt called IL Nolybdaena membranacea nitens,” and on which Qvist and others made experiments. He demonstrates that this substance, which he refers to throughout as molybdsna, was a combination of sulphur with an acid of metallic nature which he separated out. This was an accurate statement of the facts in the language of a time when the part played by oxygen in nature was still unrecognized. g In the succeeding year he published another paper (op. cit., vol. XI, p. 238), ‘< Farsiik med Blyerts, Plumbago,” in which he shows that the blyerts well known in commerce, the “molyb- daena, textura micacea et granulata ” of Cronstedt, now known as graphite, was 8 mineral sulphur or charcoal, the constituent parts of which were aerial acid (carbonic acid gas) and a considerable amount of phlogiston. This again is correct in terms of the old phlogiston phraseology, in which phlogiston is a kind of negative oxygen, so that aerial acid + phlogiston = carbon. A small quantity of iron was, he said, probably present in the form of pyrites, which yielded sulphurous fumes on heating. He showed that this blyerts was also obtained as a residuum when cast iron was dissolved in acid. The word blyerts is used throughout, plumbago only appearing in the title. pp g It is not quite clear why Scheele allocated to molybdenite the term molybdsna, which had for many years been on the whole more frequently applied to graphite. Apparently, at the time of the first paper he thought that the Swedish term blyerts was sufficient for the better known mineral. Even as late as 1779 we find mol bdeneand plombagine used as synonyms. A. G. Monnet, “Nouieau Systeme Je Min6ralo-ie ” Bouillon, 1779, p. 180. Again, in the “Miueialogie” of J. F. Gmelin, pibkshed at Nuernberg in 1780 (p. 85), graphite IS still (and apparently molybdenite as well) referred to only as Wasserbley, and considered to consist of talc, with much iron, often sulphur, and more rarely tin. 1 It appears that Romi! de Lisle was engaged in the study of raphite at the same time as Scheele, and had obtained results which, if righi& interpreted, would have disclosed the real constitution of the mineral. He was not convinced by Scheele’s work, and expressed his belief that Scheele’s molybdrena was identical with mine de fer micacbe grise, viz. micaceons iron ore, an oxide of iron, and that his plumbago owed its action on nitre ‘‘ au fer noirgtre phlogistiqn6, en un mot Q 1’6thiops,martial natif et it la matiere grasee qui s’y rencontrent.” See B. G. Sage, ‘‘ Elhens de Min6ralogie Docimastique,” 2nd edition, 1777, vol. i, p. 194, and vol. ii, p. 207 ; P. J. Macquer, “ Dictionnaire de Chimie,” 2nd edition, 1778, under molybd&e ; Rome de Lisle, “ Crystallographie,” 1783, vol. ii, p. 501, and “ Description MQthodique,” 1773, p. 165. * y p p In the second volume, published in 1783, of a German translation of the Latin edition of Wallerius, cited above, the latter is followed more or less closely, so that we find the words molybdaena and plumbago em loyed in the same way as before, but a brief reference is made to Scheele and gergman, and the new distinction between the terms (pp. 235-9, 297). p p q p * Molibdena and piombaggine were adopted in the same senses in Italian in a note to the article “ Molibdena” in the translation of Maequer’s “ Dictionnaire de Chimie,” by G. A. Scopoli, vol. vii, Paris, 1784, p. 69. 1 The latter writer states (p. 452) that Yelletier demonstrated that when plumbago ‘ is pure, it neither produces any fixed or inflammable air ; both which, when found, are entirely owing to the substances that are inised with it.” As a matter of fact, in the paper referred to, ‘‘ Sur l’analyse de la Plombagine et de la Molybdene” (Obs. sur Phys., vol. xsvii, 1785, pp. 343 and 434), Pelletier states that plombagiue should be reqarded as ‘‘ une substance in- flammable particulihe,” and adds that ‘‘ les Lstibstances dans lesquelles l’air fixe ne paroit pas entrer, donnent apr& leur d6composition des indices de cet Gtre ” (p. 357). THE MBlNINGS AND SYNONYMS OF PLUMBAGO. Afterwards, when he wanted a Latin term for graphite, plumbago was the most commonly known word that still remained available. However this may be, his usage decided the future application of the terms.’ 174 THE MEANINGS AND SYNONYMS OF PLUMBAGO. A translation of the second paper into French by ‘ M. Mgn. de Dijon’ appeared in “ Observations sur la Physique,” vol. xix, 1782, p. 162. In this translation the word plombagine is used throughout for graphite. The next volume, published in the same year, contained (p. 342) a translation by ‘Madame P. . . . de Dijon ’ of the earlier paper, and in this molybdine is used in the same way as molybdsena in the original.’ English translations of these papers, by Thomas Beddoes, appeared in the ‘‘ Chemical Essays of Charles William Scheele,” published in 1786, and in these the terms molybdena and plumbago are used in like manner (pp. 227, 243). (‘ In the (‘ Sciagraphia” of Tobernus Bergman, yet another Swedish mineralogist, published at Leipzig and Dessavia in 1782, we find (p. 93), (‘ plumbago” described among inflammable bodies as “ Phlogiston acido aBreo satiatum,” with the comment “ Compositionem genuiuam detexit D. Scheele,” and “ molyb- dsena ” as ‘‘ Phlogiston acido, tam vitnolico, quam molybdsenae adunatum, vel, quod eoden; recidit, sulphur cum acido molybdsense conjunctum.” An English translation, by William Withering, appeared in 1783, where we read (p. 64) : “Phlogiston saturated with aerial acid. . . . Plumbago. Black-lead,” and “Phlogiston united to the acid of vitriol and of molybdsena; or what amounts to the same, sulphur joined to the acid of molybdsena. . . . Molybdsena [Latin], Nolybdsena [English].” In tlie “ Elements of Mineralogy,” by Richard Kirwan, F.R.S., THE MEAXINGS A N D sYxoNYm OF PLUMBAGO. 175 published in 1784, we have a fuller explanation of these terms. “ Plumbago, Reissbley, of the Germans, Blyertz, of the Swedes.” “ In a strong heat and open fire it is wholly volatile, leaving only a IittIe iron, which seems to be only accidentally found in it, and a few grains of silex. It is probable that 100 grains of it contain 33 of aerial acid, and 67 of phlogiston” (p. 158). “Molyb- dena, Molybdena membranacea, Cronst. . , . Wasserbley of the Germans.” ‘L It resembles plumbago ” (p. 357). THE MBlNINGS AND SYNONYMS OF PLUMBAGO. 337-40), under the heading ‘( Graphit,” after a long list of authorities and synonyms come the following notes :-“ Ehedem war es entweder schlechthin zum Wasserblei (Molybdaen) gerechnet, oder nur specifisch von ihm unterschieden.” ‘( Die neuern Mineralogen nennen dieses Fossil durchgangig im Lateinischen plumbago ; da aber dieser Nahme schon Ton Aelteren, dem Bleischweif gegeben ist; so hat Herr Werner ihn mit graphites vertauscht, weil sein haufiger Gebrauch zu Bleistiften diesen sehr passend macht. Man konnte daher auch im Deutschen leicht, den sonst gewiihnlichen Ausdruk Reissblei, in Schreibblei umandern.” Hoffmann.” Here, under ‘‘ Brediche Wesen,” we find (p. 380) a mineral species “ Graphit,” with a note (p. 395) Von andern wird es Reisbley, wie auch (sehr unschicklich) Plumbago genannt,” and under ‘‘ Metallarten,” ‘( Molybdan,” ‘ I Wasserblei ” (p. 386). a d u de eta a te , ( o ybda , Wasse b e (p. 386). I n the same year was published the “Museum Leskeanum, Regnum Minerale, quod ordine sptematico disposuit atque de- scripsit D. L. Gustavus Karsten.” Here (vol. ii, pt. 1, pp. 337-40), under the heading ‘( Graphit,” after a long list of authorities and synonyms come the following notes :-“ Ehedem war es entweder schlechthin zum Wasserblei (Molybdaen) gerechnet, oder nur specifisch von ihm unterschieden.” ‘( Die neuern Mineralogen nennen dieses Fossil durchgangig im Lateinischen plumbago ; da aber dieser Nahme schon Ton Aelteren, dem Bleischweif gegeben ist; so hat Herr Werner ihn mit graphites vertauscht, weil sein haufiger Gebrauch zu Bleistiften diesen sehr passend macht. Man konnte daher auch im Deutschen leicht, den sonst gewiihnlichen Ausdruk Reissblei, in Schreibblei umandern.” It would be interesting to know if Werner, in choosing the term graphites, had the Grafio of Ferrante or the graphis plumbea of Boulenger in his mind. Curiously enough, we find the almost identical word graffites in the ‘ Speculum Lapidum ’ of Camillus Leonardus (Venice, 1502, p. 328), as a synonym of Galactides, an ash or milk-coloured stone, apparently similar t o chalk, found in the rivers Nile and Athaleus. On p. 562 of the same volume we find Molybdan and Wasserblei used as synonyms, with molybdaenum as the Latin and molybdena as the English equivalents. g q I n the “ Delectus Opusculorum ad Scientiam Naturalem spectantiurn,” published at Leipzig in 1790, is included the Systema Regni Mineralis, Anni YDCCLXXXVIII, of Werper. Here, on p. THE MBlNINGS AND SYNONYMS OF PLUMBAGO. Similar views are expressed in the ‘Handbuch der Nineralogie,” by John Fibig, published at Nainz and Frankfort in 1787 (pp. 52, 273), and the second English edition of Cronstedt’s work by John Hyacinth de Magellan (1788) (pp. 451, 863).’ p g p There was still a certain confusion of ideas, and the fact that plumbago was essentially a form of carbon was apparently still unrealized. Its recognition, however, could not have been long delayed for those who appreciated the importance of Lavoisier’s discovery of the true nature of combustion and the part played in it by oxygen, which was now taking the place in chemical theory of its shadowy correlative phlogiston, had not a new misunder- standing arisen. In a paper read before the AcadBmie des Sciences by Vandermonde, Monge, I% Berthollet in 1786 (Obs. sur Phys., xxix, 1786, pp. 283-4; Mem. Acd. Xoy. Sci., 1786, pp, 193-6), thefe authors concluded from the production of fixed air by the chemical action of plumbago on oxide of lead or arsenic, that plumbago contained carbon ; other experiments showed that it contained iron ; they therefore declared that plumbago was a compound of carbon and iron, and for some thirty years this view of its composition was generally accepted. Meantime, however, the great mineralogist and geological pioneer Werner had bestowed on the mineral a new name, which, it would seem, first appeared in 1789 in the “ Bergmlnnisehes Journal,” in an article entitled “ Mineral System des Herrn Inspektor Werner3 mit dessen Erlaubnis herausgegeben yon C. A. S. THE XEANIKGS AND SYNOXYMS OF PLUMBAGO. 176 Hoffmann.” Here, under ‘‘ Brediche Wesen,” we find (p. 380) a mineral species “ Graphit,” with a note (p. 395) Von andern wird es Reisbley, wie auch (sehr unschicklich) Plumbago genannt,” and under ‘‘ Metallarten,” ‘( Molybdan,” ‘ I Wasserblei ” (p. 386). I n the same year was published the “Museum Leskeanum, Regnum Minerale, quod ordine sptematico disposuit atque de- scripsit D. L. Gustavus Karsten.” Here (vol. ii, pt. 1, pp. THE MBlNINGS AND SYNONYMS OF PLUMBAGO. 555, we find Graphites (Reisbley Plumbago), and on p. 560 Molybdsenum galenare (Wasserbley). In each case there i s a reference to Karsten’s Xuseum Leskeanum. Apparently this classified list of minerals mas first compiled in 1788, but there is no evidence that it was ever published in that year. Graphite was now used as synonymous with plumbago, both by those who adhered to the old system of chemical nomenclature and those who adopted the new views. J. I?. Gmelin, in his “ Grundriss der Mineralogie,” published at Gottingen in 1790, writes (p. 381) : ‘‘ Reissblei (Schreibblei, Loschblei, TGpferblei, Graphit, Eisen- schwarze, Bleierz, Plumbago) enthLlt ausser etwas (&) Eisen, bloss THE MEANINGS AND SYNONYMS O F PLUJIBAGO. 177 veste Luft [fixed air or carbonic acid gas] und brennbares Wesen [phlogiston]” ; while in a catalogue published by Ignaz von Born at Vienna in the same year we find (vol. ii, pp. 295-9), “Plombagine; Carbure de Fer.” “ Mr. cle Fourcroy regarde la Plombngine comme du charbon form6 dans l’inthrieur du globe ou enfoui dans la terre.” “ Plombagine grise. Graphite. , . . Elle est compos6e de Carbone et d’un dixihme de Fer.” ‘‘ Barrodal prds de Kesmig” is given as a locality. “ A plan of a course of lectures on Nineralogy,” by John Hailstone, Cambridge, 1792, contains the following (p. 72) : “Plumbago. Base of fixed Air united to a small portion of Iron, Black Lead, Graphite.” This is, so far as I am aware, the first use of the word graphite in English.’ I n the thirtennth edition of the “ Systema Naturs” (1793), ‘ graphites’ is used as the generic and plumbago as the specific name of graphite (p. 284), and plumbago is not employed in any other sense. Molybdenite becomes molybdsns vulgaris (p. 309): Meantime Pelletier (op. cit., p. 442) and P. J. Hjelm had separated the metal contained in molybdenite, and the latter had given it the name of molybdenum (<‘ Kongl. Sven. Acad. Nya, Hand.,” vol. ix, 1788, p. 288). In 1796 the second volume of the second edition of Kirwan’s ( 6 Elements of Mineralogy” was published in Dublin. On p. 58 of vol. ii we find: “Carbon, combined with one-tenth, or one- eighth of its weight of Xetallic Iron, Plumbago. Graphite of Werner, Reisbley of others. (‘ Blyertz of the Swedes.” On p. g 3 On p. 154 we find as the third species of iron “Mineralized by Carbon. plombaginous, or mcaceous iron ore. Xisen Glimmer of Werner. . . . the single scales are somewhat Transparent, and transmit a reddish light.” This i, what is now known as specular iron ore, a variety of hematite (Fez03). It contains no carbon and is in no way allied to plumbago (see p. 166 a.te). See also “A System of Mineralogy,” founded chiefly on the plan of Cronstedt, by J. G. Schmeisser, London, 1794, p. 303, and “ A Systematic Arrangement of Minerals,” by William Babington, London, 1795, p. 25. ‘‘ y g p 2 In ‘‘ A General System of Nature,” by Sir Charles Linneus, London, H O G , pp. 237, 309, Molybdenum is made the generic name of the mineral in analogy with the procedure of Linneus in the case of the compounds of the other metals, and following the editions of 1765 and 1775. See also “A System of Mineralogy,” founded chiefly on the plan of Cronstedt, by J. G. Schmeisser, London, 1794, p. 303, and “ A Systematic Arrangement of Minerals,” by William Babington, London, 1795, p. 25. 2 In ‘‘ A General System of Nature,” by Sir Charles Linneus, London, H O G , pp. 237, 309, Molybdenum is made the generic name of the mineral in analogy with the procedure of Linneus in the case of the compounds of the other metals, and following the editions of 1765 and 1775. 3 On p. 154 we find as the third species of iron “Mineralized by Carbon. plombaginous, or mcaceous iron ore. Xisen Glimmer of Werner. . . . the single scales are somewhat Transparent, and transmit a reddish light.” This i, what is now known as specular iron ore, a variety of hematite (Fez03). It contains no carbon and is in no way allied to plumbago (see p. 166 a.te). THE MBlNINGS AND SYNONYMS OF PLUMBAGO. 319, under the heading (‘ Molybdenite (Molybdenum of Hjelu~),~’ an account is given of the metal molybdenum. The first mineral species under this heading is described as “ Mineralized by sulphur. Molybdena, or Molybden, Wasserbley of the Germans, Blegerz of the Swedes ” (p. 323). 178 THE MEANINGS AND SYNONYMS OF PLUMBAGO. I n 1807 Brongniart, in his Mineralogy (ii, p. 92), apparently misunderstanding Karsten, applied his term molybdenite to the mineral sulphide instead of the metal, and it has since continued in general use in this sense, both in French and English. In Germany the older form Molybdan has continued to be used for the sulphide, but ‘ Molybdiin glane,’ first employed by Karsten (Tab., 1808, p. 70), is also in use in Germany. The name ‘ edler Molgbdiinglanz ’ was given by A. Breithaupt (“ Vollst. Char. Nin. Syst.,” 1832, pp. 273, 233) to an auriferous rariety of nagyagite, a mineral containing sulphur, tellurium, and antimony, and the terms argent molybdique (Born, op. cit., 1790, ii, p. 419) and Molybdan Silber (Werner, “Letz. Min. Syst.,” 1817, pp. 18, 48) were applied to a variety of tetradymite (a sulphotelluride of bismuth), containing dyer. These and other tellurium minerals, which are similar in appearance to molybdenite, are often associated with gold, and the references to the occurrence of gold with ‘molybdaena’ in early writers (e.g., Berlichius, loc. cit., and Briinnich in his notes on Cronstedt’s Mineralogy, German edition of 1770, p. 181, and appendix to the English edition of 1772, p. 14) render it probable that they were included under that term. y The mineral known as plumbago or graphite continued to be comidered a carbide of iron till Karsten in 1826 (“ Arch. Bergbau u. Huttenk.,” vol. xii, pp. 91-6) and Sefstrom in 1828 (“ Jern Contorets Ann.,” vol. xii, pt. 1, 1829, p. 145) showed that the old view, that the iron was only present as an impurity in the form of iron pyrites, was correct, and that the mineral was merely a pure form of carbon. The name of graphite appears to have come earlier into general use in Germany, where, however, Reiszblei still survives, than in France or England. In France mine dc plomb and plombagine are still widely used, though graphite is the recognized scientific expression for the mineral. 1 See also “ Graphite,” by F. Cirkel, Ottaura, 1907, in which ,plumbago. is nnly used in quotations, though the subject is treated from the ecunomlc standpoint. THE MBlNINGS AND SYNONYMS OF PLUMBAGO. In English we have the choice of the three terms black lead, plumbago, and graphite, but the French form plombagine has been occasionally used. At the present day the term black lead is still popular, while plumbago is almost confined to the language of commerce and of the arts, including mining. Graphite has long been firmly established in scientific literature, and is gradually extending its sphere of employment. This is particularly the case in America, as is illustrated by the fact that while the term plumbago was employed as a heading in the first volume (1892) of the “Mining Industry,” annually 179 THE MEANINGS AND SYNOPIYMS OF PLUMBAGO. published in New York, this dcsignation was changed to graphite in subsequent issues.‘ I n Italian both graffito and piombaggine are still in use, and in Spanish grafito and plombagina, as well as lipiz, ljpiz plomo, alquifol (properly galena), carbon, chacal, mina de plomo, and piedra mineral de plomo (E. Halse, Dict. Span. Min. Terms, 1908, p. 176). In tracing the history of these words I have had often to pass from country to country, and from one idiom to another ; for the literature of science is to some extent at least international in character, so that it is impossible t o give an intelligible account of its technical terms if the attention is confined to one state or a single language. The stream of speculation and research to which we owe our knowledge of the laws of nature and our control, such as it is, over its forces, has wandered far on its way to where we stand, and has paid but little heed t o frontiers of any kind, even the narrow seas that have in so many ways fostered our individuality among the peoples of Europe.
https://openalex.org/W4377029624	https://hal.science/hal-04029554/file/Mesmar%20et%20al.%2C%202022%20%28Origanum%20breast%20cancer%29%28Front%20in%20Pharmacol%29.pdf	English	null	Ethanolic Extract of Origanum syriacum L. Leaves Exhibits Potent Anti-Breast Cancer Potential and Robust Antioxidant Properties	null	2,023	cc-by	16,884	Ethanolic extract of Origanum syriacum L. leaves exhibits potent anti-breast cancer potential and robust antioxidant properties OPEN ACCESS EDITED BY Hina Siddiqui, University of Karachi, Pakistan REVIEWED BY Angela Bisio, University of Genoa, Italy Saima Rasheed, University of Karachi, Pakistan CORRESPONDENCE Abdullah Shaito, abdshaito@qu.edu.qa Marc Maresca, m.maresca@univ-amu.fr Elias Baydoun, eliasbay@aub.edu.lb SPECIALTY SECTION This article was submitted to Experimental Pharmacology and Drug Discovery, a section of the journal Frontiers in Pharmacology RECEIVED 14 July 2022 ACCEPTED 12 September 2022 PUBLISHED 10 October 2022 Joelle Mesmar1, Rola Abdallah1, Kamar Hamade2, Serine Baydoun3, Najlaa Al-Thani4, Abdullah Shaito5, Marc Maresca6, Adnan Badran7 and Elias Baydoun1 1Department of Biology, American University of Beirut, Beirut, Lebanon, 2UMRT INRE 1158 BioEcoAgro, Laboratorie BIOPI, University of Picardie Jules Verne, Amiens, France, 3Breast Imaging Section, Imaging Institute, Cleveland Clinic Foundation, Cleveland, OH, United States, 4Research and Development Department, Barzan Holdings, Doha, Qatar, 5Biomedical Research Center, College of Medicine, and Department of Biomedical Sciences at College of Health Sciences, Qatar University, Doha, Qatar, 6Aix-Marseille University, CNRS, Centrale Marseille, iSm2, Marseille, France, 7Department of Nutrition, University of Petra, Amman, Jordan Background: Breast cancer (BC) is the second most common cancer overall. In women, BC is the most prevalent cancer and the leading cause of cancer- related mortality. Triple-negative BC (TNBC) is the most aggressive BC, being resistant to hormonal and targeted therapies. Hypothesis/Purpose: The medicinal plant Origanum syriacum L. is a shrubby plant rich in bioactive compounds and widely used in traditional medicine to treat various diseases. However, its therapeutic potential against BC remains poorly investigated. In the present study, we screened the phytochemical content of an ethanolic extract of O. syriacum (OSEE) and investigated its anticancer effects and possible underlying mechanisms of action against the aggressive and highly metastatic human TNBC cell line MDA-MB-231. Methods: MTT, trans-well migration, and scratch assays were used to assess cell viability, invasion, or migration, respectively. Antioxidant potential was evaluated in vitro using the DPPH radical-scavenging assay and levels of reactive oxygen species (ROS) were assessed in cells in culture using DHE staining. Aggregation assays were used to determine cell-cell adhesion. Flow cytometry was used to analyze cell cycle progression. Protein levels of markers of apoptosis (BCL-2, pro-Caspase3, p53), proliferation (p21, Ki67), cell migration, invasion, or adhesion (FAK, E-cadherin), angiogenesis (iNOS), and cell signaling (STAT3, p38) were determined by immunoblotting. A chorioallantoic Membrane (CAM) assay evaluated in ovo angiogenesis. TYPE Original Research PUBLISHED 10 October 2022 DOI 10.3389/fphar.2022.994025 TYPE Original Research PUBLISHED 10 October 2022 DOI 10.3389/fphar.2022.994025 TYPE Original Research PUBLISHED 10 October 2022 DOI 10.3389/fphar.2022.994025 1 Introduction glycosides, terpenes, and phenols (Mesmar et al., 2022). These bioactive compounds bestow the plant with various pharmacological properties including antioxidant, anti- inﬂammatory, anticancer, antimicrobial, and neuroprotective effects, among others (Alwafa et al., 2021). Importantly, its extracts have been documented to inhibit the proliferation of human BC MCF-7 cells (Al-Kalaldeh et al., 2010; Husein et al., 2014) and leukemic TH-1 cells (Ayesh et al., 2014). This prompted the investigation of the effects of the plant in the context of the aggressive TNBC subtype, using MDA-MB- 231 BC cells as an in vitro model of TNBC. Cancer is a leading cause of death worldwide, having claimed an estimated 10 million deaths in 2020. Breast cancer (BC) is the most common cause of new cancer cases and the ﬁfth leading- cause of cancer-related deaths (Who, 2021). Moreover, the incidence of BC has increased signiﬁcantly in recent years to become the world’s most prevalent cancer (Breast Cancer, 2021). Despite signiﬁcant advancements in treatment regimens and modalities, treatment of most types of breast cancer is still limited to surgery, chemotherapy, and irradiation. Hormone replacement therapy can be used for breast cancer subtypes that are positive for the estrogen receptor (ER) or progesterone receptor (PR), while targeted therapies using antibodies, like trastuzumab, is effective against breast cancers that over-express human epidermal growth factor receptor (HER2). Triple-negative breast cancer (TNBC) accounts for 10–20% of BC cases. Lacking the overexpression of HER2 and being negative for ER and PR, TNBC does not respond to targeted or hormone replacement therapies. As such, TNBC is an aggressive BC subtype that is associated with poor prognosis (Breastcancer.org), mandating that alternative treatment approaches be sought. In this regard, therapeutic approaches using plant sources have been gaining interest and popularity (Howes, 2018). Contextually, women have an inclination for the use of natural products and herbal remedies as these are claimed to be safer alternatives without signiﬁcant side effects compared with conventional medicines (Cassidy, 2003). Furthermore, plants have a long history in the treatment of cancer and have been a source of several anticancer drugs (Cragg and Newman, 2005; Buyel, 2018). In this study, we screened the phytochemical constituents of an ethanolic extract of O. syriacum (OSEE) and tested its effect on the malignant phenotype of MDA-MB-231 cells, aiming to uncover the possible molecular mechanisms behind its anticancer activity. We report that OSEE has a potent antioxidant activity. KEYWORDS herbal medicine, phytochemical content, breast cancer, metastasis, oxidative stress, reactive oxygen species, ROS, Origanum syriacum L 2 Materials and methods Origanum syriacum L. is an aromatic perennial shrub native to the Mediterranean region and widely used in culinary practices. It has also been traditionally used in folk medicine to relieve stomach pain and in the treatment of colds and toothaches (Alwafa et al., 2021). In recent years, it has been reported to be rich in bioactive compounds such as ﬂavonoids, 1 Introduction Importantly, OSEE inhibited the proliferation of TNBC cells by causing a G0/G1 phase arrest, concomitant with a decrease of Ki67 levels and an increase of p21 levels. OSEE signiﬁcantly inhibited MDA-MB-231 cell growth and metastatic properties by inhibiting proliferative signaling, activating suppressors of cell growth, enhancing apoptotic cell-death machinery, reducing migration and invasion of MDA-MB1-cells, in addition to inhibiting angiogenesis in a process that correlated with inhibition of iNOS. Mechanistically, OSEE inhibited STAT3 signaling and activated the p38 MAPK pathway, implicating a crosstalk between p21, p53, iNOS, and reactive oxygen species (ROS). Ethanolic extract of Origanum syriacum L. leaves exhibits potent anti-breast cancer potential and robust antioxidant properties Results: We demonstrated that OSEE had potent radical scavenging activity in vitro and induced the generation of ROS in MDA-MB-231 cells, especially at higher OSEE concentrations. Non-cytotoxic concentrations of OSEE attenuated cell proliferation and induced G0/G1 cell cycle arrest, which was associated with phosphorylation of p38 MAPK, an increase in the levels of tumor suppressor protein p21, and a decrease of proliferation marker protein Ki67. Additionally, only higher concentrations of OSEE were able to attenuate inhibition of proliferation induced by the ROS scavenger N-acetyl cysteine (NAC), indicating that the anti-proliferative effects CITATION Mesmar J, Abdallah R, Hamade K, Baydoun S, Al-Thani N, Shaito A, Maresca M, Badran A and Baydoun E (2022), Ethanolic extract of Origanum syriacum L. leaves exhibits potent anti- breast cancer potential and robust antioxidant properties. F t Ph l 13 994025 Front. Pharmacol. 13:994025. doi: 10.3389/fphar.2022.994025 COPYRIGHT © 2022 Mesmar, Abdallah, Hamade, Baydoun, Al-Thani, Shaito, Maresca, Badran and Baydoun. 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. 01 Frontiers in Pharmacology Frontiers in Pharmacology frontiersin.org Mesmar et al. 10.3389/fphar.2022.994025 10.3389/fphar.2022.994025 of OSEE could be ROS-dependent. OSEE stimulated apoptosis and its effector Caspase-3 in MDA-MB-231 cells, in correlation with activation of the STAT3/ p53 pathway. Furthermore, the extract reduced the migration and invasive properties of MDA-MB-231 cells through the deactivation of focal adhesion kinase (FAK). OSEE also reduced the production of inducible nitric oxide synthase (iNOS) and inhibited in ovo angiogenesis. Conclusion: Our ﬁndings reveal that OSEE is a rich source of phytochemicals and has robust anti-breast cancer properties that signiﬁcantly attenuate the malignant phenotype of MD- MB-231 cells, suggesting that O. syriacum may not only act as a rich source of potential TNBC therapeutics but may also provide new avenues for the design of novel TNBC drugs. 2.3 LC-MS Test for phenolic compounds: 0.5 g of the plant extract was mixed with 5 ml of ethanol and ultrasonicated for 15 min at 30°C. Test for phenolic compounds: 0.5 g of the plant extract was mixed with 5 ml of ethanol and ultrasonicated for 15 min at 30°C. The mixture was ﬁltered and 2 ml of distilled water added to the ﬁltrate followed by a few drops of 5%-FeCl3. The presence of phenolic compounds was determined by the appearance of a dark green color (Keo et al., 2017). 2.1 O. syriacum ethanolic extract Leaves of O. syriacum were collected from South of Lebanon in the spring season (April-June) of 2020 and 2021. The plant was identiﬁed as Origanum syriacum L. by Mohammad Al Zein, a Frontiers in Pharmacology 02 frontiersin.org Mesmar et al. 10.3389/fphar.2022.994025 10.3389/fphar.2022.994025 Test for cardiac glycosides: 5 ml of ethanol was added to 0.5 g of the plant extract and ultrasonicated for 15 min at 30°C. The mixture was ﬁltered and the ﬁltrate was evaporated to dryness. A few milligrams of the dried extract were dissolved in 1 ml of glacial acetic acid and few drops of 2%-FeCl3, and then 1 ml of concentrated H2SO4 was added to the side of the test tube. The presence of a brown ring indicated the presence of cardiac glycosides (Keo et al., 2017). plant taxonomist at the Biology Department, American University of Beirut (AUB), and a voucher specimen has been deposited at the Post Herbarium (AUB), under number MSA 2020–1. The leaves were rinsed and air-dried in the dark at room temperature, then ground into a ﬁne powder and suspended in 80% ethanol [20 ml of distilled water and 80 ml of absolute ethanol (Fisher Scientiﬁc; U.K)] for 72 h in the dark. The suspension was then ﬁltered, dried using a rotary vacuum evaporator and lyophilized. The obtained powder was dissolved in 80% ethanol at a concentration of 200 mg/ml and stored at 4°C. Test for terpenoids: 0.5 g of the plant extract was added to 5 ml of chloroform and ultrasonicated for 15 min at 30°C. The mixture was ﬁltered and 2 ml of concentrated H2SO4 added to the side of the test tube. The presence of a reddish-brown color indicated the presence of terpenoids (Keo et al., 2017). 2.3.1 Sample preparation The mixture was ﬁltered and 2 ml of distilled water added to the ﬁltrate followed by a few drops of 5%-FeCl3. The presence of phenolic compounds was determined by the appearance of a dark green color (Keo et al., 2017). Sample was ﬁltered through 0.22 µm PTFE membrane ﬁlters and placed in glass vials for further LC-MS analysis. 2.2 Phytochemical analysis Test for anthraquinones: 0.5 g of the plant extract was added to 4 ml of benzene. The mixture was ﬁltered, and 10% ammonia solution was added. After shaking, the presence of a red or violet color indicated the presence of anthraquinones (Basiru et al., 2013). Test for tannins: 5 ml of distilled water was added to 0.5 g of the plant extract and ultrasonicated for 15 min at 80°C. The mixture was ﬁltered, cooled down to room temperature, and ﬁve drops of 0.1%- FeCl3 added to the ﬁltrate. Brownish green or blue-black coloration indicated the presence of tannins (Keo et al., 2017). Test for anthocyanins: 5 ml of ethanol was added to 0.5 g of the plant extract and ultrasonicated for 15 min at 30°C. Then, 1 ml of NaOH was added to 1 ml of the extract and heated for 5 min at 100°C. The presence of a bluish-green color indicated the presence of anthocyanin (Bassal et al., 2021). Test for resins: 5 ml of distilled water was added to 0.5 g of the plant extract and ultrasonicated for 15 min at 30°C. Then, the mixture was ﬁltered. The presence of resins was indicated by turbidity of the ﬁltrate (Keo et al., 2017). Test for essential oils: 5 ml of ethanol was added to 0.5 g of the plant extract and ultrasonicated for 15 min at 30°C. Then, 100 µl of 1 M NaOH was added to the ﬁltrate followed by a few drops of 1 M HCl. The formation of a white precipitate indicated the presence of essential oils (Keo et al., 2017). Test for saponins: 5 ml of distilled water was added to 0.5 g of the plant extract and ultrasonicated for 15 min at 80°C. The mixture was ﬁltered, cooled down to room temperature, and then shaken until the formation of a stable persistent froth, which indicated the presence of saponins (Keo et al., 2017). 2.9 Flow cytometry analysis of cell cycle Cells were grown in 10-mm tissue-culture plates for 24 h before the addition of OSEE or ethanol at a concnetration equavlent to that present in OSEE as a vehicle control. After incubation, cells were harvested, washed twice, resuspended in 500 µl PBS, ﬁxed with an equal volume of 100% ethanol, and incubated at −20°C for at least 12 h. Cells were then pelleted, washed twice with PBS and permeabilized in 0.1% Triton X-100/ PBS and incubated for 15 min on ice. Afterwards cells were pelleted, resuspended in PBS containing 40 μg/ml propidium iodide and 25 μg/ml RNase A, and incubated at 37°C for 5 min. 2.5 Cell viability assay MDA-MB-231 cells (5 × 103) were seeded in 96-well plates and allowed to grow until they reached 30–40% conﬂuence. The cells were then treated with increasing concentrations of OSEE and incubated for a total period of 72 h. Cell viability was measured by the reduction of 3-(4,5- dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich, St. Louis, MO, United States). Cell growth was determined as the proportional viability of the treated cells compared with the ethanol vehicle-treated cells, the viability of which is set to be 100%. In cell viability assays with N-acetyl cysteine (NAC; Sigma-Aldrich, St. Louis, MO, United States), 5 mM NAC was added to the cells for 30 min before OSEE treatment. Assays were performed in triplicate and repeated three times. Data are presented as mean values ± standard error of the mean (SEM). 2.3.2 LC-MS data acquisition 0.5 ml of different concentrations of OSEE (50, 100, 200, 400, and 600 μg/ml) was mixed with 0.5 ml of DPPH solution (0.5 mM in methanol) and 3 ml of methanol. The blank consisted of 0.5 ml of 80% ethanol, 0.5 ml of DPPH solution and 3 ml of methanol. Mixed samples were then kept in the dark for 30 min and the OD was measured at a wavelength of 517 nm using a spectrophotometer. The DPPH-scavenging activity of each concentration of the extract was calculated using the formula: % radical-scavenging activity = [(OD blank—OD plant extract at each concentration)]/(OD blank)] X 100. Ascorbic acid was used as a standard. D9312, Sigma-Aldrich Co.,) is a free radical used as a colorimetric probe to evaluate the antioxidant properties of plant extracts and constituents: the color of the solution changes from purple to pale yellow. 0.5 ml of different concentrations of OSEE (50, 100, 200, 400, and 600 μg/ml) was mixed with 0.5 ml of DPPH solution (0.5 mM in methanol) and 3 ml of methanol. The blank consisted of 0.5 ml of 80% ethanol, 0.5 ml of DPPH solution and 3 ml of methanol. Mixed samples were then kept in the dark for 30 min and the OD was measured at a wavelength of 517 nm using a spectrophotometer. The DPPH-scavenging activity of each concentration of the extract was calculated using the formula: % radical-scavenging activity = [(OD blank—OD plant extract at each concentration)]/(OD blank)] X 100. Ascorbic acid was used as a standard. TABLE 1 Chromatographic gradient conditions for the analysis of O. Syriacum ethanolic crude extract. TABLE 1 Chromatographic gradient conditions for the analysis of O. Syriacum ethanolic crude extract. Time (min) Methanol (%) Water (%) 0 10.0 90.0 5 20.0 80.0 8 40.0 60.0 11 50.0 50.0 13 60.0 40.0 16 80.0 20.0 17 90.0 10.0 19 10.0 90.0 21 10.0 90.0 2.7 Dihydroethidium staining MDA-MB-231 cells were seeded in 12-well plates and incubated until they reached 50% conﬂuence. The cells were then treated for 24 h with the indicated concentrations of OSEE; media containing less than 1% ethanol was used as the vehicle control. After incubation, the medium was removed and the cells were washed twice with ice-cold phosphate-buffered saline (PBS). DHE stain (6 μM) was added and the cells were incubated in the dark for 45 min. Then the stain was removed, the cells were washed once with cold PBS, and visualized using a ZEISS Axio Observer. Human breast cancer cells MDA-MB-231 (American Type Culture Collection, Manassas, VA) were maintained in DMEM high-glucose medium supplemented with 10% fetal bovine serum (FBS) (both from Sigma-Aldrich, St. Louis, MO, United States) and 1% penicillin/streptomycin (Lonza, Switzerland) and kept in a humidiﬁed chamber (37°C and 5% CO2). 2.8 Microscopic analysis of apoptotic morphological changes Cells were grown in 6-well tissue-culture plates in the absence or presence of the indicated concentrations of OSEE. Morphological changes characteristic of apoptotic cells were determined after 24 and 48 h using an inverted phase-contrast microscope (objectives 10×, 20×, and 40×). 2.3.2 LC-MS data acquisition Test for ﬂavonoids: 1 ml of 2% NaOH solution was mixed with 0.2 g of the plant extract. This produced a concentrated, yellow-colored solution. Then, few drops of diluted acid were added to the mixture, which made the solution colorless, indicating the presence of ﬂavonoids (Mir et al., 2013). The LC-MS analysis was performed using a single quadripole LC-MS-2020 mass spectrometer (Shimadzu Corporation, Kyoto, Japan), which was equipped with an electrospray ion source (ESI). UPLC separation was performed using a Kinetex C18 (1.7 µm, 100 mm × 2.1 mm, Phenomenex, Torrance, CA, United States) column. The column temperature was maintained at 40°C. The injected volume was 10 µL. Water and methanol, both supplemented with 0.1% formic acid, were used as mobile phases. Test for quinones: 0.5 g of the plant extract was added to 5 ml of ethanol and ultrasonicated for 15 min at 30°C. The mixture was ﬁltered and 1 ml of concentrated H2SO4 was added to 1 ml of ﬁltrate. The appearance of a red color indicated the presence of quinones (Keo et al., 2017). A stepwise gradient method, presented in Table 1, was used for elution, at a ﬂow rate of 0.4 ml/min. Test for steroids: 5 ml of ethanol was added to 0.5 g of the plant extract and ultrasonicated for 15 min at 30°C. The mixture was ﬁltered and the ﬁltrate was evaporated to dryness. A few milligrams of the dried extract were dissolved in 1 ml of chloroform and 1 ml of glacial acetic acid, and then 1 ml of concentrated H2SO4 was added to the side of the test tube and mixed with the solution. The presence of steroids was indicated by appearance of a green color (Keo et al., 2017). MS data were collected in the negative ion mode, over a m/z range of 50–1,500. The parameters of electrospray ionization (ESI) source were set as follows: ESI probe temperature 350°C, DL temperature 250°C, heat block temperature 200°C, ESI probe voltage 4.5 kV, detector voltage 1.6 kV, DL voltage 100 V, Q-array RF voltage 60 V, and nebulizing gas ﬂow 1.5 L/min. Frontiers in Pharmacology 03 frontiersin.org 10.3389/fphar.2022.994025 Mesmar et al. D9312, Sigma-Aldrich Co.,) is a free radical used as a colorimetric probe to evaluate the antioxidant properties of plant extracts and constituents: the color of the solution changes from purple to pale yellow. 2.11 Trans-well migration chamber assay The migratory ability of MDA-MB-231 cells was also assessed with trans-well inserts (8 μm pore size; BD Biosciences, Bedford, MA, United States). Cells were seeded at a density of 1.0 × 105 cells per well, into the upper chamber of the insert, and treated with less than 1% ethanol, as a vehicle control, or the indicated concentrations of OSEE. DMEM supplemented with 10% fetal bovine serum was placed into the bottom wells in the system as a chemo-attractant and then the plates were incubated at 37°C for 24 h. Cells were then washed, and non- penetrating cells were removed from the upper surface of the ﬁlter with a sterile cotton swab. Cells that had migrated through to the lower surface of the insert were ﬁxed with 4% formaldehyde, stained with DAPI, and counted under a ﬂuorescence microscope. Assay was repeated three times and data were presented as mean values ± SEM. 2.15 Whole-cell extracts and western blotting analysis For whole-cell lysates, cells were washed twice with PBS and lysed in 2% SDS, 60 mM Tris lysis buffer (pH 6.8) and centrifuged at 5,000 g for 10 min. The protein concentration of the supernatant was determined using the Bradford protein assay kit (Biorad, Hercules, CA, United States) and 25–30-μg aliquots were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis before being transferred to a polyvinylidene diﬂuoride membrane (Immobilon PVDF; Biorad) and blocked for 1 h at room temperature with 5% non-fat dry milk in TBST (TBS and 0.05% Tween 20). Immunodetection was performed by incubating the membrane with speciﬁc primary antibodies at 4°C overnight. Horseradish peroxidase- conjugated anti-IgG was used as secondary antibody and immunoreactive bands were detected using the ECL substrate kit (Thermo Scientiﬁc, Rockford, IL, United States), according to the manufacturer’s instructions. All primary and secondary antibodies were purchased from Cell Signaling (Cell Signaling Technology, Inc., Danvers, MA, United States). 2.10 Wound-healing assay MDA-MB-231 cells were grown in 12-well tissue-culture plates until conﬂuent. A scrape was made through the conﬂuent monolayer using a sterile 200-μL plastic pipette tip. The culture medium was then removed, the cells were washed twice with PBS (Sigma-Aldrich, St. Louis, MO, United States) to remove cellular debris, and incubated at 37°C in fresh medium in the presence or absence of the indicated concentrations of OSEE. Photomicrographs of the wound were taken at baseline (0 h) and for the 4–10 h period considered, using an inverted phase-contrast microscope (objective 10×). The width of the wound was expressed as the average ± SEM between the measurements taken at time zero and the corresponding time points. 2.6 DPPH (α, α-diphenyl-β-picrylhydrazyl) antioxidant activity assay The antioxidant activity of the ethanolic extract of O. syriacum leaves was measured using the DPPH-radical- scavenging assay as previously described but with some modiﬁcations (Moraes-De-Souza et al., 2008). DPPH (cat# Frontiers in Pharmacology 04 frontiersin.org Mesmar et al. 10.3389/fphar.2022.994025 10.3389/fphar.2022.994025 Cell samples were then analyzed with the BD FACSCanto II Flow Cytometry System (Becton Dickinson) and data acquired using the FACSDiva 6.1 software. surface of the ﬁlter with a sterile cotton swab. Cells that had penetrated through the Matrigel to the lower surface of the insert were ﬁxed with 4% formaldehyde, stained with DAPI, and counted under a ﬂuorescence microscope. Assay was repeated three times and data were presented as mean values ± SEM. 2.13 Adhesion assay MDA-MB-231 cells were grown in the presence or absence of OSEE for 24 h and then seeded onto collagen-coated 24-well tissue-culture dishes in duplicate. Cells were incubated at 37°C for 1 h and unattached cells were removed by gently washing the wells twice with PBS. The number of adherent cells was determined by the MTT reduction assay, as described above. 2.14 Chorioallantoic membrane assay Fertilized chicken eggs were incubated at 38°C and 60% relative humidity for 10 days. Afterwards, the highly vascularized CAM was dropped by drilling a 1-cm2 hole through the eggshell into the air sac. OSEE was then applied onto the CAM to test its effect on blood vessel growth. After 24 h, pictures of the CAM were taken and the angiogenic response was as analyzed using the AngioTool software, which quantiﬁes the length of the vessels and number of junctions. OSEE Tannins + Resins + Saponins - Phenols + Flavonoids + Quinones + Sterols and steroids + Cardiac glycosides + Terpenoids + Anthraquinones - Anthocyanins - Essential oils + (−): absent; (+): present. 2.12 Matrigel invasion assay The invasiveness of the MDA-MB-231 cells was evaluated using a BD Matrigel Invasion Chamber (8-μm pore size; BD Biosciences, Bedford, MA, United States). Brieﬂy, cells were seeded at a density of 1.0 × 105 cells per well, into the upper chamber of the insert, and treated with less than 1% ethanol, as a vehicle control, or the indicated concentrations of OSEE. DMEM supplemented with 10% fetal bovine serum was placed into the bottom wells of the chamber as a chemo-attractant and then incubated at 37°C for 24 h. Cells were then washed, and non-penetrating cells were removed from the upper Frontiers in Pharmacology 05 frontiersin.org Mesmar et al. Mesmar et al. 10.3389/fphar.2022.994025 3.1 Phytochemical screening O. syriacum has many primary and secondary bioactive metabolites (Mesmar et al., 2022). Extensive HPLC analyses and the phytochemical bioactives of O. syriacum have been reported in several studies (Alma et al., 2003; Dorman et al., 2004; Mesmar et al., 2022). Apigenin, naringenin, rosmarinic acid, carvacrol, carveol thymoquinone, thymol, and caffeic acid are some of the reported molecules (Alma et al., 2003; Dorman et al., 2004; Mesmar et al., 2022). Here we conﬁrmed the presence of several classes of phytochemical compounds in OSEE. Table 2 shows that OSEE contains tannins, phenols, ﬂavonoids, quinones, steroids, terpenoids as well as cardiac glycosides and essential oils. To conﬁrm the anti-proliferative effects of OSEE, protein lysates from OSEE-treated MDA-MB-231 cells were immunoblotted with an antibody against Ki67, a widely used biomarker for the evaluation of cell proliferation and the prognosis of many cancers. Particularly, Ki67 is highly expressed in TNBC, which is associated with its aggressive pathologic features and poor clinical outcomes (Yang C et al., 2018). Figure 2B, shows that treatment of MDA-MB-231 cells with 100 and 200 μg/ml OSEE caused a remarkable decrease in Ki-67 protein levelsby 0.83- and 0.67-fold, compared with vehicle-treated control cells, respectively (Figure 2B). The decrease in Ki67 protein levels seems to be concentration- dependent (Figure 2B). These data conﬁrm data from Figure 2A, suggesting that OSEE does indeed interfere with the cell proliferation process in MDA-MB-231 cells. 2.16 Statistical analysis Results were evaluated using Student’s t-test. For the comparison of more than two means, ANOVA was used using one-way ANOVA (with Dunnett’s post hoc test) or two-way ANOVA (with Tukey–Kramer’s post hoc test). Data were presented as mean ± SEMand a p-value of <0.05 was considered as statistically signiﬁcant. Several of the bioactives reported to be present in O. syriacum have been shown to have potent anti-BC effects. Naringenin, apigenin, carvacrol, thymoquinone, thymol, and rosmarinic acid were shown to reduce the malignant phenotype of BC cell lines (Kanno et al., 2005; Demain and Vaishnav, 2011; Lee et al., 2019; Messeha et al., 2020; Sampaio et al., 2021). Knowing that the unfractionated plant extract may often have more potent activities than a single or a few of its phytochemicals, it was thought prudent to investigate the anti-cancerous effects of O. syriacum leaves in a triple-negative human BC cell line (TNBC), MDA-MB-231. To this end, we examined the anti-proliferative activity of OSEE against MDA-MB-231 cells. The effect of various concentrations (0, 50, 100, 200, 400 and 600 μg/ml) of the extract on the proliferation of human TNBC MDA-MB- 231 cells was assessed at 24, 48, and 72 h of treatment. Results showed that OSEE treatment decreased cell viability in a concentration- and time-dependent manner (Figure 2A). For example, at 48 h of treatment, cell viability using 50, 100, 200, 400 and 600 μg/ml of OSEE to treat MDA-MB-231 cells was 77.4 ± 5.1, 68.4 ± 9.5, 45.5 ± 7.9, 28.2 ± 2.9, and 20.2 ± 6.9% that of control cells, respectively (Figure 2A). The half-maximal inhibitory concentration (IC50) was 875, 179.4, and 125.4 μg/ ml at 24, 48, and 72 h, respectively. Based on these IC50 values, 100 and 200 μg/ml OSEE were used in further experiments. 3.2 LC-MS of O. Syriacum crude extract Metabolites were principally identiﬁed by matching masses and retention times of pure standards. Six compounds were identiﬁed as shown in Table 3 and Figure 1. TABLE 2 Phytochemical screening of O. syriacum ethanolic crude extract. Metabolite OSEE Tannins + Resins + Saponins - Phenols + Flavonoids + Quinones + Sterols and steroids + Cardiac glycosides + Terpenoids + Anthraquinones - Anthocyanins - Essential oils + (−): absent; (+): present. Frontiers in Pharmacology 3.4 OSEE has potent antioxidant activity and can increase the generation of ROS in MDA-MB-231 cells syriacum inhibits cellular proliferation of MDA-MB-231 breast cancer cells. (A) MDA-MB-231 cells were treated or not with the indicated concentrations of O. syriacum ethanolic extract (OSEE) for 24, 48, and 72 h. For vehicle control, a concnetration of ethanol equivalent to that present in OSEE was used. Cell viability was monitored using the metabolism-based MTT assay, as described in Materials and Methods. (B) MDA-MB-231 cells were incubated for 24 h with and without the indicated concentrations of OSEE. Cells were then lysed, and protein lysates were subject to Western blotting with a Ki67 antibody. Data represent the mean ± SEM of three independent experiments (n = 3) carried out in triplicate and expressed as a percentage of the corresponding control cells. Statistical analysis was performed using one-way ANOVA followed by LSD post-hoc test (p < 0.05, p < 0.005, *p < 0.001). FIGURE 2 O. syriacum inhibits cellular proliferation of MDA-MB-231 breast cancer cells. (A) MDA-MB-231 cells were treated or not with the indicated concentrations of O. syriacum ethanolic extract (OSEE) for 24, 48, and 72 h. For vehicle control, a concnetration of ethanol equivalent to that present in OSEE was used. Cell viability was monitored using the metabolism-based MTT assay, as described in Materials and Methods. (B) MDA-MB-231 cells were incubated for 24 h with and without the indicated concentrations of OSEE. Cells were then lysed, and protein lysates were subject to Western blotting with a Ki67 antibody. Data represent the mean ± SEM of three independent experiments (n = 3) carried out in triplicate and expressed as a percentage of the corresponding control cells. Statistical analysis was performed using one-way ANOVA followed by LSD post-hoc test (p < 0.05, p < 0.005, p < 0.001). 2022; Oyenihi et al., 2022). To investigate whether OSEE exerts anti- proliferative effects on MDA-MB-231 cells through ROS generation, MDA-MB-231 cells were ﬁrst pre-treated with NAC to dampen ROS generation, followed by treatment with OSEE at various concentrations. Cell proliferation and viability were then assessed for a period of 3 days. NAC, acting as a ROS scavenger, can by itself inhibit proliferation of MDA-MB-231 cells at 24 h (data not shown), 48 h, and 72 h of treatment (Figure 3C). Figure 3C also shows that treatment with NAC augmented the inhibition of proliferation of MDA-MB-231 cells treated with lower concentrations of OSEE (50 and 100 μg/ml). 3.4 OSEE has potent antioxidant activity and can increase the generation of ROS in MDA-MB-231 cells ROS are implicated in many aspects of health and disease, including signaling processes. There is a delicate balance between oxidative stress and antioxidant mechanisms inside the cell, ensuring that physiological functions are maintained, and proper defense mechanisms are in place. Any disturbance of Frontiers in Pharmacology Frontiers in Pharmacology 06 frontiersin.org Mesmar et al. 10.3389/fphar.2022.994025 TABLE 3 Compounds identiﬁed from O. Syriacum ethanolic crude extract. Compound name Elemental composition [M-H]− precursor ion (m/z) RT (min) Vicenin-1 C26H28O14 563.14 5.23 Vicenin-2 C27H30O15 593.15 4.82 Orientin C21H20O11 447.09 5.60 Isoorientin C21H20O11 447.09 5.82 Vitexin C21H20O10 431.09 6.11 Isovitexin C21H20O10 431.09 6.71 FIGURE 1 Structures of the compounds identiﬁed from O. Syriacum ethanolic crude extract using LC-MS. FIGURE 1 Structures of the compounds identiﬁed from O. Syriacum ethanolic crude extract using LC-MS. FIGURE 1 Structures of the compounds identiﬁed from O. Syriacum ethanolic crude extract using LC-MS. (Figure 3A). Despite this signiﬁcant antioxidant-radical-scavenging activity of OSEE in the test tube in vitro (Figure 3A), we tested the effect of OSEE on ROS generation in MDA-MB-231 cells in culture. Indeed, MDA-MB-231 cells treated with increasing concentrations of OSEE, showed increased DHE ﬂuorescence as the concentration of OSEE increased (Figure 3B), indicating that OSEE increases the levels of ROS generation in MDA-MB-231 cells. this balance may lead to pathological outcomes. Notably, both ROS and antioxidants have been shown to play either anti- or pro- cancerous roles. Indeed, phytochemicals and other natural products have been reported to act as anti- or pro-oxidant agents, depending on the context, in a biphasic and concentration-dependent manner. For example, dietary supplementation with the antioxidant N-acetyl cysteine (NAC) can promote cancer progression and metastasis (Liou and Storz, 2010; Chio and Tuveson, 2017; Shaito et al., 2020). O. syriacum has been reported to contain many bioactive molecules with high antioxidant potential, such aspolyphenols.In this study, OSEE antioxidant potential was evaluated in vitro using the DPPH-radical-scavenging assay. OSEE exhibited signiﬁcant free- radical-scavenging activity which was concentration-dependent Reactive oxygen species function like a double-edged sword in cancer progression, depending on their concentration in the cell and the stage of cancer. ROS have been reported to either enhance tumorigenesis and promote tumor progression by causing DNA damage and inducing pro-oncogenic pathways, or to induce cell death and apoptosis of cancer cells (Shaito et al., 2020; Oyenihi et al., Frontiers in Pharmacology 07 frontiersin.org Mesmar et al. 10.3389/fphar.2022.994025 FIGURE 2 O. 3.4 OSEE has potent antioxidant activity and can increase the generation of ROS in MDA-MB-231 cells However, NAC treatment was not able to blunt the inhibition of proliferation of MDA-MB-231 cells induced by higher concentrations of OSEE (200, 400 and 600 μg/ml); on the contrary, OSEE attenuated NAC-induced inhibition of proliferation (Figure 3C). These data indicate that the anti-proliferative effects of OSEE may depend on the levels of ROS generation inside the cell, conﬁrming the biphasic concentration-dependent effects reported for other natural antioxidants. 3.5 OSEE induces cell-cycle arrest of MDA-MB-231 cells at G0/G1 phase To investigate the mode of the anti-proliferative effect induced by OSEE in MDA-MB-231 cells, the cell-cycle distribution of these cells was assessed, using PI staining Frontiers in Pharmacology Frontiers in Pharmacology 08 frontiersin.org frontiersin.org Mesmar et al. 10.3389/fphar.2022.994025 FIGURE 3 O. syriacum has remarkable antioxidant potential and can increase the generation of ROS in MDA-MB-231 cells. (A) The antioxidant activity of the indicated concentrations of OSEE was measured by 2,2-di-phenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity assay as described in Materials and Methods. Data represent the means ± SEM of three independent experiments. (B) Fluorescent images of dihydroethidium (DHE)- stained MDA-MB-231 cells. Cells were treated with and without the indicated concentrations of OSEE for 24 h and stained with DHE, as indicated in Materials and Methods, to measure intracellular ROS production. (C) MDA-MB-231 cells were pre-treated with NAC (10 mM) for 30 min and then with OSEE at the indicated concentrations. Cell viability was measured using the MTT assay at the indicated time points. OSEE-treated cells without NAC pre-treatment were used for comparison. Values represent the means ± SEM of three independent experiments performed in triplicates and expressed as percentage of vehicle-treated control cells (p < 0.05, p < 0.005, p < 0.001). FIGURE 3 O. syriacum has remarkable antioxidant potential and can increase the generation of ROS in MDA-MB-231 cells. (A) The antioxidant activity of the indicated concentrations of OSEE was measured by 2,2-di-phenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity assay as described in Materials and Methods. Data represent the means ± SEM of three independent experiments. (B) Fluorescent images of dihydroethidium (DHE)- stained MDA-MB-231 cells. Cells were treated with and without the indicated concentrations of OSEE for 24 h and stained with DHE, as indicated in Materials and Methods, to measure intracellular ROS production. (C) MDA-MB-231 cells were pre-treated with NAC (10 mM) for 30 min and then with OSEE at the indicated concentrations. Cell viability was measured using the MTT assay at the indicated time points. OSEE-treated cells without NAC pre-treatment were used for comparison. Values represent the means ± SEM of three independent experiments performed in triplicates and expressed as percentage of vehicle-treated control cells (p < 0.05, p < 0.005, p < 0.001). 09 Frontiers in Pharmacology frontiersin.org Mesmar et al. 10.3389/fphar.2022.994025 FIGURE 4 O. syriacum induces G0/G1 cell cycle arrest in MDA-MB-231 cells. 3.5 OSEE induces cell-cycle arrest of MDA-MB-231 cells at G0/G1 phase (A) MDA-MB-231 cells were incubated with OSEE (200 μg/ml) or ethanol as a vehicle control for 24 h. Cells were then harvested, ﬁxed, stained with propidium iodide, and analyzed by ﬂow cytometry as described in Materials and Methods. Data represent the mean ± SEM of three independent experiments. Statistical analysis was performed using one-way ANOVA (p < 0.05, *p < 0.005) (B) MDA-MB-231 cells were treated with or without increasing concentrations of OSEE for 24 h. Proteins were then extracted and the levels of phosphor-p38 and p21 were analyzed by Western blotting, with total-p38 and β-actin as loading controls, respectively. Data represent the mean ± SEM of three independent experiments ( denotes a p < 0.05 and ** denotes a p < 0.005). FIGURE 4 O. syriacum induces G0/G1 cell cycle arrest in MDA-MB-231 cells. (A) MDA-MB-231 cells were incubated with OSEE (200 μg/ml) or ethanol as a vehicle control for 24 h. Cells were then harvested, ﬁxed, stained with propidium iodide, and analyzed by ﬂow cytometry as described in Materials and Methods Data represent the mean ± SEM of three independent experiments Statistical analysis was performed using one-way ANOVA (p < FIGURE 4 O. syriacum induces G0/G1 cell cycle arrest in MDA-MB-231 cells. (A) MDA-MB-231 cells were incubated with OSEE (200 μg/ml) or ethanol as a vehicle control for 24 h. Cells were then harvested, ﬁxed, stained with propidium iodide, and analyzed by ﬂow cytometry as described in Materials and Methods. Data represent the mean ± SEM of three independent experiments. Statistical analysis was performed using one-way ANOVA (p < 0.05, *p < 0.005) (B) MDA-MB-231 cells were treated with or without increasing concentrations of OSEE for 24 h. Proteins were then extracted and the levels of phosphor-p38 and p21 were analyzed by Western blotting, with total-p38 and β-actin as loading controls, respectively. Data represent the mean ± SEM of three independent experiments ( denotes a p < 0.05 and ** denotes a p < 0.005). 10 Frontiers in Pharmacology frontiersin.org Mesmar et al. 10.3389/fphar.2022.994025 followed by ﬂow cytometry, at 24 h of treatment with 200 μg /ml OSEE. Figure 4A, shows that OSEE induced an arrest at the G0/ G1 phase of the cell cycle. 3.7 OSEE inhibits the STAT3 signaling pathway To analyze the molecular signaling behind OSEE-induced apoptosis, we assessed the expression levels of p53 and STAT3 (signal transducer and activator of transcription 3 protein), in MDA-MB-231 cells treated with OSEE at 100 μg/ml and 200 μg/ ml for 24 h. STAT3 is a transcription factor with established oncogenic properties. It is activated by phosphorylation, inducing its dimerization and subsequent translocation to the nucleus, where it reportedly has been shown to inhibit endogenous expression of p53 protein. Our results showed a signiﬁcant decrease in the phosphorylation of STAT3 by 0.49 ± 0.05- and 0.44 ± 0.02-fold in cells treated with 100 μg/ml and 200 μg/ml OSEE, compared to vehicle control-treated cells (Figure 6). Moreover, a signiﬁcant increase was observed in the phosphorylation of p53 upon treatment of MDA-MB- 231 cells with 200 μg/ml of OSEE (Figure 6). These data suggest that OSEE inhibits STAT3 signaling, resulting in the activation of p53, and therefore induction of intrinsic apoptosis mediated by caspase-3. 3.6 OSEE induces intrinsic apoptosis in MDA-MB-231 cells To follow up on sub-G0 cell cycle data and to conﬁrm the apoptosis status in OSEE-treated MDA-MB-231 cells, the cells were examined 24 h after treatment with OSEE. Analysis of OSEE-treated cells using an inverted phase-contrast microscope showed an OSEE concentration-dependent decrease in the total number of cells per microscopic ﬁeld, and the appearance of apoptotic cells characterized by cell shrinkage, membrane blebbing, and nuclear abnormalities (Figure 5A). Further analysis of OSEE-treated and DAPI- stained cells showed condensation of nuclear material, chromatin lysis, and the presence of apoptotic bodies, all indicative of possible induction of apoptosis by OSEE treatment (Figure 5B). 3.5 OSEE induces cell-cycle arrest of MDA-MB-231 cells at G0/G1 phase The percentage of cells in G0/G1 phase increased in OSEE-treated cells (44.6 ± 0.8 vs 36.5 ± 2.1 in control cells), accompanied by a concomitant decrease in the percentage of cells in the S phase (14.8 ± 1.0 vs 23.5 ± 1.3 in control cells), suggesting that OSEE triggers a G1 phase arrest and inhibits entry into the S phase (Figure 4A). The cell cycle data also revealed that untreated control cells hardly exhibit any sub-G0 DNA (Figure 4A). Treatment of cells with OSEE caused a signiﬁcant increase in the sub-G0 cell population, indicative of apoptosis (Figure 4A). consequently augmented caspase activation and induced the intrinsic apoptotic cascade (Figure 5C). The B-cell lymphoma 2 antiapoptotic protein, BCL-2, also plays an important role in the intrinsic apoptosis pathway and has been shown to contribute to chemoresistance in many cancers (Yip and Reed, 2008), implying that targeting BCL-2 could have a potential role in the treatment of TNBC. In our study, OSEE-treated cells showed a decrease in BCL-2 protein levels in a concentration-dependent manner, achieving a signiﬁcant difference from the control at 200 μg/ml of OSEE (Figure 5C). These data further conﬁrm that OSEE induces cell death by targeting apoptotic mechanisms. The p38 MAPK (mitogen-activated protein kinase) pathway has been widely associated with anti-proliferative functions by regulating cell cycle progression and inducing apoptosis to maintain cellular homeostasis (Lafarga et al., 2009; Martínez-Limón et al., 2020). Western blotting analysis of the levels of the active phosphorylated form of p38 indicated signiﬁcant activation of p38 following treatment of MDA-MB-231 cells with 100 or 200 μg/ml OSEE (1.96 ± 0.11- and 2.23 ± 0.33-fold increases, respectively) (Figure 4B). Our results also show that the levels of a downstream effector of p38, the cell cycle regulator protein p21 (Lafarga et al., 2009), which inhibits progression of the cell cycle, increased remarkably upon treating the cells with OSEE (Figure 4B), further conﬁrming the cell cycle data. 3.8 OSEE increases the aggregation of MDA-MB-231 cells (B) Cells were incubated with OSEE at the indicated concentrations for 24 h and stained with 4′,6-diamidino-2- (Continued) 12 Frontiers in Pharmacology frontiersin.org Mesmar et al. 10.3389/fphar.2022.994025 FIGURE 5 (Continued) phenylindole (DAPI) to visualize nuclei. Nuclear morphological changes and apoptosis were then assessed using a ﬂuorescence microscope. Arrows indicate (1) condensation of nuclear material, (2) cell swelling and chromatin lysis, and (3) apoptotic bodies. (C) Cells were treated with and without the indicated concentrations of OSEE for 24 h. Protein levels of pro-caspase 3 and BCL-2 were determined by Western blotting. Immunoblotting for β-actin was used as a loading control. Data represent the mean of three ± SEM independent experiments (n = 3). (* denotes a p < 0.05). FIGURE 6 O. syriacum inhibits the STAT3 signaling pathway in MDA-MB-231 cells. MDA-MB-231 cells were treated with and without the indicated concentrations of OSEE for 24 h and protein lysates were examined for the phosphorylation of STAT3 and levels of phospho-p53 by Western blotting. Values represent the mean ± SEM of three independent experiments (n = 3). p < 0.05 and *p < 0.001. FIGURE 6 O. syriacum inhibits the STAT3 signaling pathway in MDA-MB-231 cells. MDA-MB-231 cells were treated with and without the indicated concentrations of OSEE for 24 h and protein lysates were examined for the phosphorylation of STAT3 and levels of phospho-p53 by Western blotting. Values represent the mean ± SEM of three independent experiments (n = 3). p < 0.05 and **p < 0.001. compared to the control cells, with signiﬁcant 43 ± 6.4 and 63.5 ± 3% increases, 1 h after OSEE treatment at 100 μg/ml and 200 μg/ ml, respectively. migration is essential in many physiological processes such as wound repair, tissue formation, and proper immune response, its deregulation contributes to the initial steps of cancer metastasis as cells spread away from the primary tumor site. The effect of OSEE on the migration of MDA-MB-231 cells was examined using assays for wound healing and trans-well migration. Figure 8A shows that OSEE decreased the migration of MDA- MB-231 cells, as demonstrated by a decrease in the ability of those cells to migrate and ﬁll the scratched area. For example, 10 h after the scratch was applied to a conﬂuent monolayer, the migration of MDA-MB-231 cells treated with OSEE at 200 μg/ml was 0.67 ± 0.4-fold that of the control cells (Figure 8A). 3.8 OSEE increases the aggregation of MDA-MB-231 cells This inhibition of migration was further conﬁrmed using the trans- well migration assay: OSEE caused a marked decrease in the cell migration ability of MDA-MB-231 cells since only 7.8 ± 0.2% of cells were able to cross from the upper to the lower chamber (Figure 8B). Cadherins are adhesion receptors that mediate homotypic cell-cell adhesion, and the loss of E-cadherin-mediated cell-cell contact is associated with malignant transformation by inducing EMT, leading to tumor metastasis. Here, MDA-MB-231 cells treated with 100 μg/ml and 200 μg/ml of OSEE showed an increase in E-cadherin protein levels in a concentration- dependent manner, by 1.14 ± 0.03- and 1.45 ± 0.11-fold that of untreated control cells, respectively (Figure 7B). 3.8 OSEE increases the aggregation of MDA-MB-231 cells Epithelial-mesenchymal transition (EMT) is a complex cellular program and a hallmark of the progression of tumor cells towards metastasis. During EMT, epithelial cells acquire a mesenchymal phenotype, characterized by the loss of cell-cell adhesion and an increase in their migratory and invasive properties. MDA-MB-231 cells have undergone a high degree of EMT, and a drug that is designed for the treatment of TNBC is expected to reverse EMT by allowing the cells to regain their epithelial properties such as cell-cell adhesion. To this end, we evaluated the effect of the OSEE extract on the cell-cell adhesion properties of MDA-MB-231 cells in suspension in a cell- aggregation assay. Figure 7A shows that OSEE caused a concentration-dependent increase in cell-cell aggregates Central to the execution of apoptosis is the processing of procaspase-3 to the active form, caspase-3. To gain further insight into the mechanism of apoptosis induced by OSEE, we examined the levels of procaspase-3 protein. The results showed a signiﬁcant decrease in procaspase-3 levels in cells treated with 100 and 200 μg/ml OSEE (0.58 ± 0.18- and 0.52 ± 0.11-fold reductions, respectively), suggesting that OSEE enhanced the proteolytic processing of procaspase-3, and Frontiers in Pharmacology 11 frontiersin.org Mesmar et al. 10.3389/fphar.2022.994025 FIGURE 5 O. syriacum induces apoptosis in MDA-MB-231 cells. (A) MDA-MB-231 cells were treated with and without the indicated concentrations of OSEE for 24 h. Morphological changes were observed by light microscopy. Arrows show (1) cell shrinkage, (2) membrane blebbing, (3) apoptotic bodies, and (4) echinoid spikes. (B) Cells were incubated with OSEE at the indicated concentrations for 24 h and stained with 4′,6-diamidino-2- (Continued) FIGURE 5 O. syriacum induces apoptosis in MDA-MB-231 cells. (A) MDA-MB-231 cells were treated with and without the indicated concentrations of OSEE for 24 h. Morphological changes were observed by light microscopy. Arrows show (1) cell shrinkage, (2) membrane blebbing, (3) apoptotic bodies, and (4) echinoid spikes. (B) Cells were incubated with OSEE at the indicated concentrations for 24 h and stained with 4′,6-diamidino-2- (Continued) FIGURE 5 O. syriacum induces apoptosis in MDA-MB-231 cells. (A) MDA-MB-231 cells were treated with and without the indicated concentrations of OSEE for 24 h. Morphological changes were observed by light microscopy. Arrows show (1) cell shrinkage, (2) membrane blebbing, (3) apoptotic bodies, and (4) echinoid spikes. 3.9 OSEE reduces the migration and the invasive properties of MDA-MB-231 cells Having established that OSEE affects cell-cell interactions, we assessed the effect of the extract on cell migration, a main characteristic of the malignant phenotype. Although cell Cell invasion is an integral component of the early stages of cancer metastasis, highlighting the ability of cancer cells that have Frontiers in Pharmacology 13 frontiersin.org Mesmar et al. 10.3389/fphar.2022.994025 FIGURE 7 O. syriacum increases the cell-cell aggregation of MDA-MB-231 cells. (A) MDA-MB-231 cells were incubated with and without the indicated concentrations of OSEE and subjected to a cell-aggregation assay as described in Materials and Methods. Micrographs of cells were taken after 1 h and the percentage of cell-cell aggregates was measured using the following equation: % aggregation = (1 – Nt/Nc) x 100, where Nt is the number of single cells in the control and Nc is the number of single cells in the treated sample. (B) MDA-MB-231 cells were incubated with and without the indicated concentrations of OSEE for 24 h, and whole-cell protein lysates were analyzed for E-cadherin protein levels by Western blotting. β-actin was used as a loading control. Data represent the mean ± SEM of three independent experiments (n = 3). p < 0.05, *p < 0.001, *p < 0.0001. FIGURE 7 O. syriacum increases the cell-cell aggregation of MDA-MB-231 cells. (A) MDA-MB-231 cells were incubated with and without the indicated concentrations of OSEE and subjected to a cell-aggregation assay as described in Materials and Methods. Micrographs of cells were taken after 1 h and the percentage of cell-cell aggregates was measured using the following equation: % aggregation = (1 – Nt/Nc) x 100, where Nt is the number of single cells in the control and Nc is the number of single cells in the treated sample. (B) MDA-MB-231 cells were incubated with and without the indicated concentrations of OSEE for 24 h, and whole-cell protein lysates were analyzed for E-cadherin protein levels by Western blotting. β-actin was used as a loading control. Data represent the mean ± SEM of three independent experiments (n = 3). p < 0.05, *p < 0.001, *p < 0.0001. reduction seems to be concentration-dependent and suggests that OSEE effectively reduces the invasive potential of MDA- MB-231 cells. spread away from the primary tumor site to invade secondary sites of metastasis. 3.9 OSEE reduces the migration and the invasive properties of MDA-MB-231 cells Values represent the average of three independent experiments and are represented as mean ± SEM (p < 0.05, **p < 0.001). FIGURE 8 O. syriacum inhibits the migration of MDA-MB-231 cells. (A) A conﬂuent culture of MDA-MB-231 cells was wounded by scratching with a pipette tip. The cells were then incubated with and without the indicated concentrations of OSEE. After 10 h, the wound was photographed using an inverted phase-contrast microscope and then measured and analyzed. Values represent the fold change in migration compared to vehicle control cells. (B) MDA-MB-231 cells were incubated overnight with and without the indicated concentrations of OSEE in Boyden chamber trans-well inserts as described in Materials and Methods. Migrating cells at the bottom of the chamber were stained with DAPI, imaged, and then counted and analyzed. Values represent the average of three independent experiments and are represented as mean ± SEM (p < 0.05, **p < 0.001). signiﬁcant as early as 10 min and was 0.12 ± 0.02-fold that of control levels after 1 h of treating MDA-MB-231 cells with OSEE. By impacting the migratory and invasive properties of MDA-MB-231 cells, OSEE can potentially reduce metastasis, the main cause of poor prognosis of TNBC tumors. OSEE applied to the surface of the highly vascularized CAM membrane for 24 h caused a signiﬁcant inhibition of new blood vessel formation (a reduction of 44 ± 5.3%, compared to the control) and a decrease in the number of junctions (a decrease of 56 ± 12.8% compared to the control) (Figure 10A). Nitric oxide is a main mediator of angiogenesis. Hence, the anti-angiogenic potential of OSEE was further investigated by testing its effect on cytokine-induced expression of inducible nitric oxide synthase (iNOS), a main producer of nitric oxide. Our results showed that OSEE treatment at 100 μg/ml and 200 μg/ml caused a signiﬁcant decrease in iNOS levels by 0.78 ± 0.06- and 0.75 ± 0.02-fold, respectively, compared to the control (Figure 10B). This indicates that OSEE interferes with the production of nitric oxide, leading to a reduction of angiogenesis. 3.9 OSEE reduces the migration and the invasive properties of MDA-MB-231 cells To this end, we examined the effect of OSEE on the invasive potential of MDA-MB-231 cells using matrigel- coated trans-well chambers in the presence or absence of OSEE (100 μg/ml and 200 μg/ml). The results showed that the number of cells that invaded the matrigel matrix to reach the bottom chamber was signiﬁcantly reduced by OSEE treatment, by as much as 65 ± 3.1% and 89 ± 1.7%, for 100 μg/ml and 200 μg/ml, respectively, compared with the control (Figure 9A). This Focal adhesion kinase (FAK) has a major role in facilitating and promoting the migration and invasiveness of tumor cells (Luo and Guan, 2010; Tai et al., 2015; Lai et al., 2018; Shen et al., 2018). Here we report that 200 μg /ml OSEE caused a 0.74 ± 0.17-fold decrease in the phosphorylation of FAK within 5 min of treatment (Figure 9B). The decrease was Frontiers in Pharmacology Frontiers in Pharmacology 14 frontiersin.org Mesmar et al. 10.3389/fphar.2022.994025 FIGURE 8 O. syriacum inhibits the migration of MDA-MB-231 cells. (A) A conﬂuent culture of MDA-MB-231 cells was wounded by scratching with a pipette tip. The cells were then incubated with and without the indicated concentrations of OSEE. After 10 h, the wound was photographed using an inverted phase-contrast microscope and then measured and analyzed. Values represent the fold change in migration compared to vehicle control cells. (B) MDA-MB-231 cells were incubated overnight with and without the indicated concentrations of OSEE in Boyden chamber trans-well inserts as described in Materials and Methods. Migrating cells at the bottom of the chamber were stained with DAPI, imaged, and then counted and analyzed. Values represent the average of three independent experiments and are represented as mean ± SEM (p < 0.05, *p < 0.001). FIGURE 8 O. syriacum inhibits the migration of MDA-MB-231 cells. (A) A conﬂuent culture of MDA-MB-231 cells was wounded by scratching with a pipette tip. The cells were then incubated with and without the indicated concentrations of OSEE. After 10 h, the wound was photographed using an inverted phase-contrast microscope and then measured and analyzed. Values represent the fold change in migration compared to vehicle control cells. (B) MDA-MB-231 cells were incubated overnight with and without the indicated concentrations of OSEE in Boyden chamber trans-well inserts as described in Materials and Methods. Migrating cells at the bottom of the chamber were stained with DAPI, imaged, and then counted and analyzed. 3.10 OSEE reduces the levels of iNOS and inhibits angiogenesis in ovo Angiogenesis plays a crucial role in tumor growth and metastasis by providing oxygen and nutrients to proliferating cells through the formation of new blood vessels. To test the effect of OSEE on angiogenesis, the chick-embryo chorioallantoic-membrane (CAM) assay was performed. The ﬁndings demonstrated that 200 μg/ml of Frontiers in Pharmacology 15 frontiersin.org Mesmar et al. 10.3389/fphar.2022.994025 FIGURE 9 O. syriacum reduces the invasive potential of MDA-MB-231 cells. (A) MDA-MB-231 cells were incubated overnight with and without the indicated concentrations of OSEE inBoyden chamber trans-well inserts pre-coated with Matrigel as described in Materials and Methods. Cells that invaded the Matrigel were stained with DAPI, imaged, and then counted and analyzed. Values represent the fold change in migration of the ethanol- treated control. The experiment was repeated three times (n = 3) and data represent the mean ± SEM (p < 0.005, **p < 0.0001). (B) Cells were treated with and without 200 μg/ml OSEE at different time points and the phosphorylation of FAK was assessed at hose points by Western blotting, using total-FAK as loading control. The Western blot is representative of three independent experiments (n = 3). p < 0.005, **p < 0.0001. FIGURE 9 O. syriacum reduces the invasive potential of MDA-MB-231 cells. (A) MDA-MB-231 cells were incubated overnight with and without the indicated concentrations of OSEE inBoyden chamber trans-well inserts pre-coated with Matrigel as described in Materials and Methods. Cells that invaded the Matrigel were stained with DAPI, imaged, and then counted and analyzed. Values represent the fold change in migration of the ethanol- treated control. The experiment was repeated three times (n = 3) and data represent the mean ± SEM (p < 0.005, **p < 0.0001). (B) Cells were treated with and without 200 μg/ml OSEE at different time points and the phosphorylation of FAK was assessed at hose points by Western blotting, using total-FAK as loading control. The Western blot is representative of three independent experiments (n = 3). p < 0.005, ***p < 0.0001. 4 Discussion Therefore, new cancer therapeutic strategies have focused on compounds with multiple targets or on combination approaches that mix or design hybrid compounds, particularly to treat aggressive hard-to-treat cancers such as TNBC (Kucuksayan and Ozben, 2017). Plants are rich in secondary metabolites with strong antitumor functions, including terpenoids, Breast cancer (BC) cells sustain proliferative signaling, evade growth suppressors, and resist cell death, providing them with a growth advantage over normal cells. In addition, cancer cells are highly migratory, can activate invasion, and induce angiogenesis. Frontiers in Pharmacology 16 frontiersin.org Mesmar et al. 10.3389/fphar.2022.994025 FIGURE 10 O. syriacum inhibits angiogenesis in ovo and reduces iNOS levels in MDA-MB-231 cells. (A) OSEE was applied to the chorioallantoic membrane (CAM) of fertilized chicken eggs as described in Materials and Methods. Upper panel of (A) shows images of CAM acquired 24 h later to score the angiogenic response. Lower panel of (A) shows analysis of the acquired images. Total vessel length and total number of junctions were quantiﬁed in both the OSEE-treated and control CAMs using the AngioTool software and represented as percentage change with respect to the control ( denotes p < 0.05 and *p < 0.01). (B) The protein levels of iNOS were determined by Western blotting in MDA-MB-231 cells treated with or without the indicated concentrations of OSEE for 24 h. The Western blot is representative of three independent experiments. The bar graph represents the quantiﬁcation of three independent Western blots and the data represent the mean ± SEM of three independent experiments (n = 3). p < 0.05. FIGURE 10 O. syriacum inhibits angiogenesis in ovo and reduces iNOS levels in MDA-MB-231 cells. (A) OSEE was applied to the chorioallantoic membrane (CAM) of fertilized chicken eggs as described in Materials and Methods. Upper panel of (A) shows images of CAM acquired 24 h later to score the angiogenic response. Lower panel of (A) shows analysis of the acquired images. Total vessel length and total number of junctions were quantiﬁed in both the OSEE-treated and control CAMs using the AngioTool software and represented as percentage change with respect to the control (* denotes p < 0.05 and *p < 0.01). (B) The protein levels of iNOS were determined by Western blotting in MDA-MB-231 cells treated with or without the indicated concentrations of OSEE for 24 h. The Western blot is representative of three independent experiments. 4 Discussion In this study, LC-MS analysis of OSEE revealed the presence of vicenin-1, vicenin-2, orientin, isoorientin, vitexin, and isovitexin, all of which are ﬂavonoid C-glycosides. Interestingly, vitexin, isovitexin, vicenin-1, and vicenin-2 are glycosides of apigenin (Engelhardt et al., 1993), which has anti-breast cancer activities and is reported to be present in O. syriacum, as discussed. In addition, these compounds were reported to have anti-cancerous activities, for example: vitexin against human leukaemia U937 cells, oesophageal cancer cells EC109; orientin against human cervical adenocarcinoma HeLa cells, oesophageal cancer EC109 cells, prostate cancer PC-3, DU-145 and LNCaP cells; isoorientin against liver hepatocellular carcinoma HepG2 cells; phenolics, and alkaloids. Our study conﬁrmed that O. syriacum contains several classes of these phytochemical compounds, e.g., phenols, ﬂavonoids, quinones, steroids, terpenoids, tannins, cardiac glycosides and essential oils. This is in agreement with other studies showing that O. syriacum ethanolic extract contains terpenoids (Kamel et al., 2001), ﬂavonoids, carotenoids, and phenols such as thymol and carvacrol (El-Moneim et al., 2014; Alonazi et al., 2021). These plant-derived natural compounds have been gaining more attention as therapeutic options because of their ability to evade resistance by the cancer cells while producing minimal side effects. In fact, natural compounds can solely target cancer cells, or can complement the effects of other chemotherapeutic drugs by sensitizing cancer cells and modulating drug-drug interactions (Scaria et al., 2020). Many natural compounds have been developed into staple anticancer drugs such as the antimitotic plactitaxel from Taxus brevifolia and vinblastine from Catharanthus roseus, pro-apoptotic pomiferin from Maclura pomifera and Dereeis Malaccensis, and anti-angiogenic ﬂavopiridiol from Dysoxylum binectariferum Hook. f and combretastatin A-4 phosphate from Combretum caffrum, among others (Greenwell and Rahman, 2015; Hassan, 2019). Plants from the Origanum genus in particular, have been shown to have strong anti-tumorigenic properties. For example, O. vulgare inhibits cell proliferation and induces apoptosis in human colon, stomach, hepatocarcinoma, and BC cell lines (Lombrea et al., 2020); O. compactum attenuates the proliferation of breast, lung and hepatoma cancer cell lines (Bouyahya et al., 2020); and O. majorana inhibits tumor growth and metastasis of numerous cancers including breast, colon, lung, pancreatic, lymphoblastic leukemia, and hepatocarcinoma (Bouyahya et al., 2021). Of particular interest, O. syriacum has been shown to exhibit an anti-proliferative effect on MCF-7 BC cells (Al-Kalaldeh et al., 2010; Husein et al., 2014) as well as human leukemia THP-1 cells (Ayesh et al., 2014). Furthermore, several phytochemicals present in O. 4 Discussion syriacum have been reported to have anti-breast cancer potential. Some examples include naringenin, apigenin, carvacrol, thymoquinone, thymol, and rosmarinic which have been reported to impact the cancerous phenotype of BC cell lines (Kanno et al., 2005; Demain and Vaishnav, 2011; Lee et al., 2019; Messeha et al., 2020; Sampaio et al., 2021). In this study, LC-MS analysis of OSEE revealed the presence of vicenin-1, vicenin-2, orientin, isoorientin, vitexin, and isovitexin, all of which are ﬂavonoid C-glycosides. Interestingly, vitexin, isovitexin, vicenin-1, and vicenin-2 are glycosides of apigenin (Engelhardt et al., 1993), which has anti-breast cancer activities and is reported to be present in O. syriacum, as discussed. In addition, these compounds were reported to have anti-cancerous activities, for example: vitexin against human leukaemia U937 cells, oesophageal cancer cells EC109; orientin against human cervical adenocarcinoma HeLa cells, oesophageal cancer EC109 cells, prostate cancer PC-3, DU-145 and LNCaP cells; isoorientin against liver hepatocellular carcinoma HepG2 cells; vicenin-2 agaisnt colorectal cancer HT29 cells, hepatocellular carcinoma HepG2, CA3, SNU-387, and HCCLM3 cells, prostate cancer cells PC-3, DU-145 and LNCaP (Nagaprashantha et al., 2011; Lee et al., 2012; Yuan et al., 2013; Guo et al., 2014; Yuan et al., 2014; Yang D et al., 2018; Huang et al., 2020). In the case of breast cancer, vitexin (Kim et al., 2018), orientin, and isoorientin (Czemplik et al., 2016) were reported to have anti-cancerous effects against MCF-7 breast cancer cells, which are not TNBC cells. Collectively, there are limited studies on the effect of these compounds in BC in general, and TNBC in particular, demanding investigation in future studies. Here, it is critical to mention that oftentimes, the whole herb or its crude extract may have more potent activity than a single or a combination of its bioactive constituents. This may partly be due to inherent synergistic effects between the various bioactive constituents, which could be lost when one or several bioactives are separately used (Rasoanaivo et al., 2011; Caesar and Cech, 2019; Zhao et al., 2020). The mechanism of this synergy has not been elucidated, yet several mechanisms may be operating in parallel (Caesar and Cech, 2019). For example, synergy could be due to the low bioavailability and poor pharmacokinetics of the individual bioactives, while the combination of bioactives may impart enhanced bioavailability (Caesar and Cech, 2019; Zhao et al., 2020). 4 Discussion The bar graph represents the quantiﬁcation of three independent Western blots and the data represent the mean ± SEM of three independent experiments (n = 3). p < 0.05. 17 Frontiers in Pharmacology frontiersin.org Mesmar et al. 10.3389/fphar.2022.994025 phenolics, and alkaloids. Our study conﬁrmed that O. syriacum contains several classes of these phytochemical compounds, e.g., phenols, ﬂavonoids, quinones, steroids, terpenoids, tannins, cardiac glycosides and essential oils. This is in agreement with other studies showing that O. syriacum ethanolic extract contains terpenoids (Kamel et al., 2001), ﬂavonoids, carotenoids, and phenols such as thymol and carvacrol (El-Moneim et al., 2014; Alonazi et al., 2021). These plant-derived natural compounds have been gaining more attention as therapeutic options because of their ability to evade resistance by the cancer cells while producing minimal side effects. In fact, natural compounds can solely target cancer cells, or can complement the effects of other chemotherapeutic drugs by sensitizing cancer cells and modulating drug-drug interactions (Scaria et al., 2020). Many natural compounds have been developed into staple anticancer drugs such as the antimitotic plactitaxel from Taxus brevifolia and vinblastine from Catharanthus roseus, pro-apoptotic pomiferin from Maclura pomifera and Dereeis Malaccensis, and anti-angiogenic ﬂavopiridiol from Dysoxylum binectariferum Hook. f and combretastatin A-4 phosphate from Combretum caffrum, among others (Greenwell and Rahman, 2015; Hassan, 2019). Plants from the Origanum genus in particular, have been shown to have strong anti-tumorigenic properties. For example, O. vulgare inhibits cell proliferation and induces apoptosis in human colon, stomach, hepatocarcinoma, and BC cell lines (Lombrea et al., 2020); O. compactum attenuates the proliferation of breast, lung and hepatoma cancer cell lines (Bouyahya et al., 2020); and O. majorana inhibits tumor growth and metastasis of numerous cancers including breast, colon, lung, pancreatic, lymphoblastic leukemia, and hepatocarcinoma (Bouyahya et al., 2021). Of particular interest, O. syriacum has been shown to exhibit an anti-proliferative effect on MCF-7 BC cells (Al-Kalaldeh et al., 2010; Husein et al., 2014) as well as human leukemia THP-1 cells (Ayesh et al., 2014). Furthermore, several phytochemicals present in O. syriacum have been reported to have anti-breast cancer potential. Some examples include naringenin, apigenin, carvacrol, thymoquinone, thymol, and rosmarinic which have been reported to impact the cancerous phenotype of BC cell lines (Kanno et al., 2005; Demain and Vaishnav, 2011; Lee et al., 2019; Messeha et al., 2020; Sampaio et al., 2021). 4 Discussion To complicate the situation even more, natural antioxidants themselves have been reported to either elevate or suppress ROS levels in cancer cells, and as a result, natural antioxidants have been reported to have either pro- or anti-cancerous effects depending on their concentration (Wang et al., 2021). Our results conﬁrm this notion since OSEE exhibited high antioxidant potential in vitro by scavenging the DPPH radicals, but elevated ROS generation in MDA-MB-231 cells in culture. This was further manifested as a biphasic anti-proliferative effect mediated by OSEE in the presence of NAC as a ROS scavenger. Indeed, our results showed that only higher concentrations of OSEE were able to mitigate the inhibition of proliferation induced by the ROS, while lower concentrations of OSEE augmented this effect, indicating that the anti-proliferative effects of OSEE can be ROS-dependent in a biphasic manner. Biphasic concentration-dependent effects have been reported for many natural antioxidants (Shaito et al., 2020). Cancer is a complex multifactorial disease mediated by multiple signaling pathways that regulate the expression of a STAT3 inhibits apoptosis by modulating key apoptosis regulators such as the pro-survival survivin (Gritsko et al., 2006), BCL-2 (Real et al., 2002), and p53 (Sp et al., 2021). As the “guardian of the genome,” p53 is critical in cell fate decisions in response to stress signals by inducing an arrest of the cell cycle, promoting DNA repair, and eliciting apoptosis. The activity of p53 is dependent on its quantity, integrity, and post-translational modiﬁcation (Lacroix et al., 2006). For example, p53 phosphorylation at the N-terminal sites has been associated with increased protein stabilization and activity (Lacroix et al., 2006). Particularly, phosphorylation at the Ser15 site has been shown to be induced by almost all kinds of stress, disrupting the interaction of p53 with its major negative regulator MDM2 and increasing its binding to acetyltransferase P300 (Ito et al., 2001). Phosphorylation of mutant p53 at Ser15 restored its conformation to the wild-type form. Prospective therapies co-targeting STAT3 and p53 are sought to overcome cancer drug resistance (Pham et al., 2020). In this regard, several plant-derived compounds have been shown to exert their anticancer properties through inhibition of STAT3- signalling pathways. For example, the naturally occurring phytoalexin resveratrol inhibits the growth, progression and metastasis of BC cells by directly affecting STAT3 and its upstream regulators (Kohandel et al., 2021). 4 Discussion Future studies should test the anti-cancerous activities of single or combined bioactives of OSEE versus the activity of the whole extract. Overall, our results show that OSEE as a crude extract has potent in vitro anti-breast cancer activities and highlight OSEE as a potential source of natural compound(s) with anti-cancerous activities. Notwithstanding, further in vivo studies are needed to validate OSEE efﬁcacy and safety in the treatment of TNBC. We demonstrated that OSEE dose-dependently inhibited MDA-MB-231 proliferation. OSEE reduced the levels of the proliferation marker Ki67, which is highly expressed in TNBC and is associated with its aggressive pathologic features and poor clinical outcomes (Yang C et al., 2018). Reducing the levels of Ki67 further conﬁrmed the potential of OSEE as a source of future TNBC therapeutics. We also showed that OSEE arrested MDA-MB-231 at the G0/G1 phase of the cell cycle. We investigated the potential correlation of the p38 MAPK (mitogen-activated protein kinase) pathway in OSEE-induced inhibition of MDA-MB-231 cell proliferation with cell cycle arrest. p38 signaling has been widely associated with anti-proliferative functions by regulating cell cycle progression and inducing apoptosis to maintain cellular homeostasis (Lafarga et al., 2009; Martínez-Limón et al., 2020). In addition, we showed that the levels of a downstream effector of p38, the cell cycle inhibitor protein p21 (Lafarga et al., 2009), increased remarkably with OSEE treatment. Overall, we showed that OSEE can inhibit proliferation of MDA-MB-231 cells and induce cell cycle arrest of TNBC cells at G0/G1 in a pathway that may involve p38/p21 signaling and downregulation of Ki67. Frontiers in Pharmacology 18 frontiersin.org Mesmar et al. Mesmar et al. 10.3389/fphar.2022.994025 and Inghirami, 2006). Activated STAT3 induces cancer transformation by affecting cellular pathways related to cell growth, apoptosis, and tumorigenesis. Interestingly, several studies have shown that STAT3 is constitutively activated in invasive BCs, but not benign tumors, indicating that STAT3 is mainly involved in tumor progression and metastasis, rather than tumor initiation (Watson and Miller, 1995; Ranger et al., 2009; Resemann et al., 2014). STAT3 has been attributed to have a key role in cell cycle regulation by mediating the progression of cells from G1 to S phase through the upregulation of D-cyclins and cell division cycle 25 A phosphatase (Cdc25A) and associated downregulation of cell cycle regulator proteins p21 and p27 (Leslie et al., 2006). 4 Discussion In agreement with the roles of STAT3, our results showed that OSEE inhibited STAT3, activated p21, and induced G0/G1 cell cycle arrest. Moreover, ROS-mediated activation of p38 is reportedly implicated in cell cycle arrest at G0/ G1 and induction of apoptosis in several cancers (Martínez- Limón et al., 2020). In this instance, p38 has been reported to activate p21, p27, or p57 (Martínez-Limón et al., 2020). Collectively, our ﬁndings suggest that OSEE exerts its anti- proliferative effects against BC by regulating several pathways, including those for STAT3, ROS, p38 MAP kinase, p21, and Ki67, satisfying the multi-target requirement of an effective anticancer therapeutic strategy, as already discussed. Reactive oxygen species are free radicals that occur in the body as by-products of mitochondrial and peroxisomal metabolism or the activity of certain enzymes like NADPH- oxidases (NOXs). ROS, at low concentrations, mediate important physiological processes, but an uncontrolled increase in their levels may precipitate pathological states. Therefore, it is critical to keep their levels under homeostatic control to avoid cellular oxidative stress resulting from an imbalance between the production of ROS and other reactive species and their elimination by cellular antioxidant systems (Posadino et al., 2019). Importantly, chronic slight elevations of ROS levels can cause cellular damage that may lead to carcinogenesis (Moloney and Cotter, 2018; Slika et al., 2022). However, this is not always the case as ROS was reported to have both pro- and anti- cancerous effects (Chio and Tuveson, 2017; Moloney and Cotter, 2018), dependent on ROS concentrations in the cell (Kong et al., 2000; De Sá Junior et al., 2017). Indeed, when ROS levels rise beyond a certain cytotoxic threshold, for example through exogenous application, they can cause the selective death of cancer cells mainly by apoptosis (Kong et al., 2000; De Sá Junior et al., 2017; Raza et al., 2017). Building on these results, targeting ROS signaling and inducing oxidative stress and/or inhibiting antioxidant processes has been envisaged as a mechanism for anticancer therapy (Chio and Tuveson, 2017; Kim et al., 2019). Nevertheless, the role of ROS in cancer therapy is full of intricacies and there are disputes over whether pro- oxidative or anti-oxidative therapies will be the effective course for the management of cancer (De Sá Junior et al., 2017). 4 Discussion Curcumin was also shown to suppress STAT3-signaling pathways and inhibit the growth of several cancers including breast, prostate, and pancreatic cancers (Hutzen et al., 2009; Lin et al., 2009; Liu et al., 2018). Here we reported similar results where OSEE induced apoptosis and downregulated the expression of STAT3. OSEE Cancer is a complex multifactorial disease mediated by multiple signaling pathways that regulate the expression of a wide array of genes implicated in tumor initiation, progression and metastasis, including the STAT3 signaling pathway. This pathway plays an important role in signaling associated with cellular proliferation, migration, invasion, and angiogenesis (Yuan et al., 2015). High levels of activated STAT3 have been observed in several types of cancer, including human BC (Levy Frontiers in Pharmacology 19 frontiersin.org Mesmar et al. 10.3389/fphar.2022.994025 FAK-signaling or whether other routes are also involved remains to be tested in future studies. Overall, by enhancing cell adhesion and attenuating cell migration and invasion, OSEE may potently inhibit TNBC metastasis. also induced the phosphorylation of p53, further supporting its multi-target properties. Phosphorylation of p53 can be associated with OSEE anticancer properties by stabilizing p53 to restore/ increase its transcriptional activities. Moreover, OSEE treatment induced an accumulation of cells in the sub-G0 phase, activation of caspase-3, and downregulation of BCL-2, indicating that OSEE potentially induces p53-dependent intrinsic apoptosis in TNBC. In total, our data indicate that OSEE-induced apoptosis of MDA- MB-231 may be executed through a pathway involving STAT3, p53, p21, caspase-3, and BCL-2. Tumors upregulate vascularization through angiogenesis in order to acquire nutrients and grow, and for later metastasis. Angiogenesis is associated with cancer invasion and metastasis (Brem et al., 1977; Brem et al., 1978). Particularly, TNBC has been associated with high microvascular density and consequently poor prognosis. Therefore, innovative cancer therapies have been directed to anti-angiogenic therapies to block tumor growth and metastasis (Braicu et al., 2016). This involves targeting pro-angiogenic factors such as inducible nitric oxide synthase (iNOS), which regulates the production of nitric oxide in response to external stimuli. Indeed, the expression of iNOS has been shown to be associated with microvascular density and to serve as a marker for the clinical staging of metastasis in gastric carcinoma (Song et al., 2002) and TNBC (Firger, 2015). Inhibition of iNOS was successful in decreasing TNBC aggressiveness, metastasis to the lungs in particular (Firger, 2015; Granados-Principal et al., 2015). 4 Discussion Here, we showed that OSEE exhibits signiﬁcant anti-angiogenic potential by reducing the formation of capillaries (decreased vessel length and junction number) on the chicken-egg CAM. This effect was correlated with a decrease in the levels of iNOS, suggesting that OSEE blocks angiogenesis through iNOS- mediated signaling pathways. Vascular endothelial growth factor (VEGF) is the predominant angiogenic factor of invasive human BC cells (Relf et al., 1997). VEGF expression is elevated in TNBC patients and is associated with poor prognosis (Linderholm et al., 2009). Extracts from O. majorana inhibit angiogenesis by downregulation of VEGF secretion (Al Dhaheri et al., 2013). It is possible that OSEE- induced inhibition of angiogenesis takes place through a similar mechanism, in addition to downregulation of iNOS. The anti- angiogenic potential of O. syriacum further cements its potential use to develop anti-TNBC therapeutics. Epithelial–mesenchymal transition (EMT) is a hallmark of cancer progression to metastasis and involves loss of cell-cell adhesion and cell-extracellular matrix (ECM) linkages, crucial steps of metastasis (Singh and Settleman, 2010), and the association of TNBC with EMT is well documented (Jang et al., 2015; Jalaleddine et al., 2019). In our study, OSEE increased the formation of cell-cell aggregates, indicating enhanced cell-cell adhesion of MDA-MB-231 cells. This was concomitant with an increase in E-cadherin levels, suggesting that OSEE interferes with tumor growth and dissemination by acting on single migratory tumor cells rather than on migration of cell-clusters. Furthermore, MDA-MB-231 cells have reduced cellular adhesion and low levels of E-cadherin, having undergone extensive EMT (Jalaleddine et al., 2019). Increased levels of E-cadherin and cellular adhesion by OSEE treatment of MDA-MB-231 cells may indicate that OSEE has reversed EMT in these cells. Given that drug resistance is correlated with the extent of EMT (Du and Shim, 2016), it may be speculated that OSEE can reverse chemoresistance. Patients with metastatic TNBC have a poor prognosis (Kassam et al., 2009). This is related to the robust invasive and migratory abilities of TNBC cells (Chang et al., 2013). The enhancement of cell migration and invasion is an essential part of the multistep process of cancer metastasis. It involves the dysregulation of cell-cell junctions and cell adhesion, and the degradation of ECM by proteases such as metalloproteinases (MMPs) (Mcgowan and Duffy, 2008; Dufour et al., 2011). In our study, wound-healing assays and trans-well migration and invasion assays were conducted to conﬁrm the anti-migratory and anti-invasive properties of OSEE. 4 Discussion Indeed, OSEE inhibited TNBC cell migration and invasion. Similarly, it has been demonstrated that O. majorana, a close relative of O. syriacum, also inhibited migration and invasion of TNBC cells through a mechanism involving the inhibition of MMP- 2 and MMP-9 (Al Dhaheri et al., 2013). The expression of MMPs has been shown to be regulated by STAT3-mediated signaling processes (Alsamri et al., 2019), inviting a future investigation of the ability of OSEE to inhibit the invasive potential of MDA-MB231 through its action on STAT3/MMPs. 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Therefore, due to its ability to modulate multiple pathways, OSEE is a potential source of candidate therapeutic anti-cancer agents. This warrants future investigation of OSEE as a source of novel compounds that can be used for multi-targeting of TNBC. Frontiers in Pharmacology 20 frontiersin.org Mesmar et al. 10.3389/fphar.2022.994025 Funding 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. This work was funded by the University Research Board of the American University of Beirut, Lebanon by a grant to EB and the University of Petra, Jordan by a grant to AB. Publication fees APC were covered by Barzan Holdings, Doha, Qatar by a grant to AS. Acknowledgments The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors. The authors would like to thank the URB of the American University of Beirut, University of Petra, and Barzan Holdings for their funding. The authors also thank Sandra Hillman for editing the manuscript and Mohammad Al Zein for taxonomic identiﬁcation of Origanum Syriacum L. Author contributions Conceptualization: EB, AS, RA, and JM designed the project; formal analysis: EB, AS, SB, NA, AB, RA, KH, MM, and JM analyzed and interpretated the data; resources: EB, AS, and AB; data curation: EB, AS, SB,NA, AB, RA, MM, KH, MM, and JM; writing: original draft preparation: EB, JM, and AS; writing:-review and editing: EB, AS, SB, NA, AB, RA, KH, MM, and JM; supervision: EB and AS; funding acquisition, EB, AS, and AB. All authors read and approved the ﬁnal manuscript. 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https://openalex.org/W3199407402	https://www.spiedigitallibrary.org/journals/journal-of-biomedical-optics/volume-27/issue-2/025002/Saliva-based-detection-of-COVID-19-infection-in-a-real/10.1117/1.JBO.27.2.025002.pdf	English	null	Saliva-based detection of COVID-19 infection in a real-world setting using reagent-free Raman spectroscopy and machine learning	medRxiv (Cold Spring Harbor Laboratory)	2,021	cc-by	15,797	Address all correspondence to Frédéric Leblond, frederic.leblond@polymtl.ca Katherine Ember ,a,b,† François Daoust ,a,b,‡ Myriam Mahfoud,a,b,‡ Frédérick Dallaire,a,b Esmat Zamani Ahmad ,a,b,c Trang Tran,a,b Arthur Plante,a,b Mame-Kany Diop,b,c Tien Nguyen ,a,b,c Amélie St-Georges-Robillard,a,b Nassim Ksantini,a,b Julie Lanthier,a,b Antoine Filiatrault,a,b Guillaume Sheehy ,a,b Gabriel Beaudoin,a,b Caroline Quach ,d,e Dominique Trudel,b,c,f,g and Frédéric Leblond a,b,c, aPolytechnique Montréal, Montreal, Canada bCenter de recherche du Center hospitalier de l’Université de Montréal, Montreal, Canada cInstitut du cancer de Montréal, Montreal, Canada dResearch Center, CHU Sainte-Justine, Montreal, Canada eUniversity of Montreal, Faculty of Medicine, Montreal, Quebec, Canada fUniversité de Montréal, Department of Pathology and Cellular Biology, Montreal, Quebec, Canada gCenter Hospitalier de l’Université de Montréal, Department of Pathology, Montreal, Quebec, Canada Katherine Ember ,a,b,† François Daoust ,a,b,‡ Myriam Mahfoud,a,b,‡ Frédérick Dallaire,a,b Esmat Zamani Ahmad ,a,b,c Trang Tran,a,b Arthur Plante,a,b Mame-Kany Diop,b,c Tien Nguyen ,a,b,c Amélie St-Georges-Robillard,a,b Nassim Ksantini,a,b Julie Lanthier,a,b Antoine Filiatrault,a,b Guillaume Sheehy ,a,b Gabriel Beaudoin,a,b Caroline Quach ,d,e Dominique Trudel,b,c,f,g and Frédéric Leblond a,b,c,* aPolytechnique Montréal, Montreal, Canada bCenter de recherche du Center hospitalier de l’Université de Montréal, Montreal, Canada cInstitut du cancer de Montréal, Montreal, Canada dResearch Center, CHU Sainte-Justine, Montreal, Canada eUniversity of Montreal, Faculty of Medicine, Montreal, Quebec, Canada fUniversité de Montréal, Department of Pathology and Cellular Biology, Montreal, Quebec, Canada gCenter Hospitalier de l’Université de Montréal, Department of Pathology, Montreal, Quebec, Canada Katherine Ember ,a,b,† François Daoust ,a,b,‡ Myriam Mahfoud,a,b,‡ Frédérick Dallaire,a,b Esmat Zamani Ahmad ,a,b,c Trang Tran,a,b Arthur Plante,a,b Mame-Kany Diop,b,c Tien Nguyen ,a,b,c Amélie St-Georges-Robillard,a,b Nassim Ksantini,a,b Julie Lanthier,a,b Antoine Filiatrault,a,b Guillaume Sheehy ,a,b Gabriel Beaudoin,a,b Caroline Quach ,d,e Dominique Trudel,b,c,f,g and Frédéric Leblond a,b,c,* aPolytechnique Montréal, Montreal, Canada bCenter de recherche du Center hospitalier de l’Université de Montréal, Montreal, Canada cInstitut du cancer de Montréal, Montreal, Canada dResearch Center, CHU Sainte-Justine, Montreal, Canada eUniversity of Montreal, Faculty of Medicine, Montreal, Quebec, Canada fUniversité de Montréal, Department of Pathology and Cellular Biology, Montreal, Quebec, Canada gCenter Hospitalier de l’Université de Montréal, Department of Pathology, Montreal, Quebec, Canada Katherine Ember ,a,b,† François Daoust ,a,b,‡ Myriam Mahfoud,a,b,‡ Frédérick Dallaire,a,b Esmat Zamani Ahmad ,a,b,c Trang Tran,a,b Arthur Plante,a,b Mame-Kany Diop,b,c Tien Nguyen ,a,b,c Amélie St-Georges-Robillard,a,b Nassim Ksantini,a,b Julie Lanthier,a,b Antoine Filiatrault,a,b Guillaume Sheehy ,a,b Gabriel Beaudoin,a,b Caroline Quach ,d,e Dominique Trudel,b,c,f,g and Frédéric Leblond a,b,c,* aPolytechnique Montréal, Montreal, Canada bCenter de recherche du Center hospitalier de l’Université de Montréal, Montreal, Canada cInstitut du cancer de Montréal, Montreal, Canada dResearch Center, CHU Sainte-Justine, Montreal, Canada eUniversity of Montreal, Faculty of Medicine, Montreal, Quebec, Canada fUniversité de Montréal, Department of Pathology and Cellular Biology, Montreal, Quebec, Canada gCenter Hospitalier de l’Université de Montréal, Department of Pathology, Montreal, Quebec, Canada ‡These authors contributed equally to the work †First author Journal of Biomedical Optics 025002-1 February 2022 • Vol. 27(2) 1.3 Saliva as a Biosample There are two primary ways of collecting SARS-CoV-2 RNA from a patient: nasopharyngeal (NPG) swabs and saliva sampling.6 NPG swabs are invasive and uncomfortable. There is also an increased risk for healthcare workers to be exposed to viruses due to patients sneezing or coughing.14 Meanwhile, saliva can be self-collected using non-invasive means, making it a much more attractive option for frequent screening. Saliva has been used successfully as a diagnostic tool for other coronavirus infections as the oral and nasal cavities can act as points of entry for respiratory viruses.15 SARS-CoV-2 may also enter the saliva from debris of the NPG epithelium, which drains into the oral cavity or from infection of the salivary glands.16 Consequently, using saliva samples paired with a reagent-free alternative approach could be an option for COVID-19 detection. Abstract Significance: The primary method of COVID-19 detection is reverse transcription polymerase chain reaction (RT-PCR) testing. PCR test sensitivity may decrease as more variants of concern arise and reagents may become less specific to the virus. Aim: We aimed to develop a reagent-free way to detect COVID-19 in a real-world setting with minimal constraints on sample acquisition. The machine learning (ML) models involved could be frequently updated to include spectral information about variants without needing to develop new reagents. Approach: We present a workflow for collecting, preparing, and imaging dried saliva super- natant droplets using a non-invasive, label-free technique—Raman spectroscopy—to detect changes in the molecular profile of saliva associated with COVID-19 infection. Results: We used an innovative multiple instance learning-based ML approach and droplet segmentation to analyze droplets. Amongst all confounding factors, we discriminated between COVID-positive and COVID-negative individuals yielding receiver operating coefficient curves with an area under curve (AUC) of 0.8 in both males (79% sensitivity and 75% specificity) and females (84% sensitivity and 64% specificity). Taking the sex of the saliva donor into account increased the AUC by 5%. Conclusion: These findings may pave the way for new rapid Raman spectroscopic screening tools for COVID-19 and other infectious diseases. © The Authors. Published by SPIE under a Creative Commons Attribution 4.0 International License. Distribution or reproduction of this work in whole or in part requires full attribution of the original pub- lication, including its DOI. [DOI: 10.1117/1.JBO.27.2.025002] © The Authors. Published by SPIE under a Creative Commons Attribution 4.0 International License. Distribution or reproduction of this work in whole or in part requires full attribution of the original pub- lication, including its DOI. [DOI: 10.1117/1.JBO.27.2.025002] Keywords: coronavirus disease-19; Raman spectroscopy; biofluids; saliva; screening. Paper 210270RR received Aug. 25, 2021; accepted for publication Jan. 20, 2022; published online Feb. 9, 2022. 025002-1 February 2022 • Vol. 27(2) Ember et al.: Saliva-based detection of COVID-19 infection in a real-world setting. . . 1.2 RT-PCR as a Gold Standard The current gold standards for SARS-CoV-2 testing are nucleic acid amplification tests (NAATs) using saliva or oro-nasopharyngeal swabs in which viral ribonucleic acid (RNA) is amplified and detected using tools, such as reverse transcription polymerase chain reaction (RT-PCR).6 This uses nucleic acid primers, enzymes, and cycles of heat to amplify a specific genomic sequence (from the SARS-CoV-2 genome in this case), enabling it to be detected more easily.7 The sen- sitivity and specificity for saliva-based NAATs are ∼83% and 99%, respectively, and the sensi- tivity and specificity for nasopharyngeal swab-based NAATs are ∼84% and 99%, respectively.6 Despite the fact that RT-PCR has successfully been used in testing for respiratory diseases, this method can show lower sensitivity for SARS-CoV-2 detection before presentation of symptoms.8 Identifying asymptomatic patients early on can help prevent and control the spread of COVID-19, so RT-PCR may fall short as a tool for mass serial screening of asymptomatic populations.7,9 Additionally, this method of testing typically necessitates time-consuming transport of samples to clinical laboratories where complex tailored reagents are used.10 These reagents may be in short supply and lose specificity as the virus mutates, which has already been shown to be the case with influenza.11 To date, the Center for Disease Control has identified seven variants of concern or variants of interest12 and more will undoubtedly arise. We need to investigate reagent-free, on-site, rapid screening approaches to reliably detect cases to control outbreaks and limit community spread of the disease.13 1.1 Current State of the COVID-19 Pandemic COVID-19 has precipitated the deaths of 3.9 million people worldwide as of the end of June 2021 and is the world’s most costly health crisis to date. The cost of the pandemic to the United States economy is projected to amount to $16 trillion.1 In a 19-month period, the severe acute respiratory syndrome (SARS)-coronavirus 2 (CoV-2) virus has infected over 182 million people and numbers of confirmed cases continue to grow.2 Key difficulties in controlling the spread of SARS-CoV-2 are the long pre-symptomatic period (median period of 5 days3), the wide range of non-specific symptoms (such as, coughing, sneezing, fever, and headaches),4 and the fact that asymptomatic individuals may be contagious.5 As governments worldwide have issued lock- downs and travel bans, rapid viral screening has become a crucial method to limit the spread of SARS-CoV-2. Journal of Biomedical Optics Journal of Biomedical Optics 025002-2 1.4 Raman Spectroscopy Raman spectroscopy is a method of assessing the molecular composition of samples in a reagent- free manner. It is a light-based technique, which measures the inelastic light scattered by matter, also called Raman scattering.17 This phenomenon was predicted in 1928 by Smekal18 and observed experimentally in 1928 by Raman and Krishnan,19 but the potential biomedical appli- cation of Raman spectroscopy did not emerge until 1970.20 Raman scattering occurs when there Journal of Biomedical Optics 025002-2 February 2022 • Vol. 27(2) 025002-2 February 2022 • Vol. 27(2) 025002-2 February 2022 • Vol. 27(2) Ember et al.: Saliva-based detection of COVID-19 infection in a real-world setting. . . is an exchange of energy between a sample and a monochromatic laser source emitting either visible or near-infrared light. The exchange of energy results in photons of scattered light with wavelengths shifted compared to the excitation source in a way that depends on molecular struc- ture and bonding. The difference in wavelength of the Raman scattered light compared to the incident light is called the Raman shift, and two different effects can be observed: the Stokes shift (red shift) or anti-Stokes shift (blue shift).17,21 The Raman shift is usually measured in wave- numbers (cm−1), which are units that are inversely proportional to wavelength. Since the ground state is more populated at thermal equilibrium, the Stokes shift is more prevalent and commonly used in Raman spectroscopy.21,22 p py In a Raman spectrometer, a detector is used to measure light that has been inelastically scat- tered from a sample after it has passed through a series of filters, and these measurements are converted into a Raman spectrum. This is a plot of the intensity of the scattered light against the Raman shift in wavenumbers.23 Intensity of a Raman peak at a particular Raman shift increases as the concentration of molecules responsible for the peak increases. Therefore, a Raman spec- trum can be thought of as a molecular fingerprint giving information about the molecules present in the sample through the analysis of the position, height, and width of peaks present in the spectrum. Raman spectroscopy can identify biomolecular features (such as lipids, proteins, nucleic acids, and amino acids) within biological samples and discriminate between organs, tissues, and biofluids based on disease state. 1.5 Metabolic Changes As the concentration of analytes is correlated with the intensity, width, and specificity of Raman peaks, Raman spectroscopy can be used to detect changes in concentration of any Raman-active molecules, e.g., proteins, lipids, and small metabolites.34 While not all metabolic impacts of SARS-CoV-2 are known, some have been identified35 or can be anticipated from diseases with similar mechanisms of action. The SARS virus, SARS-CoV-1, has been shown to induce long- term metabolic changes in formerly infected patients.36 Studies of serum from COVID-positive versus COVID-negative individuals show changes in lipid and tryptophan metabolism,35 and membrane-bound mucin type I and secreted mucin type 5A have been found to be elevated in the airways of COVID patients.37 The transmission vectors of the SARS-CoV-2 virus have been identified through the propagation of aerosols of biofluids, such as saliva carrying a viral load.38,39 ACE2 is a receptor involved in the mechanism of entry of the virus into cells, and there is a high expression of angiotensin-converting enzyme II (ACE2) in epithelial cells of the oral cavity so it may be possible to detect the virus itself.40 Furthermore, it has been hypothesized that COVID-19 may cause bacterial infection and other diseases of the salivary gland, which may in turn bring about changes in molecules within saliva.41 1.4 Raman Spectroscopy In recent years, clinical applications of this light-based technique have gained traction in oncology,24–26 inflammatory diseases,27,28 trans- plantation,29 and virology.30,31 For example, Camacho et al.30 developed surface enhanced Raman sensors for the detection of the Zika virus by functionalizing core-shell nanoparticles with Zika ZIKV NS1 antibodies and reported a sensitivity of 10 ng∕ml. Machine learning (ML) techniques can be used to classify biological samples based on their Raman spectra. Typically, Raman spectra from a single sample are averaged or are individually labeled before supervised ML is applied. However, this does not allow for spectral heterogeneity within a sample (e.g., due to heterogeneous spatial distribution of molecules). Multiple instance learning (MIL) could provide a method for analyzing heterogeneous samples, e.g., dried drop- lets. Instead of labeling each individual spectrum, multiple spectra from a single sample are grouped into a “bag”, which is then labeled.32,33 Journal of Biomedical Optics Journal of Biomedical Optics 025002-3 1.6 Point-of-Care Rapid Screening In a pandemic context, fast near real-time screening is required to track and contain the propa- gation of a virus. Airports, schools, workplaces, and remote communities would benefit from a rapid, reagent-free screening technique. The sensitivity and the label-free approach of Raman spectroscopy makes it a candidate for fast, robust, low-cost, and transportable means of viral screening to complement the diagnostic capacity of the gold standards. Any COVID screening ournal of Biomedical Optics 025002-3 February 2022 • Vol. 27(2) 025002-3 February 2022 • Vol. 27(2) Ember et al.: Saliva-based detection of COVID-19 infection in a real-world setting. . . tool should be applicable to all users, regardless of symptoms, age, sex, time of sample collec- tion, or diet. This poses a challenge when developing a label-free Raman spectroscopic approach as saliva composition is affected by time of day, sex, age, and potentially other underlying health conditions.42–48 A 2021 study (n ¼ 30 for COVID positive volunteers) found that Raman micro-spectroscopy could detect COVID-19 infection in saliva with 84% sensitivity and 92% specificity.49 However, the clinical cohort was restricted to elderly volunteers who had presented to hospitals, and the study design required saliva collection prior to breakfast. About 36% of positive cases were severely or critically ill, and a further 30% had symptoms and evidence of pneumonia upon imaging. Another recent study (n ¼ 29 for COVID positive volunteers) determined infrared spectroscopy could be used to discriminate between COVID-infected and non-infected saliva with 93% sensitivity and 82% specificity.50 As for the previous study, both the COVID-positive and COVID-negative cohorts consisted of hospitalized, symptomatic patients requiring treatment. Most label-free tests would be aimed at screening non-hospitalized people who may be symptomatic or asymptomatic with different levels of severity of COVID-19 infection. Sample collection information and other clinical characteristics apart from viral load were not reported for this dataset, including age, sex, and comorbidities. To be applicable in a setting outside of hospitals, a label-free screening tech- nique must be applicable to people of all ages, with or without symptoms, regardless of diets, smoking status, with samples collected at any time of day. Other label-free analytical techniques include mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. 1.6 Point-of-Care Rapid Screening An MS-based technique for COVID-19 has been developed, however, this requires a specific protein to be used to capture the virus and samples must be transported to a testing lab.51 NMR spectrometers are typically large, costly, and require trained personnel. Although benchtop spectrometers are available,52 there have yet to be any studies applying these to COVID-19. Here, we present the results of a study demonstrating that Raman spectroscopy combined with ML is a candidate for real-world COVID-19 screening in the general population. The study design was developed to ensure that the number of samples collected allowed us to match COVID-positive and COVID-negative samples in terms of potential confounding factors includ- ing sex at birth, age, COVID symptoms, body mass index (BMI), and prescription drugs taken. We studied saliva samples taken from volunteers at a COVID-19 testing clinic, including asymptomatic volunteers and those with respiratory and non-respiratory symptoms. Samples were taken from people aged >10 years old to <61 years old. We found that we could detect COVID-19 infected saliva with 79% sensitivity and 75% specificity in males, and 84% sensi- tivity and 64% specificity in females. This ML-based technique could be adjusted to increase sensitivity and reduce specificity (or vice versa) where required. It is also the first published application of multiple instance learning either via embedded instance selection (MILES) or discriminative mapping (MILDM) to Raman spectral data, allowing us to account for different molecular content of Raman spectra acquired from the same sample. Journal of Biomedical Optics urnal of Biomedical Optics 025002-4 February 2022 • Vol. 27(2) 2.1 Sample Collection The experimental workflow is shown in Fig. 1. A total of 37 COVID-19 positive and 513 COVID-19 negative samples were collected from the Pointe-Saint-Charles COVID-19 testing clinic in accordance with ethical guidelines from the Centre Hospitalier de l’Université de Montréal (CHUM) Research Ethics Board (project number: 20.133). Volunteers presenting between 10 am and 2 pm were asked to complete a questionnaire reporting their symptoms and associated biological and environmental factors that could affect saliva composition, e.g., age, sex at birth, and comorbidities. When applicable, volunteers were asked to remove any lipstick or lip balm using makeup removal wipes (About Face Cleansing Wipes, Micronova Manufacturing Inc., Torrance, California). Each volunteer was instructed to first rinse their mouth three times with bottled water to remove food debris. Water has very weak Raman signal. Volunteers then waited 5 to 10 min for saliva to accumulate before spitting in a 50-ml Falcon tube to collect a minimum of 1.5 ml of the biofluid. Tubes were then immediately stored in a refrigerator February 2022 • Vol. 27(2) 025002-4 Ember et al.: Saliva-based detection of COVID-19 infection in a real-world setting. . . Fig. 1 Workflow from saliva collection to determination of COVID infection status from Raman spectra. Volunteers donated between 1 and 5 ml of saliva into a 50-ml tube. The liquid was pipetted into a 1.5-ml microcentrifuge tube which was centrifuged. The saliva supernatant and pellet were then stored separately at −80°C but supernatant only is shown here for clarity. Supernatant was thawed, vortexed and mounted on an aluminum slide. After 45 min of drying, spectra were acquired using a Renishaw InVia Raman spectrometer. This figure was created with BioRender.com. Fig. 1 Workflow from saliva collection to determination of COVID infection status from Raman spectra. Volunteers donated between 1 and 5 ml of saliva into a 50-ml tube. The liquid was pipetted into a 1.5-ml microcentrifuge tube which was centrifuged. The saliva supernatant and pellet were then stored separately at −80°C but supernatant only is shown here for clarity. Supernatant was thawed, vortexed and mounted on an aluminum slide. After 45 min of drying, spectra were acquired using a Renishaw InVia Raman spectrometer. This figure was created with BioRender.com. at 4°C. Samples were transported to the Centre de Recherche du CHUM (CRCHUM) on ice. Samples were classified as being from asymptomatic, non-respiratory symptomatic, or symp- tomatic volunteers based on symptoms reported in the questionnaire. 2.1 Sample Collection “Respiratory” symptoms included those that might result in significant amounts of mucus in saliva, such as runny nose, difficulty breathing, sore throat, coughing, or wheezing. “Non-respiratory” symptoms included all other symptoms including headache, muscle aches, and tiredness. The classification of each sample as being obtained from a COVID-19 positive or negative volunteer was based on PCR tests based on either NPG swabs or saliva tests, depending on test availability. Meta-analysis by Butler-Laporte et al.6 demonstrates sensitivity of saliva-based tests to be ∼83%, and sensitivity of NPG swabs to be ∼84%. Specificity of both is ∼99%. Journal of Biomedical Optics 2.5 Raman Microspectroscopy of Individual Model Saliva Components Solid compounds placed on aluminum slides were imaged using the Renishaw InVia micro- scope. About 25 spectra were taken from each compound using the 50× lens between 105 and 1725 cm−1, and 2601 and 3359 cm−1. 2.3 Model Saliva Preparation To ensure we could relate morphology of dried droplets of saliva to major contributors to the Raman signal and to assign major peaks, we took spectra from dried model saliva. This was made using concentrations listed in Table S2 in the Supplementary Material (adapted from Sarkar et al.53). As for human saliva supernatant, it was frozen at −80°C and thawed at room temperature for 30 min, vortexed for 40 s and 10 μl of sample were pipetted onto an aluminum slide and dried. Bovine submaxillary mucin (Sigma Aldrich, St. Louis, Missouri, product A2153) was used to generate the model saliva due to limitations in availability of human mucin. To address this limitation, Raman spectra from a limited available quantity of human mucin type I (Sino Biologic, Beijing, China, product 12123-HCCH) were also acquired for more accurate human mucin peak assignment in human saliva supernatant samples. As mentioned in Sec. 1.5, this type of mucin has been found to be elevated in the airways of COVID patients. For other biomolecules not present in our recipe such as lipids and nucleic acids, we assigned peaks from literature values in Table S3 in the Supplementary Material. 2.4 Raman Microspectroscopy of Dried Saliva Samples Dried saliva supernatant samples and model saliva were imaged using an inVia™confocal Raman microscope (Renishaw, Gloucester) in reflection mode. Brightfield montage images of each droplet were obtained with a 5× lens (N PLAN, numerical aperture ¼ 0.12, air immer- sion). Brightfield montage images of the central crystalline region and edge region were obtained using a 50× long working distance objective (N PLAN, numerical aperture ¼ 0.50, air immer- sion). For Raman spectral acquisitions, the excitation consisted of a 785-nm 40-mW laser in line- focus mode (3 μm × 8 μm spot size) with a 1200 l∕mm grating. A dual set of matched dielectric edge filters (785-nm Rayleigh edge filters) were used to remove the laser light. Spectra were acquired in the fingerprint region (between 602 and 1726 cm−1) (Fig. 4) from three separate morphological regions within each droplet: “edge” (the perimeter of the droplet), “on crystal” (inside visible crystals in the center of the droplet), and “off crystal” (outside visible crystals in the center of the droplet). The fingerprint region is composed of peaks due to a broad range of molecular vibrations (e.g., C=C, C–N, and P=O), any of which could be perturbed in a disease state. Ten spectra were acquired in each region and 8 to 10 repeat measurements were taken from each point. Spectra were taken from random on crystal and off crystal regions in the densest region of the droplet, and in a zig–zag pattern within the edge to capture spectra from the very edge and slightly closer to the center. Each measurement was made ensuring 60% to 70% of the Raman microscope sensor dynamical range was used to minimize the impact of shot noise, resulting in an acquisition time of 2 to 10 s for edge and 15 to 40 s for center spectra (on crystal and off crystal), depending on the level of sample autofluorescence. Wire 4.4 by Renishaw was used to visualise the data and compile the brightfield image of the sample. 2.2 Pre-Imaging Sample Processing Protocol Saliva samples were processed and imaged at biosafety containment level 2 (BSL2) in biosafety cabinets. About 1 ml of whole saliva samples were transferred to 1.5-ml microcentrifuge tubes and centrifuged at 4000 rpm for 30 min at 4°C. The supernatant was pipetted into one cryotube, mixed, and then 40 to 500 μl were aliquoted into five separate 1.8-ml cryotubes. The part of the supernatant near the pellet was discarded, and the pellet was retained in the microcentrifuge tube. Pellet and supernatant samples were stored at −80°C. Prior to Raman spectroscopy interrogation, saliva supernatant samples were thawed at room temperature for 30 min, vortexed for 40 s, and 10 μl were pipetted onto an aluminum slide. Droplets were allowed to dry at room temperature for at least 45 min. Journal of Biomedical Optics 025002-5 February 2022 • Vol. 27(2) February 2022 • Vol. 27(2) Journal of Biomedical Optics Journal of Biomedical Optics Journal of Biomedical Optics 2.7 Data Preparation and Feature Selection For data quality reasons, a limited number of spectra (from three patients for on crystal, from five patients for edge) were removed before training the ML models. Exclusion was based on abnor- mally high levels of residual stochastic noise (after background removal) or the presence of unusual spectral shapes unrelated to biomolecular content, e.g., incomplete lipstick removal (Fig. S1 in the Supplementary Material). For each spectrum, a Gaussian fitting procedure was applied to each peak of biological origin fitted to extract its position, its height, and its width; a total of 24 peaks were extracted using this procedure. These, along with the relative intensity of 700 individual bands in a spectrum, represent the data from which ML models could be trained (Fig. 2). Standardization of the data, where each feature is individually normalized to exhibit a mean of 0 and unit variance, was performed before the feature selection and classification steps. A feature selection technique was applied to reduce the size of the feature set prior to model ML model training. Feature reduction is necessary because some spectral regions either provide no useful information for the classification or are perhaps too correlated. Algorithms typically run with at least one parameter, known as a hyperparameter, that needs to be initialized. A vari- ance-based algorithm called SelectKBest57 was used to coarsely reduce the number of features below 10% of the total number of features, to speed up the multivariate technique in the next step. Features presenting a large variance across the complete dataset have a higher weight and by sorting them according to their weight, only the k-best features are retained, where k was varied between 5 and 80 for spectral features, and 5 and 72 for peak features. A random forest classifier (RF)58 with 200 estimators is then used to further reduce the feature set; a multivariate Fig. 2 ML schematic workflow. The ML workflow consists of a 5-fold CV embedded in a 5-time repeat loop creating different splitting of the training and validation sets. A 5-time repeat 5-fold CV allowed assessment of variance in AUCs produced by the models, thus reducing the bias induced when splitting the dataset while allowing computation time to remain reasonable. AUCs were sta- ble using this procedure. Feature selection, bagging, mapping, and training steps are repeated for each fold. Raman spectra are represented by spectral peaks and fitted peaks. 2.6 Spectral Data Processing The following data pre-processing steps were applied to every individual measurement: (1) removal of cosmic rays with an in-house algorithm using the first derivative to detect narrow peaks (∼2 cm−1) with very large intensities; (2) smoothing using a Savitzky–Golay filter54 of order 3 with a window size of 11; (3) background subtraction of signals produced by the aluminum slides and autofluorescence using a custom adaptation of the rolling ball algorithm;55 (4) cropping the region below 1100 cm−1 due to the large variances at lower wavenumber shifts (likely due to wide ranges in healthy salivary salt concentrations);56 (5) averaging of the repeat measurements taken at a given spatial point; and (6) standard normal variate (SNV) normalization.26 Journal of Biomedical Optics 025002-6 February 2022 • Vol. 27(2) 025002-6 February 2022 • Vol. 27(2) February 2022 • Vol. 27(2) 025002-6 Ember et al.: Saliva-based detection of COVID-19 infection in a real-world setting. . . 2.8 Classification Model and Statistical Analyses A multiple-instance learning (MIL) approach was favored to be at the core of the classification algorithm. In such a scenario, all the spectra (instances) belonging to a given patient are rep- resented by a bag to which a label is associated (COVID positive or COVID negative) rather than labeling the instances individually (Fig. 2). Labels associated with each patient (bag) correspond to the result from their PCR test. More specifically, the selected method was an MIL via embedded instance selection (MILES) algorithm that computes a similarity measure between instances to map them into a different feature space.32 Instead of being represented by multiple instances from the original feature, made of spectral and peak features, each bag was represented by a single instance that lay in a new feature space containing the similarity measures. Under MILES, the mapping was done using an intermediate instance pool (IIP)—a matrix that contains all the instances. It was also possible to create a discriminative instance pool (DIP), which is a subset of the IIP represented only by the best instances. In the new feature space, each instance representing a bag had the dimensions of the number of instances forming the IIP or DIP matrix. The method using a DIP is called an MIL with MILDM algorithm.33 The similarity function has a hyperparameter, σ2, that varies between 1 and 70. For high σ2 values, the similarity function tends to 1, and 0 for small σ2 values. The size of the DIP is defined by a hyperparameter, mdip, that varies between 5% and 60% of the total number of spectra. Following the mapping, for both MILES and MILDM algorithms, the same feature selection algorithm used for peak and peak features is applied to the new mapped feature space. The number of features in the MILES and MILDM spaces are defined by hyperparameters kMILES and kMILDM that vary between 5% and 60% of the total number of spectra and between 5 and mdip, respectively. An RF classifier with 200 estimators is applied subsequently. Once the bags have been mapped to the new feature space, either with MILES or MILDM, a standard support vector machine (SVM) algorithm is used for the training.59 Because we used a linear SVM, the only hyperpara- meter is the regularization parameter C, which corresponds to the penalty term and was varied between 1 and 50. 2.7 Data Preparation and Feature Selection Each instance is represented by the relevant features which are selected using a combination of a variance-based algorithm, acting as a broad skimmer, and an RF. Each bag is then mapped from a multiple in- stance representation to a single instance representation through an instance similarity measure; the mapping function being different for MILES and MILDM. A linear SVM algorithm is used to train each model and output a classification probability for patients in the validation set. After each CV procedure, a receiver operating ROC curve is computed with a corresponding AUC value to assess the model performance. The final output is the ROC and AUC averaged over the five repetitions, ensuring further stability. Computational time for each classification scenario was 30 min or less. Fig. 2 ML schematic workflow. The ML workflow consists of a 5-fold CV embedded in a 5-time repeat loop creating different splitting of the training and validation sets. A 5-time repeat 5-fold CV allowed assessment of variance in AUCs produced by the models, thus reducing the bias induced when splitting the dataset while allowing computation time to remain reasonable. AUCs were sta- ble using this procedure. Feature selection, bagging, mapping, and training steps are repeated for each fold. Raman spectra are represented by spectral peaks and fitted peaks. Each instance is represented by the relevant features which are selected using a combination of a variance-based algorithm, acting as a broad skimmer, and an RF. Each bag is then mapped from a multiple in- stance representation to a single instance representation through an instance similarity measure; the mapping function being different for MILES and MILDM. A linear SVM algorithm is used to train each model and output a classification probability for patients in the validation set. After each CV procedure, a receiver operating ROC curve is computed with a corresponding AUC value to assess the model performance. The final output is the ROC and AUC averaged over the five repetitions, ensuring further stability. Computational time for each classification scenario was 30 min or less. February 2022 • Vol. 27(2) 025002-7 Ember et al.: Saliva-based detection of COVID-19 infection in a real-world setting. . . approach is more robust to prevent selecting highly correlated features. Again, this was done independently for spectral and peak features. In the end, the final number of features will range between 10 and 152, with an average of 90. Journal of Biomedical Optics 2.8 Classification Model and Statistical Analyses As the values for the hyperparameters of the feature selection and classification algorithms are not known a priori, many combinations need to be considered. A combination is formed by randomly selecting a value for each hyperparameter within their respective range. A 5-fold cross validation (CV) procedure is used to assess the classification performance for each hyperpara- meter combination. Once applied on the validation set, the trained model will output a classi- fication probability, continuous between 0 and 1, for each sample in the validation set. This procedure is repeated until all folds have been used once as a validation set. The model per- formance is assessed with a receiver operating characteristic (ROC) curve from which we can compute the accuracy, sensitivity, and specificity associated with the point with the minimal distance to the upper left corner and the area under curve (AUC). The ROC curve is computed by comparing the classification probabilities from the validation sets to their pathological labels, either 0 (COVID negative) or 1 (COVID positive), by varying the threshold value between 0 and 1. To test the repeatability of the results, all steps were repeated five times, allowing the data to be split differently between the folds. The final performance assessment is an average ROC curve with its own AUC associated. This procedure is repeated until all the desired hyperparameter combinations have been considered and the set of hyperparameters with the highest performance corresponds to the final model. To assess overfitting of classification models, the steps were repeated using random class labels instead of the class labels associated with the data. The models were run 96 times using random class labels and 96 times using the true labels, and histograms of the AUC generated by each model were plotted. Data processing, feature selection, and classification models were carried out using Python and the Scikit-learn library.57 February 2022 • Vol. 27(2) Ember et al.: Saliva-based detection of COVID-19 infection in a real-world setting. . . Ember et al.: Saliva-based detection of COVID-19 infection in a real-world setting. . . 3.1 Saliva Collection and Preparation Protocol, Volunteer Demographics We developed a technique for obtaining Raman spectra from saliva supernatant in a way that minimized person-to-person variation (Fig. 1). Raman spectroscopy is sensitive to all Raman- active molecules within a sample and so minimizing exogenous particles is critical. We had previously found that the Raman spectrum of chromophores was present in volunteers who donated saliva while wearing lipstick (Fig. S1 in the Supplementary Material). Therefore, we provided volunteers with lipstick removal wipes if necessary and implemented a mouth washing step to remove food debris. After a waiting time of 5 min, whole saliva was collected from 550 volunteers at a COVID-19 testing site and processed in a containment level 2 facility (clinical characteristics listed in Table 1). Saliva was centrifuged to remove further food particles and the supernatant stored at −80°C. The supernatant was allowed to thaw at room temperature. It was then vortexed, dried on an aluminum slide, and analyzed using a Raman microspectrom- eter (InVia, Renishaw). Spectra obtained from aqueous phase saliva supernatant were primarily due to fluorescence with no visible Raman peaks, but upon drying we could obtain Raman spec- tra with clearly distinguishable Raman peaks. The methodology is described in the materials and methods section in more detail. Clinical characteristics of the volunteers are reported in Table 1. The viral loads of our samples ranged from a cycle threshold of 15.5 (very high) to 36.3 (very low) (Table S1 in the Supplementary Material). Not all viral loads were made available to us. 3.2 Human Saliva Supernatant Forms Morphological Regions with Distinct Raman Spectra 3.2 Human Saliva Supernatant Forms Morphological Regions with Distinct Raman Spectra Raman microspectrometers are spectrometers coupled to microscopes to yield Raman spectra with a spatial resolution that can be modulated by the use of objectives with different magnifications.60 A single Raman spectrum can be obtained from a discrete point within a sample and the area of excitation in these experiments was 3 μm by 8 μm, using a 50× objective (N PLAN, numerical aperture ¼ 0.50, air immersion). In brightfield images (5× and 50×) taken with the system, we observed that drops of human saliva supernatant most frequently dried with a translucent crystalline region in the center and a slightly more opaque peak around the edge [Figs. 3(a)–3(c)]. This is a common phenomenon observed in dried water-based droplets due to the coffee-ring effect in which suspended particles accumulate at the edge of the droplet due to capillary flow.61 In some saliva droplets, there was also a region between the crystalline center and edge where there were no crystals and Raman signal was minimal, which could be attributed to a lower concentration of Raman-active molecules. To determine which part of the droplet should be interrogated for discrimination between COVID-negative and COVID-positive samples, we took measurements from both the center [Fig. 3(b)] and the edge [Fig. 3(c)] of the dried droplet. The central region was divided into on crystal—where measurements were taken from visible crystals—and off crystal—where mea- surements were taken from the milieu in-between crystals. Spectra were taken at 10 points in each of the edge, on crystal, and off crystal regions with 8 to 10 successive acquisitions taken from each point to increase signal-to-noise ratio by averaging the spectra [Fig. 3(d)]. In the on crystal and off crystal central regions of human saliva, we observed strong peaks at 1003 and 1045 cm−1 as well as 853 and 925 cm−1 in some volunteers. In the edge region, we observed strong peaks at 1003, 1449, and 1665 cm−1 (Table S3 in the Supplementary Material). These different Raman signatures indicate that the molecular content of the center and the edge of dried saliva supernatant is different. As a droplet dries, certain molecules accumulate at the edge and others in the center. 3 Results 3.1 Saliva Collection and Preparation Protocol, Volunteer Demographics 3.2 Human Saliva Supernatant Forms Morphological Regions with Distinct Raman Spectra The strong peak at 1003 cm−1 is common in Raman studies of biological materials as it corresponds to the ring breathing mode of phenylalanine and is often one of the strongest peak in proteins.62 3.3 Raman Peak Assignment by Comparison of Human Saliva to Model Saliva 3.3 Raman Peak Assignment by Comparison of Human Saliva to Model Saliva To confidently assign Raman peaks to donor spectra, we used a saliva model adapted from Sarkar et al.53 containing salts, bovine serum albumin, mucin, and other metabolites. The precise Journal of Biomedical Optics February 2022 • Vol. 27(2) 025002-9 Ember et al.: Saliva-based detection of COVID-19 infection in a real-world setting. . . Table 1 Clinical characteristics of the total volunteer cohort. Characteristics were taken from questionnaires given to volunteers. There were 513 COVID negative volunteers and 37 COVID positive volunteers. The number on the left in each column is the number of individuals with each characteristic, and the number in parentheses on the right is the percentage of the total number of COVID negative or positive volunteers. About 38 COVID-19 negative and 33 COVID-19 positive samples were analyzed due to time constraints and accessibility to biosafety level 2 con- tainment facilities. Data from the remaining volunteers were not used in this paper but samples have been retained for future studies. Table 1 Clinical characteristics of the total volunteer cohort. Characteristics were taken from questionnaires given to volunteers. There were 513 COVID negative volunteers and 37 COVID positive volunteers. The number on the left in each column is the number of individuals with each characteristic, and the number in parentheses on the right is the percentage of the total number of COVID negative or positive volunteers. About 38 COVID-19 negative and 33 COVID-19 positive samples were analyzed due to time constraints and accessibility to biosafety level 2 con- tainment facilities. Data from the remaining volunteers were not used in this paper but samples have been retained for future studies. 3.3 Raman Peak Assignment by Comparison of Human Saliva to Model Saliva Total COVID-19 negative Analyzed COVID-19 negative Analyzed COVID-19 positive Total number of volunteers 513 38 33 Age range, n (%) — — — 0–20 58 (11) 6 (15) 7 (21) 21–40 255 (50) 17 (45) 13 (39) 41–60 135 (26) 12 (32) 11 (33) 61–80 60 (12) 3 (8) 2 (6) 81+ 1 (0) 0 (0) 0 (0) Not given 4 (1) 0 (0) 0 (0) Sex at birth, n (%) — — — Female 225 (44) 18 (47) 18 (55) Male 206 (40) 20 (53) 15 (45) Prefer not to say 82 (16) 0 (0) 0 (0) Symptoms, n (%) — — — Respiratory symptoms 187 (36) 24 (61) 21 (64) Non-respiratory symptoms 30 (6) 1 (3) 3 (9) None 279 (54) 13 (34) 5 (15) Not reported 17 (3) 4 (11) 4 (12) Disease, n (%) — — — Other disease 124 (24) 10 (26) 3 (9) None 385 (76) 18 (74) 30 (91) Nicotine consumption, n (%) — — — Smoking 96 (19) 4 (11) 3 (9) Vaping 32 (6) 2 (5) 0 (0) Alcohol consumption, n (%) 323 (63) 24 (63) 18 (54) BMI 25.4 27.4 24.6 Prescription medication or vitamins taken 294 (57.3) 27 (71) 24 (73) composition of the model is listed in Table S2 in the Supplementary Material. We dried the droplet in the same way as the human saliva supernatant samples and observed similar morphol- ogy in brightfield images [Figs. 4(a)–4(c)] as in human saliva. Although there are some particles of solid compounds, which had not completely dissolved in the model saliva, the branched 025002-10 February 2022 • Vol. 27(2) February 2022 • Vol. 27(2) Ember et al.: Saliva-based detection of COVID-19 infection in a real-world setting. . . Fig. 3 Raman spectroscopy of a representative droplet of human saliva supernatant. (a)–(c) Brightfield images for (a) whole droplet dried on aluminum slide (5×); (b) crystalline region (50×); and (c) edge (50×) with acquisition points shown with different symbols: diamonds (on crystal), crosses (off crystal), and triangles (edge). (d) Average spectra from one saliva sample for on crystal (top), off crystal (middle), and edge (bottom) regions, respectively, with shaded areas representing the interspectral variance within the specimen. Each spectrum is an average of multiple acquisitions obtained with a 785-nm laser using a Renishaw InVia Raman microscope. 3.3 Raman Peak Assignment by Comparison of Human Saliva to Model Saliva This is consistent with findings in serum and other biofluids—proteins move toward the edge of the droplet in the drying process while salts may be spread throughout the droplet.63,64 3.3 Raman Peak Assignment by Comparison of Human Saliva to Model Saliva The scale bar in A is 1 mm in length, whereas the scale bar in B and C are 0.1-mm long. Fig. 3 Raman spectroscopy of a representative droplet of human saliva supernatant. (a)–(c) Brightfield images for (a) whole droplet dried on aluminum slide (5×); (b) crystalline region (50×); and (c) edge (50×) with acquisition points shown with different symbols: diamonds (on crystal), crosses (off crystal), and triangles (edge). (d) Average spectra from one saliva sample for on crystal (top), off crystal (middle), and edge (bottom) regions, respectively, with shaded areas representing the interspectral variance within the specimen. Each spectrum is an average of multiple acquisitions obtained with a 785-nm laser using a Renishaw InVia Raman microscope. The scale bar in A is 1 mm in length, whereas the scale bar in B and C are 0.1-mm long. crystalline region resembles that of human saliva supernatant. The edge region of model saliva also resembles that of human supernatant in terms of size and the fact that it lacks crystals. also resembles that of human supernatant in terms of size and the fact that it lacks crystals. Furthermore, the Raman peaks from the center and the edge of model saliva supernatant [Fig. 4(d)] resembled those of human saliva, suggesting a similar molecular composition. We took spectra of the pure constituents of the model saliva to determine which molecules give rise to peaks in the model saliva and whether these peaks were also present in human saliva supernatant (Figs. S3–S6 in the Supplementary Material). By comparing spectra of human saliva supernatant to model saliva, we could identify Raman peaks due to salts, proteins, and nucleic acids (Table S3 in the Supplementary Material). We found that the spectra from the edge of the drop were primarily composed of peaks due to proteins (phenylalanine at 1003 cm−1, amide III at 1200 to 1340 cm−1, amide I at 1605 to 1665 cm−1). We found that the peak due to nitrate, 1045 cm−1, was strong in spectra taken from the central crystalline region of both model saliva and real human saliva super- natant. In the central region compared to the edge, the peak at 1003 cm−1 was stronger relative to amide bands, suggesting a contribution to this peak by the salt urea. 3.4 Predictive Modeling for Segmented Saliva Samples To fully explore the potential of all regions of the dried saliva supernatant matrix for detection of COVID-19, we developed predictive models based on edge, on crystal, and off crystal regions. Journal of Biomedical Optics 025002-11 February 2022 • Vol. 27(2) 025002-11 February 2022 • Vol. 27(2) February 2022 • Vol. 27(2) 025002-11 Ember et al.: Saliva-based detection of COVID-19 infection in a real-world setting. . . Fig. 4 Raman spectroscopy from a droplet of model saliva composed of a mix of salts, bovine serum albumin, mucin, and other metabolites. (a)–(c) Brightfield images for (a) whole droplet dried on aluminum slide (5×); (b) crystalline region (50×); and (c) edge (50×) with acquisition points shown with different symbols: diamonds (on crystal), crosses (off crystal) and triangles (edge). (d) Average spectra from one saliva sample for on crystal (top), off crystal (middle), and edge (bottom) regions respectively with shaded areas representing the interspectral variance within the specimen. Each spectrum is an average of multiple acquisitions obtained with a 785-nm laser using a Renishaw InVia Raman microscope. The scale bar in A is 1 mm in length, whereas the scale bar in B and C are 0.1-mm long. Fig. 4 Raman spectroscopy from a droplet of model saliva composed of a mix of salts, bovine serum albumin, mucin, and other metabolites. (a)–(c) Brightfield images for (a) whole droplet dried on aluminum slide (5×); (b) crystalline region (50×); and (c) edge (50×) with acquisition points shown with different symbols: diamonds (on crystal), crosses (off crystal) and triangles (edge). (d) Average spectra from one saliva sample for on crystal (top), off crystal (middle), and edge (bottom) regions respectively with shaded areas representing the interspectral variance within the specimen. Each spectrum is an average of multiple acquisitions obtained with a 785-nm laser using a Renishaw InVia Raman microscope. The scale bar in A is 1 mm in length, whereas the scale bar in B and C are 0.1-mm long. The large number of COVID-negative saliva samples allowed us to match each COVID-positive sample with a negative sample having approximately the same characteristics in terms of sex at birth, age, COVID symptoms, BMI, and prescription drugs taken (Table 1). This ensured that the clinical characteristics of the samples analyzed were approximately the same between the 33 positive (15 males, 18 females) and 38 negative (20 males, 18 females) samples. Journal of Biomedical Optics 3.6 Discrimination between PCR-Positive and PCR-Negative Samples Regardless of Sex at Birth Among all confounding factors including sex at birth, we could discriminate between COVID positive and negative saliva supernatant samples using ML on Raman spectra taken from the edge region with an AUC of 0.76 [Figs. 7(a) and 7(b)]. This predictive model was built using MILDM (n ¼ 71, 33 COVID-positive and 38 COVID-negative), yielding a sensitivity of 73% and a specificity of 71%. This reduced the AUC by 0.04 relative to the model built in males alone using the same part of the droplet. Half of the top molecular features are shared between the two models [Figs. 5(c) and 7(c)]. We could discriminate between COVID-19 positive and negative on crystal regions with an AUC of 0.69 using MILES (n ¼ 69, 31 COVID-positive, and 38 COVID- negative) [Figs. 7(d) and 7(e)]. This resulted in a reduction in AUC of 0.11 compared to the model built in females only using the same part of the droplet [Fig. 6(e)]. More than 65% of the top features are shared between the two models [Figs. 5(c) and 7(c)]. We could not build a reliable model using spectra from the off crystal region (AUC ¼ 0.57). This was also true for models built for males and females considered separately (Figs. S13 and S14 in the Supplementary Material). Key features used in the edge model for females and males together [Fig. 7(c)] included peaks corresponding to mucin (1146 and 1248 to 1249 cm−1), carotenoids (1146, 1170, and 1580 cm−1), as well as multiple bands that corresponded to the amide I and III regions in proteins and nucleic acids. In the on crystal region, the top features used to produce the clas- sification model included peaks associated with lipids (1123 to 1124, 1265 to 1267, 1447 to 1449, and 1661 to 1663 cm−1), proteins (all peaks except 1643 cm−1), nucleic acids, and urea or uric acid (1643 cm−1). One of the key peaks for the on crystal region is the peak at 1339 to 1340 cm−1 corresponding to protein and nucleic acids. This is also a peak that is much lower and less distinct in edge regions, suggesting that the regions yield complementary information. Furthermore, only the peaks at 1447 to 1449 cm−1 and 1661 to 1663 cm−1 were shared fea- tures between edge and on crystal predictive models for both sexes together [Figs. 7(c) and 7(f)]. Journal of Biomedical Optics 025002-13 February 2022 • Vol. 3.5 Separating Samples Based on Sex at Birth Increases Accuracy of COVID Detection 3.5 Separating Samples Based on Sex at Birth Increases Accuracy of COVID Detection As sex hormones can affect metabolism and immune responses and thus the molecular content of saliva, we separated samples based on sex at birth. In males, we could discriminate between COVID positive and negative saliva supernatant samples with an ROC curve AUC of 0.80 using ML on Raman spectra taken from the edge region [Figs. 5(a) and 5(b)]. This predictive model was built using MILDM (n ¼ 35, 15 COVID positive and 20 COVID negative samples), yielding a sensitivity of 79% and a specificity of 75%. Key features used in model building included peaks that can be assigned to carbohydrates, carotenoids, proteins, and nucleic acids [Fig. 5(c)]. In males, models built using the on crystal region had a lower AUC than the edge models (0.72 with MILDM, 15 COVID positive and 20 COVID negative samples) [Figs. 5(d)–5(f)]. For females, using on crystal spectra, we could discriminate between COVID positive and negative saliva samples with an AUC of 0.80 [Figs. 6(d) and 6(e)]. This model was built using MILES (n ¼ 36, 18 COVID-positive and 18 COVID-negative), with a sensitivity of 84% and a specificity of 65%. Key features included peaks that can be assigned to lipids, proteins and nucleic acids [Fig. 6(f)]. In females, models built using the edge region had a lower AUC than the on crystal models (0.67 with MILES, n ¼ 36, 16 COVID-positive, 18 COVID-negative) [Figs. 6(a)–6(c)]. This is reflected by the observation that the mean edge spectra of COVID- positive and COVID-negative females have much greater overlap than the mean edge spectra from males, suggesting the molecular composition of the protein-rich edge region of saliva is not altered as severely in females as it is in males. The best model in males (built using the edge) only shared 1203 to 1206 cm−1 and 1603 to 1605 cm−1 with the best model in females only. All other features were different. 3.4 Predictive Modeling for Segmented Saliva Samples Volunteer matching was done to reduce the impact of potential confounding factors. However, there was a slightly higher percentage of analyzed samples from volunteers with comorbidities in the COVID negative group. Comorbidities included optic neuritis, cancer, hypertension, diabetes, anxiety, allergies, and migraines. ML algorithms were trained using spectra from 33 COVID positive and 38 COVID negative samples. The area of laser excitation was small (24 μm2) relative to the area of the droplet (approximately 12 mm2). Due to the crystallization process and dilution of biomolecules within saliva, not all spectra necessarily carry molecular information relevant to the COVID status of the patient. As a result, an ML approach needed to be adapted whereby each spectrum acquired from a saliva droplet from a volunteer could be treated independently (Fig. 2). The ML approaches that were employed are based on MIL; specifically, MIL via embedded instance selection (MILES) and MIL with discriminative bag mapping (MILDM). The implementation details of techniques are presented in the Experimental section. For each classification scenario pre- sented below, results are shown using an ROC curve for both MILES and MILDM. February 2022 • Vol. 27(2) 025002-12 Ember et al.: Saliva-based detection of COVID-19 infection in a real-world setting. . . 3.6 Discrimination between PCR-Positive and PCR-Negative Samples Regardless of Sex at Birth 27(2) February 2022 • Vol. 27(2) Ember et al.: Saliva-based detection of COVID-19 infection in a real-world setting. . . Ember et al.: Saliva-based detection of COVID-19 infection in a real-world setting. . . Fig. 5 ML model discriminating between COVID-negative and positive saliva supernatant from males using (a)–(c) edge and (d)–(f) on crystal Raman spectra from dried droplets. (a) and (d) Upper frame shows SNV-normalized, background corrected Raman spectra from all volunteers Main features used in model building designated by dotted lines Mean spectra Fig. 5 ML model discriminating between COVID-negative and positive saliva supernatant from males using (a)–(c) edge and (d)–(f) on crystal Raman spectra from dried droplets. (a) and (d) Upper frame shows SNV-normalized, background corrected Raman spectra from all volunteers. Main features used in model building designated by dotted lines. Mean spectra from COVID-negative volunteers (n ¼ 20, at least eight spectra per volunteer) are shown in black and spectra from COVID-positive volunteers (n ¼ 15, at least eight spectra per volunteer) are shown in red. Variance is not shown for reasons of clarity. Bottom frame shows the stand- ardized Raman spectra, where each individual feature has 0 mean and unit variance. (b) and (e) ROC for these models with sensitivity and specificity. (c) and (f) List of features used in model building and their assignments as determined using compounds in model saliva and from literature. Fig. 5 ML model discriminating between COVID-negative and positive saliva supernatant from males using (a)–(c) edge and (d)–(f) on crystal Raman spectra from dried droplets. (a) and (d) Upper frame shows SNV-normalized, background corrected Raman spectra from all volunteers. Main features used in model building designated by dotted lines. Mean spectra from COVID-negative volunteers (n ¼ 20, at least eight spectra per volunteer) are shown in black and spectra from COVID-positive volunteers (n ¼ 15, at least eight spectra per volunteer) are shown in red. Variance is not shown for reasons of clarity. Bottom frame shows the stand- ardized Raman spectra, where each individual feature has 0 mean and unit variance. (b) and (e) ROC for these models with sensitivity and specificity. (c) and (f) List of features used in model building and their assignments as determined using compounds in model saliva and from literature. Fig. 5 ML model discriminating between COVID-negative and positive saliva supernatant from males using (a)–(c) edge and (d)–(f) on crystal Raman spectra from dried droplets. (a) and (d) Upper frame shows SNV-normalized, background corrected Raman spectra from all volunteers. Main features used in model building designated by dotted lines. Ember et al.: Saliva-based detection of COVID-19 infection in a real-world setting. . . Mean spectra from COVID-negative volunteers (n ¼ 20, at least eight spectra per volunteer) are shown in black and spectra from COVID-positive volunteers (n ¼ 15, at least eight spectra per volunteer) are shown in red. Variance is not shown for reasons of clarity. Bottom frame shows the stand- ardized Raman spectra, where each individual feature has 0 mean and unit variance. (b) and (e) ROC for these models with sensitivity and specificity. (c) and (f) List of features used in model building and their assignments as determined using compounds in model saliva and from literature. Fig. 5 ML model discriminating between COVID-negative and positive saliva supernatant from males using (a)–(c) edge and (d)–(f) on crystal Raman spectra from dried droplets. (a) and (d) Upper frame shows SNV-normalized, background corrected Raman spectra from all volunteers. Main features used in model building designated by dotted lines. Mean spectra from COVID-negative volunteers (n ¼ 20, at least eight spectra per volunteer) are shown in black and spectra from COVID-positive volunteers (n ¼ 15, at least eight spectra per volunteer) are shown in red. Variance is not shown for reasons of clarity. Bottom frame shows the stand- ardized Raman spectra, where each individual feature has 0 mean and unit variance. (b) and (e) ROC for these models with sensitivity and specificity. (c) and (f) List of features used in model building and their assignments as determined using compounds in model saliva and from literature. 025002-14 025002-14 February 2022 • Vol. 27(2) Journal of Biomedical Optics Ember et al.: Saliva-based detection of COVID-19 infection in a real-world setting. . . Ember et al.: Saliva-based detection of COVID-19 infection in a real-world setting. . . Fig. 6 ML model discriminating between COVID-negative and positive saliva supernatant from f l i ( ) ( ) d d (d) (f) t l R t f d i d d l t ( ) d Fig. 6 ML model discriminating between COVID-negative and positive saliva supernatant from females using (a)–(c) edge and (d)–(f) on crystal Raman spectra from dried droplets. (a) and (d) Upper frame shows SNV-normalized, background corrected Raman spectra from all volun- teers. Main features used in model building designated by dotted lines. Mean spectra from COVID-negative volunteers (n ¼ 18, at least nine spectra per volunteer) are shown in black, and spectra from COVID-positive volunteers (n ¼ 18 for edge, n ¼ 16 for on crystal, at least nine spec- tra per volunteer) are shown in red. Variance is not shown for reasons of clarity. Bottom frame shows the standardized Raman spectra, where each individual feature has 0 mean and unit vari- ance. (b) and (e) ROC for these models with sensitivity and specificity. (c) and (f) List of features used in model building and their assignments as determined using compounds in model saliva and from literature. Fig. 6 ML model discriminating between COVID-negative and positive saliva supernatant from females using (a)–(c) edge and (d)–(f) on crystal Raman spectra from dried droplets. (a) and (d) Upper frame shows SNV-normalized, background corrected Raman spectra from all volun- teers. Main features used in model building designated by dotted lines. Mean spectra from COVID-negative volunteers (n ¼ 18, at least nine spectra per volunteer) are shown in black, and spectra from COVID-positive volunteers (n ¼ 18 for edge, n ¼ 16 for on crystal, at least nine spec- tra per volunteer) are shown in red. Variance is not shown for reasons of clarity. Bottom frame shows the standardized Raman spectra, where each individual feature has 0 mean and unit vari- ance. (b) and (e) ROC for these models with sensitivity and specificity. (c) and (f) List of features used in model building and their assignments as determined using compounds in model saliva and from literature. 025002-15 February 2022 • Vol. 27(2) Journal of Biomedical Optics Journal of Biomedical Optics Fig. 7 ML model discriminating between COVID-negative and positive volunteer saliva superna- Ember et al.: Saliva-based detection of COVID-19 infection in a real-world setting. . . Ember et al.: Saliva-based detection of COVID-19 infection in a real-world setting. . . Ember et al.: Saliva-based detection of COVID-19 infection in a real-world setting. . . Fig. 7 ML model discriminating between COVID-negative and positive volunteer saliva superna- tant using (a)–(c) edge and (d)–(f) on crystal Raman spectra from dried droplets. (a) and (d) Upper frame shows SNV-normalized, background corrected Raman spectra from all volunteers. Main features used in model building designated by dotted lines. Mean spectra from COVID-negative volunteers (n ¼ 38 for both edge and on crystal, at least nine spectra per volunteer) are shown in black and mean spectra from COVID-positive volunteers spectra (n ¼ 33 for edge, 31 for on crys- tal, at least nine spectra per volunteer) are shown in red. Variance is not shown for reasons of clarity. Bottom frame shows the standardized Raman spectra, where each individual feature has 0 mean and unit variance. (b) and (e) ROC for these models with sensitivity and specificity. (c) and (f) List of features used in model building and their assignments as determined using compounds in model saliva and from literature. Fig. 7 ML model discriminating between COVID-negative and positive volunteer saliva superna- tant using (a)–(c) edge and (d)–(f) on crystal Raman spectra from dried droplets. (a) and (d) Upper frame shows SNV-normalized, background corrected Raman spectra from all volunteers. Main features used in model building designated by dotted lines. Mean spectra from COVID-negative volunteers (n ¼ 38 for both edge and on crystal, at least nine spectra per volunteer) are shown in black and mean spectra from COVID-positive volunteers spectra (n ¼ 33 for edge, 31 for on crys- tal, at least nine spectra per volunteer) are shown in red. Variance is not shown for reasons of clarity. Bottom frame shows the standardized Raman spectra, where each individual feature has 0 mean and unit variance. (b) and (e) ROC for these models with sensitivity and specificity. (c) and (f) List of features used in model building and their assignments as determined using compounds in model saliva and from literature. 3.7 Assessing Effects of Potential Confounding Factors Building separate models for females and males resulted in higher predictive accuracy than mod- els including both sexes, so we investigated whether we could discriminate between samples based on sex at birth in COVID-negative samples only. The predictive model produced an ROC with an AUC of 0.70 on edge (n ¼ 20 males, n ¼ 18 females, MILDM) yielding 69% sensitivity, 67% specificity (Figs. S15a–S15c in the Supplementary Material) and 0.80 on crystal yielding 72% sensitivity, 76% specificity (MILES) (Figs. S15d–S15f in the Supplementary Material). We had also hypothesized that Raman spectroscopy could be used to discriminate between samples from volunteers with respiratory symptoms compared to those without respiratory symptoms, perhaps as the mucus content of saliva could differ between the two. However, we found that we could not build models with high predictive accuracy to discriminate between samples based on whether reported symptoms were considered respiratory symptoms or not (Figs. S16a–S16f in the Supplementary Material). We used samples from all volunteers and grouped non-respiratory symptomatics and asymptomatic samples together into a non-respira- tory group and compared the corresponding spectra to those from respiratory symptometics (n ¼ 44). Using spectra taken from all volunteers, the AUC was 0.57 in models built using edge spectra (n ¼ 44 respiratory, n ¼ 23 non-respiratory) and the AUC was 0.56 using MILDM on on crystal spectra (n ¼ 43 respiratory, n ¼ 23 non-respiratory). 025002-16 025002-16 February 2022 • Vol. 27(2) Journal of Biomedical Optics Journal of Biomedical Optics Ember et al.: Saliva-based detection of COVID-19 infection in a real-world setting. . . Ember et al.: Saliva-based detection of COVID-19 infection in a real-world setting. . . Ember et al.: Saliva-based detection of COVID-19 infection in a real-world setting. . . 3.8 Overfitting Assessment ML models were compared to models generated using random labels to ensure that models generated using true classification labels were not cases of overfitting. Full results are shown in Tables S4 and S5 in the Supplementary Material and AUC histograms of models generated using random and true classification labels are shown in Figs. S7–S13 in the Supplementary Material. These show that the models produced using the true labels have consistently higher AUCs than those produced using random labels apart from for COVID detection in males using the on crystal region [Fig. S7b in the Supplementary Material, Fig. 5(d)] and the models pro- duced for symptom classification (Fig. S12 and S16 in the Supplementary Material), which have already been discussed. Journal of Biomedical Optics 4 Discussion 3 and Table S3 in the Supplementary Material). This is in agreement with electron microscopy studies of dried solutions of lysozyme and salt mixtures in which lysozyme was shown to accumulate at the edge of dried droplets.65 Although many studies claim to use Raman spectroscopy of saliva to diagnose diseases, this is the first published instance to our knowledge in which both edge and center regions (both on crystal and off crystal) have been used in a single study, yielding complementary information in terms of interrogated biomolecular vibrational modes. Moreover, we could not find any other publications in which a saliva model was used to confidently assign molecular features in a Raman spectrum. The observation that the best model for males is built using different features and spectra from different parts of the droplet compared to females suggests that COVID-19 may elicit different changes in the biomolecular profile of saliva between the sexes. Studies have shown that there are differences in the immune response of males and females to COVID, with females mounting a more robust T cell response, whereas males had higher cytokine levels and are more severely impacted by COVID-19.66 Furthermore, ACE2 is expressed in lower levels in liver and lung tissue of women compared to men, which could result in different impacts on metabolism.67 We found that sex at birth was a confounding factor in saliva-based COVID-19 detection using label-free Raman spectroscopy as the AUC was reduced in models built from males and females together compared to those built from males and females separately. We could also discriminate between saliva supernatant samples based on sex at birth from COVID-negative females and males with an AUC of 0.80 using the on crystal region. This is consistent with a study by Muro et al.68 in which Raman spectroscopy was used to discriminate between 60 samples of whole saliva from males and females. Moreover, the biomolecular composition of saliva has been shown to be different in females and males by NMR spectroscopy.69 Levels of glycine, lactic acid, and acetate were all higher in saliva taken from males in that study while with Raman spectroscopy, we observed differences in peaks associated with proteins and nucleic acids, and a peak associated with lactic acid (Fig. 15d–15f in the Supplementary Material). 4 Discussion We were able to discriminate between COVID-positive and COVID-negative saliva samples in a primer-free, label-free way using Raman spectroscopy and ML. This is the first published case to our knowledge of MILES or MILDM applied to Raman spectroscopy. We achieved a sensitivity of 79% and specificity of 75% compared to PCR tests for detecting COVID-19 infection in non- hospitalized males with a range of symptoms, ages, medical conditions, and viral load. We achieved a sensitivity of 84% and specificity of 65% in detecting COVID in non-hospitalized females with similar real-world variables. Our novel application of MILES and MILDM techniques allowed us to account for possible differences in molecular content between Raman spectra within the same dried droplet. Spatially distinct Raman spectra from a COVID-positive sample may not all contain molecular informa- tion associated with COVID positivity and studies have shown that SARS-CoV-2 virions aggregate when dried.50 MIL techniques enable assessment of individual spectra rather than assessment of average spectra from each sample, which could result in a loss of information. Application of such a technique could be very useful in Raman microspectroscopic classifica- tion. Our proposed rapid screening platform may not rely on microscopy so other ML techniques will be investigated. In a pandemic control context, these ML models could be distributed rapidly and frequently through software updates while development and worldwide distribution of new primers to address variants can take valuable time, which could otherwise be used in limiting the spread of infection. We found that for males, the best classification model was built using the edge region using features associated with carbohydrates, carotenoids, proteins, and nucleic acids, whereas for February 2022 • Vol. 27(2) Ember et al.: Saliva-based detection of COVID-19 infection in a real-world setting. . . females, the best classification model was built using the on crystal region using features asso- ciated with lipids, proteins and nucleic acids. The off crystal region—the central region of the saliva droplet outside of the crystals—was not useful, suggesting that compounds associated with COVID-19 were perhaps less concentrated in this region than within the crystals themselves or the edge region. We have found that the Raman spectrum of the edge region of dried saliva supernatant differs from that of the central region, with the central region containing peaks due to salts and proteins, whereas the edge region is primarily composed of peaks due to proteins (Fig. 4 Discussion ) It seems we cannot discriminate between samples from volunteers with respiratory symp- toms compared to volunteers without respiratory symptoms with high sensitivity or specificity using Raman spectroscopy. This may be because the majority of mucus was pelleted along with food debris during the saliva centrifugation process or because mucus content does not differ significantly in saliva supernatant between respiratory and non-respiratory samples. However, as this study occurred during December 2020–February 2021, some volunteers may have exaggerated their symptoms during reporting to obtain a PCR test for non-essential rea- sons, e.g., travel or meeting family members during the holiday season. Unreliable reporting of symptoms is a key factor to be taken into account during infectious disease research and test- ing. With symptoms assessed by a trained medical professional and an approximate time since exposure given, we may be able to more accurately determine whether the sensitivity and specificity of our test is affected by whether the volunteer is pre-symptomatic, has respiratory symptoms, non-respiratory symptoms or no symptoms. We also aim to conduct a study in which COVID-tested volunteers could be tested for other respiratory diseases to check the specificity of our test in the face of other respiratory illnesses or simultaneous infections. Another limitation of the study is the mixed PCR tests—saliva and NPG—employed by the COVID testing unit, which was outside of our control. Although previous studies have shown good agreement between the two,6 it would be informative to compare saliva PCR, NPG PCR, and Raman saliva tests. February 2022 • Vol. 27(2) 5 Conclusions Therefore, we have devel- oped in parallel a rapid single-point Raman spectroscopy platform similar to the probe we have already developed and commercialized for detecting brain cancer25,70,71 for use in future studies oped in parallel a rapid single-point Raman spectroscopy platform similar to the probe we have already developed and commercialized for detecting brain cancer25,70,71 for use in future studies for biofluids. This can image a whole droplet within a few seconds and is portable, affordable, and suitable for high throughput on-site screening. PCR tests typically take at least 2.5 h from sample collection to result.10 As sensitivity and specificity of ML models can be tweaked along the ROC curve, sensitivity could be traded for slight losses in specificity in a Raman-based screening test. In a pandemic control context, a high sensitivity screening technique would be desirable, as potential positives could have a follow-up PCR test to confirm positivity. Our rapid screening technique could be followed by a more specific PCR test or be employed as a reagent-free alternative to lateral flow tests, depending on government policy. The platform could also be used for detection of other infectious diseases or COVID variants simply by retraining the ML algorithms on new samples. This would also enable us to test the generalizability of our methodology on multiple spectrometers. Saliva is a complex medium and multiple factors can affect the Raman spectrum. However, even while taking into account sex at birth, the maximum AUC in our study was 0.80. This suggests that there are further confounding factors. We are currently expanding our analysis to image the remaining 475 COVID-negative samples to take such factors into account using the single point platform. We will be thoroughly assessing the effects of more confounding fac- tors such as age, diet, smoking, and comorbidities on the salivary Raman fingerprint and evalu- ating whether these variables can impact the accuracy of detecting COVID-19. In the future, with our more rapid device, we aim to carry out further testing using independent test sets from differ- ent testing centers, allowing us to gather more spectral data and associated information about confounding variables. This will enable us to look into factors that could affect hormone levels such as pregnancy, medical conditions, prescription medications and surgeries, and assess their impact on the Raman spectrum of saliva. Disclosures There are no conflicts to declare. 5 Conclusions In summary, our volunteer-matched study demonstrates that there are molecular differences between saliva of COVID-positive and COVID-negative individuals that may be detected using Raman microspectroscopy amongst confounding factors. We next aim to create a single point Raman spectroscopy platform that could be easily integrated into the viral screening workflow. 025002-18 Journal of Biomedical Optics 5 Conclusions In conclusion, we have shown that Raman spectroscopy can be used to detect biomolecular changes between COVID-positive and COVID-negative saliva supernatant and that accounting for the sex of the saliva donor can increase the accuracy of predictive models. However, lim- itations of our Raman microspectroscopy approach include the fact that it was only possible to sample <1% of the full sample area and imaging times were too long for rapid, high throughput COVID screening (17 min per edge region and 1 h for on crystal). It is likely that in this study, we may not have captured the full molecular profile of every saliva drop. Therefore, we have devel- oped in parallel a rapid single-point Raman spectroscopy platform similar to the probe we have already developed and commercialized for detecting brain cancer25,70,71 for use in future studies for biofluids. This can image a whole droplet within a few seconds and is portable, affordable, and suitable for high throughput on-site screening. PCR tests typically take at least 2.5 h from sample collection to result.10 As sensitivity and specificity of ML models can be tweaked along the ROC curve, sensitivity could be traded for slight losses in specificity in a Raman-based screening test. In a pandemic control context, a high sensitivity screening technique would be desirable, as potential positives could have a follow-up PCR test to confirm positivity. Our rapid screening technique could be followed by a more specific PCR test or be employed as a reagent-free alternative to lateral flow tests, depending on government policy. In conclusion, we have shown that Raman spectroscopy can be used to detect biomolecular changes between COVID-positive and COVID-negative saliva supernatant and that accounting for the sex of the saliva donor can increase the accuracy of predictive models. However, lim- itations of our Raman microspectroscopy approach include the fact that it was only possible to sample <1% of the full sample area and imaging times were too long for rapid, high throughput COVID screening (17 min per edge region and 1 h for on crystal). It is likely that in this study, we may not have captured the full molecular profile of every saliva drop. Journal of Biomedical Optics References 1. D. M. Cutler and L. H. Summers, “The COVID-19 pandemic and the $16 trillion virus,” JAMA - J. Am. Med. Assoc. 324(15), 1495–1496 (2020). 2. “Global situation,” World Health Organisation Coronavirus (COVID-19) Dashboard, 2021, https://covid19.who.int/. 3. M. A. Johansson et al., “SARS-CoV-2 transmission from people without COVID-19 symp- toms,” JAMA Netw. Open 4(1), e2035057 (2021). 4. M. C. Grant et al., “The prevalence of symptoms in 24,410 adults infected by the novel coronavirus (SARS-CoV-2; COVID-19): a systematic review and meta-analysis of 148 stud- ies from 9 countries,” PLoS One 15(6), e0234765 (2020). 5. A. J. Ing, C. Cocks, and J. P. Green, “COVID-19: in the footsteps of Ernest Shackleton,” Thorax 75(8), 693–694 (2020). 6. G. Butler-Laporte et al., “Comparison of saliva and nasopharyngeal swab nucleic acid amplification testing for detection of SARS-CoV-2: a systematic review and meta-analysis,” JAMA Intern. Med. 181(3), 353–360 (2021). 7. B. Böger et al., “Systematic review with meta-analysis of the accuracy of diagnostic tests for COVID-19,” Am. J. Infect. Control 49(1), 21–29 (2021). 8. L. 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To et al., “Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study,” Lancet Infect. Dis. 20(5), 565–574 (2020). y f 15. D. Herrera et al., “Is the oral cavity relevant in SARS-CoV-2 pandemic?” Clin. Oral Investig. 24(8), 2925–2930 (2020). 16. L. M. Czumbel et al., “Saliva as a candidate for COVID-19 diagnostic testing: a meta- analysis,” Front. Med. 7, 465 (2020). 17. D. W. Shipp, F. Sinjab, and I. Notingher, “Raman spectroscopy: techniques and applications in the life sciences,” Adv. Opt. Photonics 9(2), 315 (2017). 18. A. Acknowledgments We would like to acknowledge Julie Dionne and Axel Bergman from the TransMedTech team. We would like to thank Laurence Knafo for biohazard training and access to containment level 2 facilities. We would like to thank the following personnel for assistance with sample collection: Gabriel Marocco, and questionnaire data entry: Jérémie Kerouac, Thomas Regouffre, Jean- Francois Martin. We would like to thank Mirela Birlea and André-Anne Grosset for help with acquisition of chemicals for model saliva. We would also like to thank all staff at the Pointe St-Charles testing center, especially Jacynthe, David and Samia. The authors acknowledge funding from the Canada First Research Excellence Fund (TransMedTech Institute, IVADO), the Natural Sciences and Engineering Research Council of Canada (Alliance and Discovery grant programs) and the Canada Foundation for Innovation (Exceptional Opportunities Fund 025002-19 February 2022 • Vol. 27(2) Ember et al.: Saliva-based detection of COVID-19 infection in a real-world setting. . . program). The first author was funded by TransMedTech Institute through a postdoctoral fellowship. program). The first author was funded by TransMedTech Institute through a postdoctoral fellowship. Code, Data and Materials Availability Anonymized Raman spectral files are available upon request. References Smekal, “Zur Quantentheorie der Streuung und Dispersion,” Naturwissenschaften 16(31), 612–613 (1928). 19. C. V. Raman and K. S. Krishnan, “A new type of secondary radiation,” Nature 121(3048), 501–502 (1928). 20. R. C. Lord and N. T. Yu, “Laser-excited Raman spectroscopy of biomolecules. I. Native lyso- zyme and its constituent amino acids,” J. Mol. Biol. 50(2), 509–524, Academic Press (1970). 21. D. A. Long, The Raman Effect: A Unified Treatment of the Theory of Raman Scattering by Molecules, Wiley, Chichester (2002). Journal of Biomedical Optics 025002-20 February 2022 • Vol. 27(2) February 2022 • Vol. 27(2) Journal of Biomedical Optics 025002-20 025002-20 025002-20 Ember et al.: Saliva-based detection of COVID-19 infection in a real-world setting. . . 22. E. Le Ru and P. 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Solonenko, “Morphology of dried drop patterns of saliva from a healthy individual depending on the dynamics of its surface tension,” Surfaces 2(2), 395–414 (2019). 65. H. M. Gorr et al., “Salt-induced pattern formation in evaporating droplets of lysozyme solutions,” Colloids Surf. B 103, 59–66 (2013). 66. T. Takahashi et al., “Sex differences in immune responses that underlie COVID-19 disease outcomes,” Nature 588(7837), 315–320 (2020). 67. T. Tukiainen et al., “Landscape of X chromosome inactivation across human tissues,” Nature 550(7675), 244–248 (2017). 68. C. K. Muro, L. De Souza Fernandes, and I. K. Lednev, “Sex determination based on Raman spectroscopy of saliva traces for forensic purposes,” Anal. Chem. 88(24), 12489–12493 (2016). 69. I. Takeda et al., “Understanding the human salivary metabolome,” NMR Biomed. 22(6), 577–584 (2009). 70. M. Jermyn et al., “Raman spectroscopy detects distant invasive brain cancer cells centimeters beyond MRI capability in humans,” Biomed. Opt. Express 7(12), 5129 (2016). 71. J. Desroches et al., “Characterization of a Raman spectroscopy probe system for intraoper- ative brain tissue classification,” Biomed. Opt. Express 6(7), 2380 (2015). 72. F. Salahioglu and M. J. Went, “Differentiation of lipsticks by Raman spectroscopy,” Forensic Sci. Int. 223, 148–152 (2012). 73. K. Czamara et al., “Raman spectroscopy of lipids: a review,” J. Raman Spectrosc. 46(1), 4–20 (2015). 74. N. Tiwari et al., “Study of adsorption behavior of aminothiophenols on gold nanorods using surface-enhanced Raman spectroscopy,” J. Nanophoton. 5(1), 053513 (2011). Katherine Ember is a postdoctoral fellow at the LRO at Polytechnique Montréal and the CRCHUM medical research center. She has carried out research into epigenetics using fluores- cence microscopy, liver damage using NMR and SRS spectroscopy, and brain cancer using Katherine Ember is a postdoctoral fellow at the LRO at Polytechnique Montréal and the CRCHUM medical research center. She has carried out research into epigenetics using fluores- cence microscopy, liver damage using NMR and SRS spectroscopy, and brain cancer using 025002-22 February 2022 • Vol. 27(2) Journal of Biomedical Optics 025002-22 February 2022 • Vol. 27(2) Ember et al.: Saliva-based detection of COVID-19 infection in a real-world setting. . . Raman spectroscopy and spearheaded the COVID-19 detection project with Prof. Leblond. She has also won or been shortlisted for nine science communication awards and gives biophysics lectures at Polytechnique Montréal to physics and engineering students. Ember et al.: Saliva-based detection of COVID-19 infection in a real-world setting. . . François Daoust is a fast-track PhD student in biomedical engineering from 2019 and his project is about clinical integration of a large field of view Raman imaging system to guide brain cancer resection surgery. He received his bachelor’s degree in engineering physics with major in photonics in 2017 and his MScA degree in biomedical engineering in 2018. Myriam Mahfoud is a research assistant at the LRO. She holds a bachelor’s degree in engineer- ing physics and a master’s degree in chemical engineering. She is passionate about using the theories and laws of physics to answer current issues in the fields of health, energy, and new technologies. She is working on the COVID-19 project, which consists of detecting the virus by analyzing human saliva using Raman spectroscopy. Frédérick Dallaire is a postdoctoral researcher at the LRO with expertise in machine learning, data processing, and feature engineering algorithms. He is mainly involved in the application of data science and statistical analysis in the biomedical field and surgical guidance. He received his PhD in particle physics with the Atlas Experiment at CERN in 2017. Esmat Zamani Ahmad is a PhD student working with Prof. Frédéric Leblond at the Laboratory of Radiological Optics (LRO) at Polytechnique Montréal in Montréal, Québec. His research focuses on Surface-Enhanced Raman Spectroscopy (SERS) applied to biofluids. His interests include plasmonics, nonlinear optics, and machine learning. He received his bachelor’s degree in engineering physics with major in photonics in 2020. Trang Tran joined at the LRO lab as a research associate. With more than 6 years in research and specialising in neurosciences and biomedical spectroscopy with a focus on clinical appli- cations, she currently oversees several clinical research projects in the laboratory including her main project researching the use of Raman spectroscopy on patients with focal cortical dysplasia. Her interests lie at the intersection of molecular biology and neurosurgery. Arthur Plante is a postdoctoral researcher at the LRO. In his PhD in particles physics, he worked on the PICASSO and PICO dark matter experiments located at SNOLAB, Sudbury. During his graduate studies, he became an expert in simulating particle-matter interactions, theo- retical dark matter interactions, and data analysis. His current work focuses on using and devel- oping Bayesian inference methods using Markov Chain Monte Carlo (MCMC) and feature engineering to identify Raman disease biomarkers. Journal of Biomedical Optics Ember et al.: Saliva-based detection of COVID-19 infection in a real-world setting. . . Mame-Kany Diop is a PhD student in the lab of Dominique Trudel at the Université de Montréal (UdeM) and CRCHUM. She has applied her biomedical knowledge to prostate cancer prognosis and treatment and the COVID-19 detection project. She received her BSc degree in biomedical sciences at UdeM where she researched nephrogenic diabetes and received her MSc degree in pathology and cellular biology at UdeM. Tien Nguyen received his BSc in physics at the Université de Sherbrooke. He joined the LRO as a research intern where he applied his scientific skills to healthcare and then became a graduate student under the supervision of Frédéric Leblond and co-supervision of Dominique Trudel. His research activities revolved around developing ML models based on hyperspectral and histologic images for tumor margins detection and biochemical recurrence predictions. He now works for Brainbox AI in AI operations. Amélie St-Georges-Robillard is a research associate in Frédéric Leblond’s and Thomas Gervais’ laboratories. She received her bachelor’s degree in engineering physics and her mas- ter’s degree in surface coatings for cell culture. She received her doctorate at Polytechnique Montréal on quantitative fluorescence imaging of tumor spheroids trapped in microfluidic chips for the treatment of cancer. She is now co-head of the Microfluidics Core Facility of the TransMedTech Institute based at the CHUM Research Center. February 2022 • Vol. 27(2) Journal of Biomedical Optics Ember et al.: Saliva-based detection of COVID-19 infection in a real-world setting. . . Ember et al.: Saliva-based detection of COVID-19 infection in a real-world setting. . . Nassim Ksantini is a research associate in the LRO working on detection of diseases in bio- fluids. He has worked on development of a time-domain Raman spectroscopic technique based on time-correlated single photon counting (TCSPC) technology to isolate the Raman spectrum from fluorescence. He received his bachelor’s degree in electrical engineering from the École de Technologie Superieure (ETS) of Montreal. During his diploma final internship, he worked on a TCSPC device for fluorescence lifetime measurement. Julie Lanthier carried out a research internship at the LRO using Raman microspectroscopy of biological constituents of saliva and is currently studying for a bachelor’s degree in engineering physics at Polytechnique Montréal. She received her bachelor’s degree in neuroscience in 2017 from the Université de Montréal and has carried out research into utilization and analysis of EEG signals applied to adolescents with idiopathic scoliosis. Ember et al.: Saliva-based detection of COVID-19 infection in a real-world setting. . . Antoine Filiatrault carried out a research internship at the LRO using Raman microspectro- scopy of biological constituents of saliva and is currently studying for a bachelor’s degree in biomedical engineering at Polytechnique Montréal. Guillaume Sheehy holds a baccalaureate and a master’s degree in engineering physics from Polytechnique Montréal. He is currently a PhD student in biomedical engineering working on depth resolved Raman spectroscopy techniques for the assessment of breast cancer surgical margin. He has been working with Dr. Leblond since a research internship in 2014. Gabriel Beaudoin is a bachelor’s student at the LRO studying engineering physics. His research interests lie in the integration of engineering and physics for medical purposes. He is currently working on spatial frequency domain imaging (SFDI) technology. Caroline Quach is a professor in the Departments of Microbiology, Infectious Diseases & Immunology and of Pediatrics at the University of Montreal. She is an adjunct professor in the Department of Epidemiology, Biostatistics & Occupational Health at McGill University. She is the physician in charge of infection prevention and control at CHU Sainte-Justine where she also works as a pediatric infectious diseases specialist and medical microbiologist. Dominique Trudel is a genitourinary and molecular pathologist in the Department of Pathology of CRCHUM and an associate clinical professor in the Department of Pathology and Cell Biology at the University of Montreal. Clinician-researcher at the FRQS, junior level 2, her research is focused on intraductal carcinoma of the prostate and clinical integration of Raman spectroscopy. She has received numerous awards, including Junior Scientist of the Year from the Canadian Association of Pathologists twice. Frédéric Leblond is a professor in the Department of Engineering Physics at Polytechnique Montréal and director of the LRO. He is also a researcher at the CRCHUM medical research center and co-founder of the company Reveal Surgical. His research is related to the develop- ment of light-based medical devices to improve the accuracy surgical procedures and medical diagnostics. He holds more than 10 patents and has published more than 95 peer-reviewed articles. February 2022 • Vol. 27(2) 025002-24 025002-24 Journal of Biomedical Optics
https://openalex.org/W4392878807	https://e-journal.uniflor.ac.id/index.php/JPE/article/download/779/776	Indonesian	null	Penerapan Metode Demonstrasi Untuk Meningkatkan Hasil Belajar Ipa Pada Siswa Kelas 5A SDI Bhoanawa 1	Ekspektasi	2,021	cc-by-sa	3,095	Keywords: demonstration method, learning outcomes PENDAHULUAN dikarenakan kurangnya fasilitas belajar IPA. Fakta ini dijumpai dalam praktek pembelajaran IPA di SDI Bhoanawa 1. Ketiadaan dan kekurangan bahan maupun alat praktikum menyebabkan guru memilih menggunakan metode ceramah yang diselingi kegiatan diskusi. Pembelajaran seperti ini memberikan keuntungan dimana guru dapat memberikan materi seluas- luasnya. Akan tetapi memiliki banyak kelemahan dari sisi lain seperti siswa tidak berinisiatif untuk bertanya kepada guru tentang suatu hal yang belum di mengerti. Siswa juga sangat mudah melupakan materi yang sudah diberikan sehingga hasil belajar siswa rendah. Sejalan dengan ilmu pengetahuan dan teknologi yang semakin berkembang pesat sangat mempengaruhi perkembangan pendidikan IPA terutama di negara-negara yang sudah maju. Dalam proses pembelajaran IPA tersebut seharusnya disediakan serangkaian pengalaman berupa kegiatan nyata yang rasional atau dapat dimengerti oleh siswa dan memungkinkan terjadinya interaksi sosial. Jadi saat proses pembelajaran siswa harus terlibat secara aktif dalam kegiatan nyata, untuk itu kita sebagai guru harus mempersiapkan pembelajaran yang melibatkan aktivitas siswa secara penuh dan siswa juga dituntut untuk menguasai materi dengan baik setelah pembelajaran IPA berlangsung. Metode pembelajaran yang ideal tidak bisa dilakukan dengan gaya komunikasi searah. Proses pembelajaran harus dilakukan dengan ragam macam metode sesuai dengan materi dan kemampuan siswa yang ada. Dalam kaitanya dengan materi IPA, metode pembelajaran yang baik tidak cukup hanya dengan ceramah (Sari, 2013). Metode pembelajaran yang bisa dijadikan acuan bagi peningkatan pada hasil belajar siswa tersebut adalah metode demonstrasi. Melalui pembelajaran IPA siswa dapat menemukan berbagai hal yang terjadi dalam kehidupan sehari-hari yakni seperti seperti kejadian-kejadian nyata yang ditemukan siswa. Pendidikan IPA dapat membantu untuk mengungkapkan secara sistematis dalam mencari tahu hal- hal yang terjadi serta dapat membentuk kepribadian atau tingkah laku siswa sehingga siswa dapat memahami proses IPA dan dikembangkan di masyarakat. Pendidikan IPA bukan hanya sekedar teori akan tetapi dalam setiap bentuk pengajaran lebih ditekankan pada bukti dan kegunaan ilmu tertentu. Dalam pembelajaran IPA setelah guru menyampaikan materi pada siswa, maka guru dan siswa dapat membuat suatu presentasi praktek yang dilakukan untuk mempraktekan dan membuktikan materi yang telah diajarkan dengan memilih metode pembelajaran yang tepat serta mempergunakan media yang mendukung sebagai sarana yang efektif. Uno dan Mohamad (2013:98) menyatakan metode demonstrasi sebagai salah satu metode yang digunakan dalam proses pembelajaran aktif sebab bersentuhan dengan bagaimana siswa memperagakan sesuatu, karena metode ini memperlihatkan bagaimana ia melakukan sesuatu yang kemudian diamati dan dibahas. Penerapan Metode Demonstrasi Untuk Meningkatkan Hasil Belajar Ipa Pada Siswa Kelas 5A SDI Bhoanawa 1 Maria Gorety Lami e-mail: wetarose5@gmail.com SDI Bhoanawa 1 Ende ABSTRAK: Penelitian ini bertujuan untuk meningkatkan hasil belajar IPA melalui metode demonstrasi pada siswa kelas 5A SDI Bhoanawa 1. Jenis penelitian ini merupakan Penelitian Tindakan Kelas (PTK) yang dilaksanakan dalam dua siklus. Subjek penelitian ini adalah 20 siswa yang terdiri dari 14 orang putra dan 6 orang putri pada kelas 5A SDI Bhoanawa 1. Data penelitian dikumpulkan melalui metode observasi, tes dan dokumentasi. Data hasil penelitian dianalisis dengan menggunakan metode analisis statistik deskriptif sederhana. Hasil penelitian menunjukkan adanya peningkatan hasil belajar dari pratindakan sebesar 5%, menjadi 45% pada siklus 1 dan meningkat maksimal 100% pada siklus 2. Jadi dapat disimpulkan bahwa dengan menerapkan metode demonstrasi dapat meningkatkan hasil belajar IPA pada siswa kelas 5A SDI Bhoanawa 1. Kata kunci: metode demonstrasi, hasil belajar ABSTRACT: This study aims to improve science learning outcomes through demonstration methods in grade 5A SDI Bhoanawa 1. This type of research is a Classroom Action Research (CAR) which is carried out in two cycles. The subjects of this study were 20 students consisting of 14 boys and 6 girls in grade 5A SDI Bhoanawa 1. The research data were collected through observation, test and documentation methods. The research data were analyzed using simple descriptive statistical analysis methods. The results showed an increase in learning outcomes from pre-action by 5%, to 45% in cycle 1 and a maximum increase of 100% in cycle 2. So it can be concluded that applying the demonstration method can improve science learning outcomes in grade 5A SDI Bhoanawa 1 students. Keywords: demonstration method, learning outcomes asi: Jurnal Pendidikan Ekonomi Volume 5, Nomor 2, Desember 2020 E-ISSN 2722-3353 96 PENDAHULUAN menurut Husamah (2014 : 95) Metode demonstrasi adalah metode yang dilaksanakan untuk menampilkan suatu proses, mekanisme atau cara kerja suatu alat yang berkaitan dengan materi pembelajaran. Menurut Suryani & Agung (2012 : 60) Metode demonstrasi adalah suatu hal yang dalam penyajian bahan ajar dengan contoh menunjukan kepada siswa tentang situasi yang terjadi pada hal-hal tertentu yang di pelajari berupa tiruan yang disertai dengan penjelasan. Namun idealisme pembelajaran yang diinginkan belum sesuai kenyataan yang terjadi di lapangan. Masih banyak proses belajar IPA yang dilalui secara abstrak tanpa memperhatikan proses untuk memahami konsep IPA itu sendiri. Umumnya faktor penyebab hal tersebut Melalui metode demonstrasi dapat membantu siswa untuk mencari jawaban 96 dengan usaha sendiri berdasarkan fakta yang benar. Metode demonstrasi, dapat menghindari proses belajar dengan cara menghafal (Halawa, 2012). Hal ini dikarenakan siswa akan disuruh langsung memperhatikan materi atau bahan yang dijelaskan. Melalui metode demonstrasi pembelajaran terlihat lebih menarik dikarenakan siswa bukan semata-mata mendengar, melainkan turut melihat peristiwa yang terjadi (Bartik dkk, 2013). Dengan mengamati langsung siswa dapat memperoleh kesempatan besar untuk dapat mengimbangi teori dan kenyataan sehingga siswa dapat mengetahui kebenaran materi yang di pelajari. guru terlebih dahulu menetapkan tujuan demonstrasi. Dengan demikian dapat diketahui kecakapan apa yang diharapkan dari hasil demonstrasi tersebut, (b) guru harus mempersiapkan diri sebaikbaiknya, baik secara teoritis maupun praktek. Dengan kata lain, guru harus menguasai teori dan penggunaan bahan dan alat-alat, (c) haru diperhatikan waktu yang tersedia dalam melakukan demonstrasi, dan (d) harus diperhatikan suasana dan hubungan baik antara guru dan siswa, sehingga ada keinginan siswa untuk memperhatikan apa yang didemonstrasikan (Situmorang, dkk., 2006). Beberapa kegiatan yang perlu dilakukan guru di dalam menerapkan metode demonstrasi (Saragih dan Situmorang, 2006) antara lain: (1) Mempersiapkan sesuatu yang akan didemonstrasikan di tempat yang lebih baik, (2) Mempersiapkan tempat duduk siswa agar semua dapat mengamati dengan jelas seluruh objek yang didemonstrasikan, (3) Guru memilih tempat berdiri yang tepat agar tidak menghalangi penglihatan siswa, (4) Selama melakukan demonstrasi, guru harus memperhatikan perhatian siswa, (5) Guru perlu mengulang bagian yang dianggap perlu, (6) Guru perlu mengajukan pertanyaanpertanyaan secara lisan untuk mengetahui sejauh mana siswa memahami demonstrasi tersebut, (7) Siswa disuruh kembali menjelaskan apa yang didemonstrasikan. Untuk itu perlu adanya upaya perbaikan terhadap proses pembelajaran IPA di SDI Bhoanawa 1 dengan menerapkan metode demonstrasi. Karena dengan metode demonstrasi, penguasaan materi siswa akan lebih maksimal sehingga dapat meningkatkan hasil belajar siswa. ektasi: Jurnal Pendidikan Ekonomi Volume 5, Nomor 2, Desember 2020 E-ISSN 2722-3353 9 (2005:107) Nuryanti y ( ) mengatakan bahwa metode demonstrasi adalah cara penyajian pelajaran dengan memperagakan sesuatu proses kejadian sehingga membuat pelajaran menjadi lebih jelas dan menjadi lebih mudah memahami materi yang disampaikan. Majid (2013: 197) mengungkapkan bahwa metode demonstrasi merupakan metode penyajian pelajaran dengan memperagakan dan mempertunjukan kepada siswa tentang suatu proses, situasi, atau benda tertentu, baik sebenarnya atau hanya sekedar tiruan. Metode demonstarsi adalah metode mengajar dengan cara memperagakan barang, kejadian, aturan, dan urutan melakukan kegiatan, baik secara langsung maupun melalui penggunaan media pengajaran yang relevan dengan pokok bahasan atau materi yang sedang disajikan. A d d i d Metode demonstrasi memiliki beberapa kelebihan (Saragih dan Situmorang, 2006) diantaranya: (a) perhatian pelajar dapat diarahkan pada hal- hal yang dianggap penting, sehingga hal- hal yang dianggap penting itu dapat diamati seperlunya. Perhatian pelajar lebih mudah dipusatkan pada proses belajar dan tidak tertuju pada halhal yang tidak relevan, (b) dapat mengurangi kesalahan- kesalahan bila dibandingkan dengan kegiatan hanya mendengar ceramah atau membaca buku, karena pelajar memperoleh gambaran yang lebih jelas dari hasil pengamatannya, (c) bila pelajar Agar metode demonstrasi dapat terlaksana dengan baik, ada beberapa syarat yang perlu diperhatikan, yakni: (a) 97 ikut aktif, maka ia akan memperoleh pengamatanpengamatan praktek untuk mengembangkan kecakapannya dan pengharapan dari lingkungan sosialnya, dan (d) beberapa masalah yang menimbulkan pertanyaan pada pelajar dapat dijawab dengan lebih teliti pada waktu proses demonstrasi. Sedangkan kelemahan metode demonstrasi dalam pengajaran adalah: (a) kurang baik dilakukan apabila siswa terlalu banyak sehingga tempat duduk dan berdiri tidak mengijinkan, (b) demonstrasi kurang efektif bila waktu yang tersedia tidak cukup, (c) demonstrasi akan merupakan metode yang tidak wajar bila alat yang didemonstrasikan tidak dapat diamati dengan seksama, dan (d) demonstrasi hanya merupakan tontonan saja apabila siswa tidak terlibat dalam mempraktekkannya. sikap dan nilai. Tipe hasil belajar afektif tampak pada siswa dalam berbagai tingkah laku seperti perhatian terhadap pelajaran, disiplin, motivasi belajar, menghargai guru dan teman sekelas, kebiasaan belajar dan lain-lain (Sudjana, 2009:53).Tipe hasil belajar afektif dalam pencapaiannya diukur melalui evaluasi prestasi afektif. Dalam merencanakan penyusunan instrumen tes prestasi siswa berdimensi afektif (ranah rasa) jenis-jenis prestasi internalisasi dan karakteristik seyogianya dapat perhatian khusus. Alasannya, kedua jenis ranah rasa yang banyak mengendalikan sikap dan perbuatan siswa. Ketiga adalah tipe hasil belajar psikomotorik, yang tampak dalam bentuk keterampilan (skill), kemampuan bertindak individu (Sudjana, 2009:54). Hasil belajar psikomotorik dalam pencapaiannya diamati melalui observasi. (2005:107) Cara pandang tepat untuk mengevaluasi keberhasilan belajar yang berdimensi ranah psikomotorik (rasa krasa) adalah observasi (Syah, 2010: 154). Observasi, dalam hal ini dapat diartikan sebagai sejenis tes mengenai peristiwa, tingkah laku, atau fenomena lain, dengan pengamatan langsung. Hasil penelitian yang dilakukan Lestari (2013:51) menunjukkan bahwa metode demonstrasi dengan media realia dapat meningkatkan hasil belajar siswa. Dengan demikian maka penerapan metode demontrasi yang baik akan mampu meningkatkan hasil belajar kognitif siswa. Hasil belajar dalam penelitian ini adalah perubahan tingkah laku yang diharapkan dari siswa setelah menjalani aktivitas melalui bimbingan guru, orang tua ataupun mandiri. ektasi: Jurnal Pendidikan Ekonomi Volume 5, Nomor 2, Desember 2020 E-ISSN 2722-3353 9 METODE PENELITIAN Jenis penelitian yang digunkan dalam penelitian ini adalah penelitian tindakan kelas (classrom action research) yang mengacu pada prosedur yang dirancang Lewin. Prosedur tersebut terdiri atas tahapan perencanaan, tindakan, pengamatan dan refleksi. Subyek penelitian ini adalah siswa kelas 5A SDI Bhoanawa 1 Tahun Pelajaran 2019/2020 sebanyak 20 siswa yang terdiri dari 14 laki-laki dan 6 perempuan. Hasil Belajar Berkaitan dengan hasil belajar, ada tiga tipe hasil belajar. Pertama, tipe hasil belajar kognitif yakni pengetahuan hafalan termasuk pula pengetahuan yang sifatnya faktual, di samping pengetahuan mengenai hal-hal yang perlu diingat kembali. 8 Tipe belajar kognitif dalam pencapaiannya diukur melalui evaluasi kognitif. Keberhasilan siswa yang berdimensi kognitif (ranah cipta) dapat diukur dengan berbagai cara, tes tertulis maupun tes lisan dan perbuatan (Syah, 2010: 151). Teknik utama yang digunakan dalam mengambil data hasil belajar adalah dengan metode tes. Teknik observasi dan wawancara juga digunakan sebagai teknik pendukung dalam mengambil data. Observasi dilakukan untuk mengambil data mengenai keterlaksanaan pembelajaran dengan metode demonstrasi. Tipe hasil belajar yang kedua adalah afektif, yakni berkenaan dengan 98 Data dianalisis dengan membandingkan persentase ketuntasan setiap siklus terhadap indicator kinerja. Adapun indikator kinerja dalam penelitian ini yakni apabila ketuntasan hasil belajar telah mencapai 100%. masih belum sesuai harapan yang diinginkan. Hal ini dikarenakan acuan metode pembelajaran yang dipakai guru belum sesuai dengan kebutuhan materi pembelajaran. Untuk memperjelas kondisi tersebut maka dilakukanlah pretest yang juga akan digunakan sebagai data pembanding terhadap hasil belajar siswa setelah diterapkan metode demonstrasi. Data pretest siswa tersaji dalam Tabel 1. 1. Deskripsi pratindakan 1. Deskripsi pratindakan Berdasarkan refleksi peneliti, kondisi awal pembelajaran IPA di SDI Bhonawa 1 Tabel 1 Perolehan Pretest pratindakan No Keterangan Perolehan 1 Nilai Terendah 11 2 Nilai tertinggi 67 3 Nilai rata-rata Kelas 23 4 Jumlah siswa yang belum tuntas belajar 19 5 Jumlah siswa yang tuntas belajar 1 6 Persentase Ketuntasan Belajar 5% dilakukan dengan cara sebagai berikut: 1) Guru menyampaikan tujuan dari pembelajaran yang ingin dicapai; 2) Guru menyajikan ringkasan materi yang akan disampaikan; 3) Guru mempersiapkan bahan atau alat yang di perlukan; 4) Guru menunjuk salah seorang siswa untuk melakukan demonstrasi sesuai skenario yang telah disiapkan; 5) Seluruh siswa memperhatikan demonstrasi dan menganalisisnya; 6) Tiap siswa mengemukakan hasil analisisnya dan juga pengalaman siswa didemonstrasikan; 7) Guru membimbing siswa membuat kesimpulan. Data pretest menunjukkan bahwa masih banyak siswa belum mencapai ketuntasan. Pencapaian ketuntasan secara klasikal hanya sebesar 24% dengan nilai rata-rata sebesar 56. Data tersebut memberikan gambaran bahwa sebagian besar siswa belum memahami materi dengan baik. Untuk itu perlu diterapkan tindakan pertama melalui metode demonstrasi. 2. Deskripsi tindakan pertama Tindakan pertama dilaksanakan sesuai siklus di dalam PTK yakni perencanaan, tindakan, observasi dan refleksi. Tahap perencanaan dilakukan dengan membuat persiapan-persiapan instrumen penelitian yang terdiri atas perangkat pembelajaran, lembar observasi dan soal tes. Selain itu juga mempersiapkan alat dan bahan yang akan digunakan untuk kegiatan demonstrasi siswa. Selama pelaksanaan pembelajaran dengan metode demonstrasi, guru dibantu oleh satu orang observer yang mengobservasi keterlaksanaan penerapan metode tersebut. pedoman observasi berisikan langkah-langkah penerapan metode demonstrasi yang dinilai keterlaksaannya dengan skala Likert 1-5. Hasil penilaian kemudian dianalisis persentasenya lalu ditentukan kategorinya sesuai pedoman acuan penilaian dalam Tabel 2. Tahap selanjutnya adalah tindakan. Pada tahapan ini, peneliti menerapkan pembelajaran sesuai perencanaan yang telah dibuat di dalam RPP. Secara garis besar langkah penerapan metode demonstrasi pada tindakan pertama asi: Jurnal Pendidikan Ekonomi Volume 5, Nomor 2, Desember 2020 E-ISSN 2722-3353 99 99 Tabel 2 Pedoman acuan penilaian pembelajaran dengan metode demonstrasi Tabel 2 Pedoman acuan penilaian pembelajaran dengan metode demonstrasi No Persentase Rata-Rata (PR) Kriteria 1 80% ≤PR ≤100% Sangat Baik 2 60% ≤PR ≤80% Baik 3 40% ≤PR ≤60% Cukup Baik 4 20% ≤PR ≤40% Kurang 5 0% ≤PR ≤20% Buruk Hasil observasi keterlaksanaan pembelajaran menunjukkan bahwa pada langkah penyampaian tujuan pembelajaran, penyajian materi dan persiapan alat serta bahan mendapat skor 4 atau kategori baik. Pada tahap pelaksanaan demonstrasi dan analisis hasil demonstrasi mendapat skor 2 atau kategori kurang. Pada tahapan ini siswa terekam masih kaku dan bingung menggunakan alat dan bahan praktikum sehingga perlu dibimbing ekstra oleh guru. Tahapan komunikasi dan menyatakan kesimpulan mendapat skor 3 atau kategori cukup. Siswa juga tidak terbiasa melakukan aktivitas pada tahapan ini sehingga membutuhkan waktu yang lama untuk menyelesaikan tahapan ini. Secara klasikal persentase rata-rata keterlaksanaan pembelajaran dengan metode demonstrasi sebesar 63%. Berdasarkan Tabel 2, maka persentase sebesar 63% berada dalam kategori baik. Dengan demikian pelaksaaan pembelajaran dengan metode demontrasi telah dilaksanakan dengan baik pada siklus I. demonstrasi serta memberikan contoh mengenai cara membuat kesimpulan analisis yang benar. 3. Deskripsi tindakan 2 ektasi: Jurnal Pendidikan Ekonomi Volume 5, Nomor 2, Desember 2020 E-ISSN 2722-3353 10 3. Deskripsi tindakan 2 p Siklus 2 dilaksanakan dengan memperbaiki kekurangan-kekurangan pada siklus 1. Perencanaan yang dilakukan pada siklus 2 tidak sebanyak pada tahapan perencanaan siklus 1. Peneliti hanya perlu melengkapi langkah-langkah pada RPP sesuai hasil refleksi siklus 1. Kekurangan yang ditemukan dalam siklus 1 antara lain pada tahap pelaksanaan demonstrasi dan analisis hasil demonstrasi yang masih mendapat kategori kurang. Pada tahapan ini siswa terekam masih kaku dan bingung menggunakan alat dan bahan praktikum sehingga perlu dibimbing ekstra oleh guru. Hal ini menyebabkan proses belajar menjadi kurang efektif karena alokasi waktu yang relatif tersita. Untuk mengatasinya, sebelum memasuki langkah pembelajaran tersebut guru memberikan tindakan dengan memberikan arahan terlebih dahulu mengenai langkah-langkah demonstrasi serta alat dan bahan apa saja yang dibutuhkan dalam melakukan demonstrasi. Setelah melalui proses pembelajaran, siswa kemudian diberi tes. Dari hasil tes yang dilakukan diketahui bahwa dari 20 siswa terdapat 7 siswa yang tuntas atau mencapai 35 % dan yang belum tuntas sebanyak 13 siswa atau mencapai 65 % dan nilai rata-rata 57. Hal ini menunjukan bahwa hasil belajar siswa belum mencapai kriteria dengan baik. Maka peneliti perlu melakukan tindakan kedua dengan beberapa refleksi seperti memberikan arahan terlebih dahulu sebelum siswa melakukan demonstrasi, memberi peluang yang banyak untuk tampil bagi siswa yang kesulitan mengkomunikasikan hasil Pelaksanaan tindakan pada siklus 2 tidak jauh berbeda dengan siklus 1. Langkah-langkah tersebut dilakukan dengan cara: 1) guru menyampaikan tujuan pembelajaran, 2) guru menyajikan ringkasan materi, 3) Guru mempersiapkan bahan atau alat yang di perlukan; 4) Guru menjelaskan prosedur demonstrasi dan menunjuk salah seorang siswa untuk melakukan demonstrasi sesuai skenario yang telah disiapkan; 5) guru membantu siswa dalam menganalisis proses demonstrasi; 6) Guru membimbing siswa kategori baik. Sedangkan tahap menyimpulkan mendapat skor 4 dengan kategori sangat baik. Secara klasikal pelaksanaan pembelajaran IPA dengan metode demonstrasi di kelas 5A mendapat persentase sebesar 91% atau dalam kategori sangat baik. mengemukakan hasil analisisnya dan juga pengalaman siswa didemonstrasikan; 7) Guru membimbing siswa membuat kesimpulan. Hasil observasi keterlaksanaan pembelajaran dengan metode demonstrasi pada siklus 2 menunjukkan pada tahap penyampaian tujuan pembelajaran dan penyajian rumusan masalah mendapat skor 5 dan berada dalam kategori sangat baik. Tahap persiapan bahan dan alat juga mendapat skor 5 atau dalam kategori sangat baik. Tahap melalukan demonstrasi, menganalisis dan mengkomunikasikan mendapat skor 4 atau berada dalam Hasil tes pada siklus 2 menunjukkan peningkatan bila dibandingkan dengan siklus 1. Dari hasil tes yang dilakukan diketahui bahwa seluruh siswa telah mencapai ketuntasan dengan rata-rata 83,25. 3. Deskripsi tindakan 2 Adapun perbandingan hasil belajar anatar pratindakan, siklus 1 dan siklus 2 tersaji dalam Gambar 2. 0 20 40 60 80 100 Hasil Belajar Pra tindakan siklus 1 siklus 2 Gambar 2. Perbandingan Hasil Belajar Pratindakan, Siklus 1, Siklus 2 Gambar 2. Perbandingan Hasil Belajar Pratindakan, Siklus 1, Siklus 2 Pada pratindakan perolehan ketintasan hanya sebesar 5%. Angka tersebut meningkat menjadi 45% pada siklus 1 namun belum mencapai target yang diinginkan. Sedangkan pada siklus 2 ketuntasan klasikal mencapai 100%. Berdasarkan pencapaian ini maka dapat disimpulkan bahwa penerapan metode demonstrasi dapat meningkatkan hasil belajar siswa SDI SDI Bhoanawa. Peningkatan tersebut sejalan dengan penelitian Karseno (2016), dimana metode demonstrasi diterapkan dalam mata pelajaran Fiqih pada siswa Madrasah Ibtidaiyah Muhammadiyah Pandansari. Metode demonstrasi dapat meningkatkan hasil belajar siswa dalam dua siklus. demonstrasi dapat meningkatkan hasil belajar IPA siswa kelas 5A di SDI Bhoanawa 1. Hal ini terbukti dar peningkatan hasil tes dalam pratindakan, siklus1 dan siklus 2 berturut-turut sebesar 5%, 45% dan 100%. Daftar Pustaka Bartik, A., Abdussamad & Roswita. 2013. Peningkatan Aktivitas Pembelajaran Matematika Dengan Penerapan Metode Demonstrasi Di Kelas III SDN 11 Sungai Kunyit. Jurnal Pendidikan dan Pembelajaran Katulistiwa, 2 (7). Halawa, M.V. 2012. Penerapan Metode Demonstrasi Untuk Meningkatkan Hasil Belajar Teknik Kolase Halawa, M.V. 2012. Penerapan Metode Demonstrasi Untuk Meningkatkan Hasil Belajar Teknik Kolase KESIMPULAN Berdasarkan hasil penelitian dapat disimpulkan bahwa penerapan metode ektasi: Jurnal Pendidikan Ekonomi Volume 5, Nomor 2, Desember 2020 E-ISSN 2722-3353 10 Melalui Produk Kerajinan Tangan Dalam Mata Pelajaran SBK Di SDN Desa Lama Kec. Hamparan Perak T.P 2011/2012. Gorga : Jurnal Seni Rupa, 1 (1). DOI: https://doi.org/10.24114/gr.v1 i1.176 http://repository.iainpurwokerto.ac. id/467/ http://repository.iainpurwokerto.ac. id/467/ http://repository.iainpurwokerto.ac. Sari, D.K. 2013. Pnerapan Metode Demonstrasi Untuk Meningkatkan Hasil Belajar Siswa Pada Mata Pelajaran Ipa Tentang Pokok Bahasan Cahaya Dan Sifat-Sifatnya : Penelitian Tindakan Kelas di SDN Nanggeleng I Kelas V Semester II Ajaran 2012/2013 Kota Sukabumi. thesis, Universitas Pendidikan Indonesia. Tersedia: http://repository.upi.edu/1499/ Sari, Husamah. 2014. Pembelajaran Bauran (Blended Learning). Jakarta : Pustakarya Karseno. 2016. Penerapan Metode Demonstrasi Pada Mata Pelajaran Fiqih Di Mi Muhammadiyah Pandansari Kabupaten Banyumas. Skripsi thesis, IAIN Purwokerto. Tersedia: Uno,H.B. & Mohamad Nurdi.2013. Belajar dengan pendekatan PAILKEM. Jakarta : Bumi Aksara ektasi: Jurnal Pendidikan Ekonomi Volume 5, Nomor 2, Desember 2020 E-ISSN 2722-3353 10
https://openalex.org/W2151869093	https://cancerci.biomedcentral.com/counter/pdf/10.1186/1475-2867-12-5	English	null	Pravastatin inhibits cell proliferation and increased MAT1A expression in hepatocarcinoma cells and in vivo models	Cancer cell international	2,012	cc-by	4,786	* Correspondence: Eli.hijonamuruamendiaraz@osakidetza.net 1Department of Gastroenterology, Donostia Hospital, Instituto Biodonostia, University of the Basque Country EHU/UPV, Ciberehd, San Sebastián, Spain Full list of author information is available at the end of the article © 2012 Hijona et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Statins may have therapeutic effects on hepatocarcinoma (HCC). This type of disorder is the most common malignant primary tumour in the liver. Our objective was to determine whether pravastatin had a therapeutic effect in vitro and in vivo models. Method: We design in vitro and in vivo model. In vitro we used PLC and determine cell proliferation. In vivo, we used and animal model to determined, PCNA and MAT1A expression and transaminases levels. Results: We found that pravastatin decreases cell proliferation in vitro (cell proliferation in pravastatin group was 82%, in sorafenib group 51% and in combined group 40%) and in vivo (in pravastatin group 80%, in sorafenib group 76.4% and in combined group 72.72%). The MAT1A levels, was significantly higher in Pravastatin group (D 62%, P 94%, S 71%, P + S 91%). The transaminases levels, decreased significantly in Pravastatin group (GOT and GPT levels D 619.5 U/L; 271 U/L) (P 117.5 U/L; 43.5 U/L) (S 147 U/L; 59 U/L) (P + S 142 U/L; 59 U/L). Conclusion: The combination of pravastatin + sorafenib were more effective than Sorafenib alone. Keywords: Pravastatin, Sorafenib, Hepatocarcinoma, Statins Pravastatin inhibits cell proliferation and increased MAT1A expression in hepatocarcinoma cells and in vivo models Elizabeth Hijona1*, Jesús María Banales2, Lander Hijona1, Juan Francisco Medina2, Juan Arenas1, Marta Herreros-Villanueva1, Pablo Aldazabal1 and Luis Bujanda1 Hijona et al. Cancer Cell International 2012, 12:5 http://www.cancerci.com/content/12/1/5 Cell line and culture To develop a rat model of HCC, we used diethylnitrosa- mine (DEN). This was administered by orogastric catheter, three times a week for 19 weeks. All these animals devel- oped HCC. The rats were divided into five groups with three experimental groups, corresponding to different drug regimens (Figure 1): The human hepatoma PLC cells were obtained from the ATCC. PLCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum, penicillin G (100 U/ml) and streptomycin (100 μg/ml). 1. CONTROL (C) Group (N = 10): free access to food and water for 19 weeks 1. CONTROL (C) Group (N = 10): free access to food and water for 19 weeks Background hepatocytes, anti-apoptotic signalling and the stimula- tion of angiogenesis-associated growth factors [7]. Hepatocellular carcinoma (HCC) is the fifth most com- mon malignancy worldwide. It ranks third place in the list of malignancies leading to death [1] and the incidence of HCC has increased in eastern Asia, Europe and the United States [2]. Clinically, HCC is characterized by its invasive- ness, poor prognosis and limited therapeutic opportu- nities. In many patients, HCC is diagnosed at an advanced stage. For these patients, the US Food and Drug Adminis- tration has approved the multikinase inhibitor, sorafenib [3,4]. In recent years, two studies have been published [5,6] which demonstrate that pravastatin increases the sur- vival of patients with advanced hepatocellular cancer alone or in combination with chemoembolization. Today, statins are regarded as attractive molecules and they may affect cancer. Statins, the 3-hydroxy-3-methyl- glutaryl coenzyme A (HMG-CoA) reductase inhibitors, are a class of drugs that inhibit the rate-limiting step in the cholesterol biosynthesis pathway, cholesterol being an important structural component of cell membranes. Various studies have been reported describing an asso- ciation of statins with either an increase or a decrease in the incidence of various cancers [8,9]. On the other hand, drug resistance is the major problem of che- motherapy, which causes treatment failure leading to progressive disease. Potential mechanisms of resistance include activation of the Ras/Raf/MEK/ERK signal trans- duction cascade [10] but also increased cholesterol levels in cancer cells [11]. The molecular pathogenesis of HCC is complex and involves the abnormal clonal expansion of dysplastic One of the potential mechanisms of action of statins is the modulation of the cell cycle through the down-regula- tion of cell cycle promoters such as cyclin D1-dependant Page 2 of 7 Hijona et al. Cancer Cell International 2012, 12:5 http://www.cancerci.com/content/12/1/5 Page 2 of 7 Hijona et al. Cancer Cell International 2012, 12:5 http://www.cancerci.com/content/12/1/5 (Promega, Spain). PLC cells were seeded onto 96-well plates, 10,000 cells/ml, and treated for 4 h. Following each treatment, 20 μl of dye solution was added to each well and incubated for 4 h. Subsequently, the absorbance was recorded at 490 nm using an ELISA plate reader. The pra- vastatin and sorafenib were used at concentrations of 50 μM. (Promega, Spain). PLC cells were seeded onto 96-well plates, 10,000 cells/ml, and treated for 4 h. Following each treatment, 20 μl of dye solution was added to each well and incubated for 4 h. Background Subsequently, the absorbance was recorded at 490 nm using an ELISA plate reader. The pra- vastatin and sorafenib were used at concentrations of 50 μM. kinase (CdK) and the up-regulation of cell cycle inhibitors p21 and p27 [12-15]. It has also been observed that they favour the regulation of homeostasis of the liver by increasing the expression of methionine adenosyltransfer- ase (MAT-1) and decrease cell proliferation by reducing the levels of the Proliferating Cell Nuclear Antigen (PCNA) [16-18]. They also inhibit the activity of matrix metalloproteinases (MMPs), especially of MMP-2 and MMP-9. Further, it has been reported that statins decrease the activity of MMP-9 by 75% [15]. This activity is directly related to tumour invasion and metastasis. Animal experiments All animal studies were performed in accordance with and approved by the Institutional Animal Care and Use Committee. We used Wistar male rats (Charles River, Spain) with initial body weights between 225 and 250 g. Histology f After sacrificing the animal, the liver was sectioned to allow macroscopic assessment of the hepatic lesions. In addition, several portions (1 cm3) of the liver were removed and fixed in 10% formaldehyde for 24 h. Sub- sequently, this tissue was processed: it was embedded in paraffin and 3 and 5 μm sections taken (Microtome, Leitz) which were fixed on slides and stained with hae- matoxylin and eosin. Samples were then mounted and examined under an optical microscope to characterise the lesions. The immunohistochemistry analysis revealed that the level of MAT1A was notably lower in the DEN group, while in the other groups it was significantly higher (p < 0.05) (Figure 5A). This difference was much more signif- icant in the groups given pravastatin than in the other animals (D 62%, P 94%, S 71%, P + S 91%) (p < 0.05) (Figure 5B). Pravastatin, sorafenib and the combination of thereof decrease the levels of transaminases The expression of PCNA (Cayman Chemical, Madrid) and MAT1A (bioNova científica, Madrid) in the liver tissue was measured using specific antibodies. For this, different tissue biopsies (1 cm3) were taken, fixed in 40 g/l of for- maldehyde buffer, embedded in paraffin and cut into 4 μm sections. Paraffin was removed with xylene, and samples were dehydrated with alcohol and subsequently used for immunohistochemical analysis. The Dako EnVision Sys- tem-HRP (DAB) was used for immunohistochemical staining, while quantification was carried out using the Genetix Ariol SL-50 system. We also observed significantly lower levels of GOT, GPT, GGT and alkaline phosphatise (ALP) in the three experimental groups. Moreover, this decrease was more significant in the pravastatin groups (D 619.5 U/L; 271 U/L; 58.35 U/L; 190 U/L) (P 117.5 U/L;43.5 U/L;7 U/ L;129 U/L) (S 147 U/L;59 U/L;23 U/L;172 U/L) (P + S 142 U/L;59.5 U/L;7 U/L;137 U/L) (p < 0.05) (Figure 6). CTRL PRAV SOR PRAV+SOR 0.0 0.5 1.0 1.5 Figure 2 Levels of proliferation in PLC cells. Hijona et al. Cancer Cell International 2012, 12:5 http://www.cancerci.com/content/12/1/5 Hijona et al. Cancer Cell International 2012, 12:5 http://www.cancerci.com/content/12/1/5 Hijona et al. Cancer Cell International 2012, 12:5 http://www.cancerci.com/content/12/1/5 Page 3 of 7 51% Abs (0.51), P + S 40% (Abs 0.4) compared to the untreated cells; p < 0.05) (Figure 2). 3. PRAVASTATIN (P) Group (N = 10): Idem HCC group, with the addition of a dose of 0.6 mg/kg/d of pravastatin given daily by orogastric catheter for 19 weeks. It was found that all the rats treated with DEN showed advanced HCC at both macro and microscopic levels. In rats treated with pravastatin, sorafenib or a combination thereof, the size of the tumours was signifi- cantly smaller and, at the microscopic level, the rats treated with pravastatin and pravastatin + sorafenib con- tained a smaller number of tumour cells (p < 0.05) while the sorafenib group had dysplastic nodules (Figure 3). 4. SORAFENIB (S) Group (N = 10): Idem HCC group, with the addition of a dose of 11.4 mg/kg/d of sorafenib daily by orogastric catheter. 5. SORAFENIB + PRAVASTATIN (P + S) Group (N = 10): Idem HCC group, with the addition of a com- bined dose of 0.6 mg/kg/d of pravastatin and 11.4 mg/ kg/d of sorafenib, administered as for P and S Groups. 5. SORAFENIB + PRAVASTATIN (P + S) Group (N = 10): Idem HCC group, with the addition of a com- bined dose of 0.6 mg/kg/d of pravastatin and 11.4 mg/ kg/d of sorafenib, administered as for P and S Groups. In the immunohistochemistry images, we note that the DEN group can be seen to have significantly higher PCNA levels than the other groups (Figure 4A). In the combined P + S Group levels were less elevated than in the other experimental groups (D 91.42%, P 80%, S 76.41%, P + S 72.72%) (Figure 4B)(p < 0.001). Statistical analysis The Chi Square test was used to determine the exis- tence of differences in the qualitative variables between the groups, while for quantitative variables ANOVA and Kruskal-Wallis tests were applied depending on the dis- tribution of variables. Multiple comparisons were carried out using the Tukey and Scheffé tests and/or the Mann Whitney test. A level of significance of p < 0.05 was selected. Cell proliferation 2. HCC (D) Group (N = 10): Idem control group but this group were administered DEN three times a week. 2. HCC (D) Group (N = 10): Idem control group but this group were administered DEN three times a week. Proliferation in cell culture was measured using the Cell- Titer 96 AQueous Non-radioactive cell proliferation assay PRAVAST ATIN CTRL CTRL 19 19 weeks weeks Standar Standar Diet Diet 19 19 weeks weeks Standar Standar Diet Diet PRAVASTATINA PRAVASTATINA SORAFENIB SORAFENIB 19 19 weeks weeks Standar Standar Diet Diet 19 19 weeks weeks Standar Standar Diet Diet PRAVASTATINA PRAVASTATINA SORAFENIB SORAFENIB PRAVASTATIN+ SORAFENIB SORAFENI B 19 19 weeks weeks Standar Standar Diet Diet DEN DEN Figure 1 Animal model. Standar Standar Diet Diet PRAVASTATINA PRAVASTATINA 19 19 weeks weeks Standar Standar Diet Diet Standar Standar Diet Diet 19 19 weeks weeks Figure 1 Animal model. Pravastatin inhibis proliferation in PLC cells We observed that cell proliferation was considerably lower in all the three experimental groups with the reduction being the most clear in the group adminis- tered pravastatin and sorafenib (P 82% (Abs 0.82), S Hijona et al. Cancer Cell International 2012, 12:5 http://www.cancerci.com/content/12/1/5 Page 4 of 7 Discussion In spite of the promising results in the treatment of HCC, it still is a disease with poor prognosis, therefore it is necessary to search for new drugs. Moreover, the use of chemotherapy in patients suffering from chronic liver disease is associated with a high rate of severe adverse effects and even death directly linked to treatment. Experimental [18] and epidemiology [19] data have DEN DEN PRAV SOR PRAV+SOR SOR PRAV PRAV+SOR Figure 3 Macro and microscopic images of livers from each of the groups. 60 80 100 e nucleous DEN DEN PRAV PRAV SOR SOR PRAV PRAV--SOR SOR B A DEN DEN PRAV SOR PRAV+SOR SOR PRAV PRAV+SOR Figure 3 Macro and microscopic images of livers from each of the groups. Discussion In spite of the promising results in the treatment of HCC, it still is a disease with poor prognosis, therefore it is necessary to search for new drugs. Moreover, the use of chemotherapy in patients suffering from chronic liver disease is associated with a high rate of severe adverse effects and even death directly linked to treatment. Experimental [18] and epidemiology [19] data have DEN DEN PRAV SOR PRAV+SOR SOR PRAV PRAV+SOR Figure 3 Macro and microscopic images of livers from each of the groups. DEN DEN PRAV SOR PRAV+SOR SOR PRAV PRAV+SOR Figure 3 Macro and microscopic images of livers from each of the groups. Figure 3 Macro and microscopic images of livers from each of the groups. chemotherapy in patients suffering from chronic liver disease is associated with a high rate of severe adverse effects and even death directly linked to treatment. Experimental [18] and epidemiology [19] data have Discussion In spite of the promising results in the treatment of HCC, it still is a disease with poor prognosis, therefore it is necessary to search for new drugs. Moreover, the use of DEN PRAV SOR PRAV-SOR 0 20 40 60 80 100 % positive nucleous DEN DEN PRAV PRAV SOR SOR PRAV PRAV--SOR SOR B A Figure 4 Expression of PCNA in the different groups. DEN DEN PRAV PRAV SOR SOR PRAV PRAV--SOR SOR A A DEN PRAV SOR PRAV-SOR 0 20 40 60 80 100 % positive nucleous B Figure 4 Expression of PCNA in the different groups. B Figure 4 Expression of PCNA in the different groups. DEN PRAV SOR PRAV-SOR 0 50 100 150 MAT1A levels DEN PRAV SOR PRAV+SOR A B Figure 5 Expression of MAT1A in the different groups. Hijona et al. Cancer Cell International 2012, 12:5 http://www.cancerci.com/content/12/1/5 Page 5 of 7 Page 5 of 7 Hijona et al. Cancer Cell International 2012, 12:5 http://www.cancerci.com/content/12/1/5 Hijona et al. Cancer Cell International 2012, 12:5 http://www.cancerci.com/content/12/1/5 DEN PRAV SOR PRAV+SOR A A DEN PRAV SOR PRAV-SOR 0 50 100 150 MAT1A levels B Figure 5 Expression of MAT1A in the different groups. B Figure 5 Expression of MAT1A in the different groups. suggested that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) may have potential as che- mopreventive agents in cancer. Observational data have also indicated that statins may have protective effects against the development of cancer, for example, modify- ing the risk of oesophageal adenocarcinoma in patients with existing Barrett’s oesophagus [20]. Two studies [5,6] published in recent years have shown that pravastatin improved survival of patients with advanced hepatocarci- noma. In our study, we found that pravastatin decreases the proliferation of hepatocellular carcinoma cell lines. This finding was then confirmed in a rat model of hepa- tocarcinoma. Specifically, it was observed that the num- ber of nodules of hepatocarcinoma was lower in rats treated with pravastatin. This small number was also associated with a relatively lower level of serum transaminases. this study, we observed that pravastatin decreased the proliferation of hepatocellular carcinoma cell lines. Immunohistochemical staining of proliferating cell nuclear antigen (PCNA) was notably lower in pravasta- tin group. Expression of proliferating cell nuclear anti- gen by cells during the S and G2 phases of the cell cycle makes the protein a good cell-proliferation marker [22]. Discussion U/L GGT AST DEN 0.5LXV PRAVASTATINA 0.6mg/kg/d SORAFENIB 11.4mg/kg/d PRAVASTATINA + SORAFENIB 0 200 400 600 800 1000 U/L GGT U/L AST DEN 0.5LXV PRAVASTATINA 0.6mg/kg/d SORAFENIB 11.4mg/kg/d PRAVASTATINA + SORAFENIB 0 200 400 600 800 1000 ALT DEN 0.5LXV PRAVASTATINA 0.6mg/kg/d SORAFENIB 11.4mg/kg/d PRAVASTATINA + SORAFENIB 0 100 200 300 400 U/L ALT DEN 0.5LXV PRAVASTATINA 0.6mg/kg/d SORAFENIB 11.4mg/kg/d PRAVASTATINA + SORAFENIB 0 100 200 300 400 U/L GGT DEN 0.5LXV PRAVASTATINA 0.6mg/kg/d SORAFENIB 11.4mg/kg/d PRAVASTATINA + SORAFENIB 0 50 100 150 Figure 6 Levels of AST, ALT, GGT and ALP in the different groups. GGT AF DEN 0.5LXV PRAVASTATINA 0.6mg/kg/d SORAFENIB 11.4mg/kg/d PRAVASTATINA + SORAFENIB 0 50 100 150 200 250 U/L U/L Figure 6 Levels of AST, ALT, GGT and ALP in the different groups. treatment effectively inhibited the production of several pro-inflammatory/pro-angiogenic mediators involved in inflammation and angiogenesis in vitro studies [24]. from rats, in which MAT1A expression is low. This expression is reactivated in the human hepatoma cell line HepG2 treated with 5-aza-2’-deoxycytidine or the histone deacetylase inhibitor trichostatin, suggesting a role for DNA hypermethylation and histone deacetyla- tion in MAT1A silencing [23]. We observed a significant increase in the expression of MAT1A, suggesting that pravastatin has a protective effect against tumour progression. from rats, in which MAT1A expression is low. This expression is reactivated in the human hepatoma cell line HepG2 treated with 5-aza-2’-deoxycytidine or the histone deacetylase inhibitor trichostatin, suggesting a role for DNA hypermethylation and histone deacetyla- tion in MAT1A silencing [23]. We observed a significant increase in the expression of MAT1A, suggesting that pravastatin has a protective effect against tumour progression. Sorafenib, a multikinase inhibitor, increases survival of patients with advanced hepatocellular carcinoma [25]. In one study, median overall survival was 10.7 months in the sorafenib group and 7.9 months in the placebo group [26]. For this reason, one of our objectives was to compare the effectiveness in vitro and in vivo of pravastatin for the treatment of hepatocarcinoma. We observed that the com- bination of pravastatin and sorafenib in vitro, considerably decreased cell proliferation and the expression of MAT1A in vivo. The results were confirmed in vivo. In particular, the combination of pravastatin and sorafenib resulted in a smaller number and size of hepatocarcinoma lesions, com- pared to the administration of the two drugs separately. Discussion This protein is located in the nucleus and favours the synthesis of DNA. Another mechanism of action of pravastatin is to cause a decrease in the expression of Methionine Ade- nosyltransferase (MAT). MAT is the enzyme that cata- lyzes the synthesis of S-adenosylmethionine (AdoMet), the main donor of methyl groups in the cell [23]. In mammals MAT is the product of two genes, MAT1A and MAT2A. MAT1A promoter is hypomethylated in liver and hypermethylated in kidney and foetal rat hepa- tocytes, indicating that this modification is tissue specific and developmentally regulated. Southern blot analysis with a MAT1A promoter probe demonstrated that MAT1A expression is linked to elevated levels of chro- matin acetylation. Early changes in MAT1A methylation are already observed in precancerous cirrhotic livers The role of statins does extend beyond their lipid-low- ering effects, as they are known to improve endothelial function, participate in plaque stabilisation, immunomo- dulation, antioxidant activity, and also act as anti- inflammatory and anticancer agents. These properties have made statins particularly attractive drugs [21]. In Hijona et al. Cancer Cell International 2012, 12:5 http://www.cancerci.com/content/12/1/5 Page 6 of 7 Hijona et al. Cancer Cell International 2012, 12:5 http://www.cancerci.com/content/12/1/5 Page 6 of 7 U/L U/L GGT DEN 0.5LXV PRAVASTATINA 0.6mg/kg/d SORAFENIB 11.4mg/kg/d PRAVASTATINA + SORAFENIB 0 50 100 150 AST DEN 0.5LXV PRAVASTATINA 0.6mg/kg/d SORAFENIB 11.4mg/kg/d PRAVASTATINA + SORAFENIB 0 200 400 600 800 1000 Figure 6 Levels of AST, ALT, GGT and ALP in the different groups. U/L U/L AF DEN 0.5LXV PRAVASTATINA 0.6mg/kg/d SORAFENIB 11.4mg/kg/d PRAVASTATINA + SORAFENIB 0 50 100 150 200 250 GGT DEN 0.5LXV PRAVASTATINA 0.6mg/kg/d SORAFENIB 11.4mg/kg/d PRAVASTATINA + SORAFENIB 0 50 100 150 U/L U/L AST DEN 0.5LXV PRAVASTATINA 0.6mg/kg/d SORAFENIB 11.4mg/kg/d PRAVASTATINA + SORAFENIB 0 200 400 600 800 1000 ALT DEN 0.5LXV PRAVASTATINA 0.6mg/kg/d SORAFENIB 11.4mg/kg/d PRAVASTATINA + SORAFENIB 0 100 200 300 400 Figure 6 Levels of AST, ALT, GGT and ALP in the different groups. Hijona et al. Cancer Cell International 2012, 12:5 http://www.cancerci.com/content/12/1/5 Hijona et al. Cancer Cell International 2012, 12:5 http://www.cancerci.com/content/12/1/5 These facts open new doors for combination treatments for advanced hepatocarcinoma. The great cumulative experience in the use of statins in clinical practice is a very considerable advantage, facilitating the development of clinical trials to assess any increase in survival when com- bining these two drugs. 9. Wong WW, Dimitroulakos J, Minden MD, Penn LZ: HMG-CoA reductase inhibitors and the malignant cell: the statin family of drugs as triggers of tumor-specific apoptosis. Leukemia 2002, 16(4):508-519. 10. Weinstein-Oppenheimer CR, Henríquez-Roldán CF, Davis JM, Navolanic PM, Saleh OA, Steelman LS, Franklin RA, et al: Role of the Raf signal transduction cascade in the in vitro resistance to the anticancer drug doxorubicin. Clin Cancer Res 2001, 7:2898-2907. The most severe limitation of this study is that we cannot know whether the response in humans would be the same as in in vitro and in vivo models. Further, it is not possible to assess whether or not the adverse effects may strengthen with the use of both drugs. 11. Banker DE, Mayer SJ, Li HY, Willman CL, Appelbaum FR, Zager RA: Cholesterol synthesis and import contribute to protective cholesterol increments in acute myeloid leukaemia cells. Blood 2004, 104:1816-1824. 12. Sleijfer S, van der Gaast A, Planting AS, Stoter G, Verweij J: The potential of statins as part of anti- cancer treatment. Eur J Cancer 2005, 41:516-522. To conclude, we observe that pravastatin, alone or in combination with sorafenib has substantial antiprolifera- tive effects in hepatocellular carcinoma cell lines in in vitro and in vivo models of hepatocarcinoma. Studies on humans are required to confirm these findings. Pravastatin decreases cell proliferation in in vitro and in vivo models, the combination of pravastatin and sorafenib being more effective than the administration of sorafenib alone. 13. Tatsuta M, Iishi H, Baba M, Iseki K, Yano H, Uehara H, Yamamoto R, et al: Suppression by pravastatin, an inhibitor of p21ras isoprenylation, of hepatocarcinogenesis induced by N-nitrosomorpholine in Sprague- Dawley rats. Br J cancer 1998, 77:581-587. y 14. Paragh G, Kertai P, Kovacs P, et al: HMG Coa reductase inhibitor fluvastatin arrests the development of implanted hepatocarcinoma in rats. Anticancer Res 2003, 23:3949-3954. 15. Luan Z, Chase AJ, Newby AC: Statins inhibit secretion of metalloproteinases-1,-2, -3, and -9 from vascular smooth muscle cells and macrophages. Arterioscler Thromb Vasc Biol 2003, 23:769-775. 16. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. 25. Coimbra M, Banciu M, Fens MH, de Smet L, Cabaj M, Metselaar JM, et al: Liposomal pravastatin inhibits tumor growth by targeting cancer-related inflammation. J Control Release 2010, 148(3):303-310. Received: 16 November 2011 Accepted: 21 February 2012 Published: 21 February 2012 Published: 21 February 2012 Published: 21 February 2012 26. Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, et al: Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 2008, 359:378-390. 26. Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, et al: Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 2008, 359:378-390. Authors’ contributions EH, LH and LB have designed the project. In vitro determination have been performed by JMB, JFM, MH and EH. In vivo determination have been performed by LH, PA, JA, LH and EH. Histology study have been performed by LH, JMB, PA and LH. Statistical study have been performed by LH, LB and EH. All authors write the manuscript. All authors read and approved the final manuscript. 21. Gauthaman K, Fong CY, Bongso A: Statins, Stem cells, and cancer. J Cell Biochem 2009, 106:975-983. 21. Gauthaman K, Fong CY, Bongso A: Statins, Stem cells, and cancer. J Cell Biochem 2009, 106:975-983. 22. Johnson DG, Walker CL: Cyclins and cell cycle checkpoints. Annu Rev Pharmacol Toxicol 1999, 39:295-312. 22. Johnson DG, Walker CL: Cyclins and cell cycle checkpoints. Annu Rev Pharmacol Toxicol 1999, 39:295-312. 23. Torres L, Avila MA, Carretero MV, Latasa MU, Caballería J, López-Rodas G, et al: Liver-specific methionine adenosyltransferase MAT1A gene expression is associated with a specific pattern of promoter methylation and histone acetylation: implications for MAT1A silencing during transformation. FASEB J 2000, 14(1):95-102. p Grant support In addition, this work was supported by grants from the Department of Health of the Basque Government 2009/111003. 24. Malagari K: Drug-eluting particles in the treatment of HCC: chemoembolization with doxorubicin-loaded DC Bead. Expert Rev Anticancer Ther 2008, 8:1643-1650. Author details 1 1Department of Gastroenterology, Donostia Hospital, Instituto Biodonostia, University of the Basque Country EHU/UPV, Ciberehd, San Sebastián, Spain. 2Department of Hepatology, University of Navarra, CIMA, Ciberehd, Pamplona, Spain. 19. Karp I, Behlouli H, Lelorier J, Pilote L: Statins and cancer risk. Am J Med 2008, 121:302-309. 19. Karp I, Behlouli H, Lelorier J, Pilote L: Statins and cancer risk. Am J Med 2008, 121:302-309. 20. Nguyen DM, Richardson P, El-Serag HB: Medications (NSAIDS, statins, proton pump inhibitors) and the risk of esophageal adenocarcinoma in patients with Barrett’s esophagus. Gastroenterology 2010, 138:2260-2266. 20. Nguyen DM, Richardson P, El-Serag HB: Medications (NSAIDS, statins, proton pump inhibitors) and the risk of esophageal adenocarcinoma in patients with Barrett’s esophagus. Gastroenterology 2010, 138:2260-2266. Hijona et al. Cancer Cell International 2012, 12:5 http://www.cancerci.com/content/12/1/5 Laezza C, Mazziotti G, Fiorentino L, Gazzerro P, Portella G, Gerbasio D, et al: HMG-CoA reductase inhibitors inhibit rat propylthiouracil-induced goiter by modulating the ras-MAPK pathway. J Mol Med 2006, 84:967-973. 16. Laezza C, Mazziotti G, Fiorentino L, Gazzerro P, Portella G, Gerbasio D, et al: HMG-CoA reductase inhibitors inhibit rat propylthiouracil-induced goiter by modulating the ras-MAPK pathway. J Mol Med 2006, 84:967-973. Discussion As well as decreasing levels of PCNA and MAT1A, sorafenib also decreased the expression of Mcl-1 messenger RNA and protein, transcriptional targets of STAT3, as well as sensitizing neoplastic cells to tumour necrosis factor- related apoptosis-inducing ligand (TRAIL)-mediated apop- tosis [26]. In addition, sorafenib produces inhibition of the expression of phospho-MEK, phospho-ERK, cyclin D1, Rb and anti-apoptotic proteins Bcl-xl and Mcl-1 [27,28]. The inhibition of the products derived from mevalo- nate may be another mechanism by which pravastatin affects cell proliferation, differentiation and apoptosis. Other authors [18] have reported how the statins inhibit proliferation and induce apoptosis in oesophageal ade- nocarcinoma cells via inhibition of Ras farnesylation and inhibition of the extracellular signal-regulated kinases and Akt signalling pathways. Pravastatin reduced viable cell numbers and inhibited proliferation in a similar dose-dependent manner. Statins induced apoptosis and enhanced the antiproliferative effect of NS-398, a selec- tive cyclooxygenase (COX)-2 inhibitor, while statin treatment also increased messenger RNA (mRNA) and protein expression of the proapoptotic proteins Bax and Bad. Recently, it has also been observed that pravastatin Page 7 of 7 Hijona et al. Cancer Cell International 2012, 12:5 http://www.cancerci.com/content/12/1/5 g We thank Mariasun Zabala and Jose Ignacio Martinez for technical assistance. We thank Mariasun Zabala and Jose Ignacio Martinez for technical assistance. We thank Mariasun Zabala and Jose Ignacio Martinez for technical assistance. y g y 17. Chodon D, Banu SM, Padmavathi R, Sakthisekaran D: Inhibition of cell proliferation and induction of apoptosis by genistein in experimental hepatocellular carcinoma. Mol Cel Biochem 2007, 297:73-80. y y 17. Chodon D, Banu SM, Padmavathi R, Sakthisekaran D: Inhibition of cell proliferation and induction of apoptosis by genistein in experimental hepatocellular carcinoma. Mol Cel Biochem 2007, 297:73-80. CIBERehd is funded by the Carlos III Health Institute. 18. Ogunwabi OO, Beales IL: Statins inhibit proliferation and induce apoptosis in Barrett’s esophageal adenocarcinoma cells. Am J Gastroenterol 2008, 103:825-837. 18. Ogunwabi OO, Beales IL: Statins inhibit proliferation and induce apoptosis in Barrett’s esophageal adenocarcinoma cells. Am J Gastroenterol 2008, 103:825-837. References 1. Parkin DM, Bray F, Ferlay J, Pisan P: Estimating the world cancer burden: Globocan 2000. Int J Cancer 2001, 94:143-156. 1. Parkin DM, Bray F, Ferlay J, Pisan P: Estimating the world cancer burden: Globocan 2000. Int J Cancer 2001, 94:143-156. 27. Hikita H, Takehara T, Shimizu S, Kodama T, Shigekawa M, Iwase K, et al: The Bcl-xL inhibitor, ABT-737, efficiently induces apoptosis and suppresses growth of hepatoma cells in combination with sorafenib. Hepatology 2010, 52(4):1310-1321. 2. El-Serag HB, Mason AC: Rising incidence of hepatocellular carcinoma in the United States. N Engl J Med 1999, 340:745-750. 2. El-Serag HB, Mason AC: Rising incidence of hepatocellular carcinoma in the United States. N Engl J Med 1999, 340:745-750. 3. Llovet JM, Burroughs A, Bruix J: Hepatocellular carcinoma. Lancet 2003, 362:1907-1917. 28. Lu X, Tang X, Guo W, Ren T, Zhao H: Sorafenib induces growth inhibition andapoptosis of human chondrosarcoma cells by blocking the RAF/ERK/ MEK pathway. J Surg Oncol 2010, 102(7):821-826. 4. Lang L: FDA approves sorafenib for patients with inoperavle liver cancer. Gastroenterology 2008, 134:379. doi:10.1186/1475-2867-12-5 Cite this article as: Hijona et al.: Pravastatin inhibits cell proliferation and increased MAT1A expression in hepatocarcinoma cells and in vivo models. Cancer Cell International 2012 12:5. 5. Kawata S, Yamasaki E, Nagase T, Inui Y, Ito N, Matsuda Y, et al: Effect of pravastatin on survival in patients with advanced hepatocellular carcinoma. A randomised controlled trial. Br J Cancer 2001, 84:886-891. 6. Graf H, Jüngst C, Straub G, Dogan S, Hoffmann RT, Jakobs T, et al: Chemoembolization combined with pravastatin improves survival in patients with hepatocellular carcinoma. Digestion 2008, 78:34-38.
https://openalex.org/W4289450326	https://revistas.udec.cl/index.php/ran/article/download/7387/7202/19018, https://www.redalyc.org/journal/5608/560872306008/560872306008.pdf	es		Transformaciones del sistema monetario y financiero en la nueva etapa de desarrollo del capitalismo mundial	RAN	2,022	cc-by	15,200	R.A.N. Vol. 8 (2) 2022 ran.udec.cl Artículo de Investigación Transformaciones del sistema monetario y ﬁnanciero en la nueva etapa de desarrollo del capitalismo mundial Transformations of the monetary and ﬁnancial system in the new stage of development of world capitalism José Vargas Mendoza 1* Universidad Nacional Autónoma de México varmoren@comunidad.unam.mx Alfonso Hernández Estrada1 Universidad Nacional Autónoma de México ahdeze@comunidad.unam.mx Resumen Propósito: Se estudian las transformaciones del sistema monetario y financiero internacional en el marco de la nueva etapa del capitalismo contemporáneo. Diseño/metodología: A través del uso de variables productivas y financieras de los países tomados como referencia, se muestra el impacto de las transformaciones que se están operando en las relaciones de fuerza entre las potencias a nivel mundial y a nivel regional, y su proyección monetaria y financiera. Implicaciones: Los resultados de estas transformaciones productivas muestran que tienen impactos en la dinámica del sistema monetario y financiero internacional. Originalidad: Esta investigación explora las transformaciones que se producen en el ámbito monetario y financiero a nivel internacional cuando cambia la base tecno-productiva de las economías nacionales. Abstract Purpose: the international monetary and financial system transformations are studied within the framework of the new stage of contemporary capitalism. Design/methodology: Using the productive and financial variables of the reference countries, it is shown the impact of the transformations in the power relationships between both the World and regional levels in their monetary and financial projections. Results: It is found that productive transformations have an impact on the international monetary and financial system dynamics. Originality: This research explores the transformations that occur in the monetary and financial spheres at the international level when the national economies’ techno-productive base changes. * Autor corresponsal 1 Universidad Nacional Autónoma de México, Facultad de Economía, Circuito interior s/n Coyoacán, C.P. 04510, Ciudad Universitaria, MÉXICO INFORMACIÓN ARTÍCULO Recibido: 4 de Abril 2022 Aceptado: 2 de Julio 2022 Palabras Claves: personalidad de marca Marca Supermercados Dimensiones de personalidad Rasgos de personalidad. ARTICLE INFO Received: 4 April 2022 Accepted: 2 July 2022 Keywords: Brand personality Brand Supermarkets Personality dimensions Personality traits 279 ISSN: 0719-7713 / 0719-6245 © Universidad de Concepción https://doi.org/10.29393/RAN8-21TSJA20021 Revista Academia & Negocios Vol. 8 (2) 2022 pp. 279 - 306 INTRODUCCIÓN En las siguientes páginas se plantean las transformaciones que se han operado en el sistema monetario y financiero internacional en el marco de la nueva fase de desarrollo del capitalismo que se abrió en el mundo hacia mediados de los años ochenta del siglo XX. También se intenta explicar por qué este sistema monetario y financiero no es el que corresponde a esta nueva fase de desarrollo que tiene requerimientos estructurales que no pueden cumplirse por la forma en que las finanzas globales se desenvuelven en la actualidad. Asimismo, se plantea que es un componente fundamental de las profundas transformaciones hacia un mundo multipolar que viene desplegándose en el siglo XXI, el cual trastoca el fuerte sesgo que le ha impreso la hegemonía estadounidense desde mediados del siglo XX. Esta nueva fase se desenvuelve en el marco de un sistema crediticio financiarizado, donde los circuitos financieros muestran un contraste de mayor crecimiento ante los sectores productivos, tal como se explica en este artículo. El manuscrito hace énfasis en aspectos teóricos sobre el sistema monetario y financiero internacional, al tiempo que también muestra con cierta información empírica las tendencias del fenómeno estudiado. Este artículo se divide en cuatro apartados, el primero con una breve introducción histórica, el segundo hace énfasis en los aspectos crecientemente especulativos del sistema monetario y financiero actual, en el tercer apartado, se analizan las principales características del sistema monetario y financiero internacional y en el cuarto apartado, se contrasta la vieja institucionalidad emanada de Bretton Woods con la nueva institucionalidad impulsada por los países emergentes, principalmente por China. ANTECEDENTES Como se ha documentado en diversos estudios, durante la etapa fordista-keynesiana que abarcó propiamente de los años cuarenta hasta principios de los años ochenta del siglo XX (Ordóñez, 1996; Rivera, 1992), el capitalismo mundial se expandió durante el lapso que abarca de 1948 a 1974, siendo interrumpido por la crisis mundial de 1974-1975 que puso fin a esa etapa de expansión donde predominó el complejo tecnológico auto280 motriz-acerero y petrolero, que fue la base material de la estructura productiva de las naciones industriales y emergentes que al articularse con el método de producción de plusvalor basado en el fordismo, hicieron que esas naciones crecieran a tasas que no se volvieron a ver después de los años setenta. Al mismo tiempo, el sistema monetario y financiero internacional se movió con relativa estabilidad, porque dicho período se caracterizó también por el crecimiento de la productividad laboral que repercutió favorablemente sobre el crecimiento económico. Por ejemplo, entre 1950 y 1975 la productividad del trabajo en los Estados Unidos de América (EUA) creció a un ritmo promedio anual de 2.8%, en la Alemania Occidental lo hizo en 5.4%, Francia 5%, mientras que el país más exitoso en esta materia fue Japón que creció a una tasa media anual de 8.3% (Brenner, 1986). Como resultado del crecimiento de la productividad laboral y del dinamismo económico que experimentaron todas las naciones capitalistas, la tasa de ganancia creció en 11% durante la llamada edad de oro del capitalismo en el mundo (19501965), tal como lo señala Roberts (2020). Como consecuencia de lo anterior, la tasa de ahorro interno comenzó a elevarse y condujo a la restitución del sistema de crédito internacional hacia mediados de los años sesenta, el cual se había prácticamente paralizado desde los años treinta (Dabat, 1980), producto de la crisis histórica que vivió el capitalismo en el mundo durante esos años. Como resultado de las contradicciones económicas que se fueron acumulando a lo largo del tiempo, el orden monetario y financiero internacional, emanado de los acuerdos de Bretton Woods de julio de 1944, comenzó a agrietarse hacia 1967 cuando se devaluó la libra esterlina y luego en 1968 el franco francés, que eran las primeras manifestaciones del agotamiento de la etapa fordista-keynesiana en el mundo, y se expresaron con la crisis de la economía norteamericana que era el eje de ese orden monetario-financiero, que tuvo que romper con la paridad dólar-oro el 15 de agosto de 1971, y luego se estableció de facto la flotación generalizada de las monedas con el acuerdo entre los países industriales de marzo de 1973 (Lelart, 1996). Esto crea en los hechos un nuevo orden monetario-financiero en el mundo, donde el Estado ya no tendría injerencia en la determinación de los tipos de cambio, cuestión que fue reconocida oficialmente por el FMI en los acuer- Transformaciones del sistema monetario… / Vargas y Hernández dos de Jamaica de 1976 en el marco de su reunión anual realizada en la ciudad de Kingston. Posteriormente, en 1973 se inició la desregulación financiera con Canadá a la cabeza de este proceso, continuada por Alemania y Suiza. Luego, el primero de enero de 1974 lo hizo EUA, cuando abolió todas las restricciones a los movimientos internacionales de capital, Gran Bretaña lo hizo en 1979, Japón en 1980, Francia e Italia en 1990, España y Portugal en 1992 (Eatwell y Taylor, 2005). México inició tal proceso de desregulación bancaria en marzo de 1986 cuando eliminó el control estatal sobre las tasas de interés y los plazos asociados a los préstamos. La apertura del mercado de valores la inició en octubre de 1988 y la culminó en 1993 con la reforma a la Ley de Inversiones Extranjeras, al igual que eliminó el tipo de cambio semi-fijo el 19 de diciembre de 1994 y comenzó a abrir su sistema bancario a la competencia internacional en octubre de 1988, culminando dicho proceso en 1999, cuando se vendieron los principales bancos del Estado a los particulares (Vargas, 2013). Con estas acciones de los Estados nacionales, se abría la era del orden monetario y financiero abocado a la especulación, porque a partir de la desregulación y la apertura financiera, el mercado será el encargado de determinar los tipos de cambio y las tasas de interés, con lo cual se pasa de las economías que tenían control sobre esas variables monetarias y financieras, a economías que ya no tendrán ese control. Se abría con ello el sistema monetario y financiero especulativo internacional en oposición a lo establecido durante el orden mundial que emergió de la segunda posguerra. Es importante precisar que en cada etapa del capitalismo mundial se transforman los diversos ámbitos que conforman la estructura productiva y circulatoria del sistema y del entramado socio-institucional (Leal, 2015). Al producirse una transformación radical de la estructura productiva y de las instituciones económicas, políticas, sociales, culturales, producto de revoluciones tecnológicas, etc., implica una transformación del espacio económico, de los sistemas financieros nacionales y a nivel internacional. Lo mismo ocurrió en el marco de la etapa imperialista clásica, cuando el capital financiero tendió a acentuar los procesos de concentración y centralización del ca1 pital, mediante la construcción de grandes imperios coloniales que constituyeron empresas multinacionales y el surgimiento de grandes grupos financieros (Hardt y Negri, 2000; Harvey, 2004; Hobson, 1981; Hilferding, 1971; Lenin, 1960; Bujarin, 1979; Luxemburgo, 1981). Para estos grupos financieros, su principal fuente de financiamiento fue el sistema bancario, constituyéndose como el principal canalizador del ahorro social hacia la producción junto con las bolsas de valores. El capital crediticio (banca comercial y de inversión, mercado de capitales, cajas de ahorro, aseguradoras, etc.) era fundamental para el desarrollo productivo y la acumulación capitalista, en una lógica de subordinación del capital crediticio al productivo. Aunque en determinados momentos, el primero se independizaba relativamente del segundo (burbujas financieras), generando crisis recurrentes. El rasgo principal, a largo plazo, era que la producción mantenía el comando del proceso sobre el capital financiero, en una lógica contradictoria determinada por las fluctuaciones del ciclo económico y los cambios tecnológicos. En esta fase del capitalismo monopolista-financiero clásico surgen tres fenómenos en los principales países capitalistas de ese entonces, con desigual desarrollo espacial y temporal. a) Surge el capital por acciones, la Bolsa de Valores y la tendencia de la gran banca de inversiones que controla la gran industria en algunos países (Europa, EUA y nuevas formas de gobernanza corporativa) (Hilferding, 1971; Oman y Blume, 2005); b) un proceso de internacionalización financiera centrado en Nueva York como gran centro financiero mundial que tiende a sobrepasar a Londres. Este proceso de financiarización del capital crediticio se derrumbó en la gran crisis de 1929 (Grossman, 2000); y c) el paulatino predominio de la banca pública o regulada y la inversión corporativa sobre las finanzas privadas hasta pasada la Segunda Guerra Mundial, lo cual originó el surgimiento de la Ley Glass-Steagal (1933), que separó drásticamente la banca de depósito de la de inversión (Bolsas de valores) supeditando formalmente al sector bancario a las necesidades de la actividad industrial1, que a En el plano teórico la crisis de 1929-33 y las dificultades del nuevo capitalismo industrial-financiero llevaron a teorías del estancamiento secular del capitalismo en diferentes versiones (Hansen, 1939, Steindl, 1952) las cuales cayeron en desuso, pero la primera se reformula (Summers, 2020); y ha corrido peculiar suerte otra teoría heterodoxa (Grossman, 1929) previa a las dos mencionadas, que algunos asimilan a los prolegómenos de las teorías de las ondas largas (ver Rodríguez, 2005). 281 Revista Academia & Negocios Vol. 8 (2) 2022 pp. 279 - 306 nivel productivo lo acompañaron altos niveles de autofinanciamiento de las grandes corporaciones en EUA (Baran-Sweezy,1968). En el plano monetario culminó el declive definitivo de la libra esterlina y el ascenso del dólar como moneda dominante a nivel internacional (Eichengreen y Flandreau, 2008). En las condiciones actuales, el poder monetario y financiero internacional se ejerce a través de la denominada Troika (FMI, Banca Central Europea y el Banco Mundial) cuya expresión en inglés se llama Unholy Trinity, que puede traducirse como trinidad profana (Peet, 2005), y no menos importante es el papel del Banco de Pagos Internacionales (Bank for International Settlements, BIS), donde su función consiste en regular a los bancos centrales de Occidente, cumpliendo tras bambalinas un papel político regulador de acuerdos estratégicos de largo alcance como los tomados durante la Segunda Guerra Mundial para financiar a diferentes partes beligerantes (Dish México, 2021). EL NUEVO SISTEMA MONETARIO Y FINANCIERO ESPECULATIVO INTERNACIONAL Los procesos de desfinanciarización temporal del capitalismo tendieron a desaparecer hacia finales de la década de los sesenta, sobrepasando el corsé legal de la Ley Glass-Steagall con los préstamos sindicados, la estanflación de los años setenta y los procesos de desintermediación bancaria anteriores, los cuales dieron paso al capitalismo financiero actual. El nuevo sistema financiero, promovido por el neoliberalismo, se asoció a la desregulación general de los mercados, el cual sustituyó progresivamente al crédito bancario regulado por la desregulación y liberalización financieras, los fondos especulativos, las operaciones con derivados y la crisis mundial de 2008-2009. La década de los setenta marcó el fin de una etapa, con cambios institucionales que favorecieron el despliegue inicial de la revolución informática y de las telecomunicaciones en EUA, como respuesta a la pérdida de competitividad productiva internacional de esa nación ante Japón y Alemania, y se expresaron en la caída de la rentabilidad en todas las economías capitalistas2 y en la estanflación desde 1974-1975 (Yaffe y Bullock, 1978). A partir de entonces, EUA ingresaría a un proceso continuo de déficits de cuenta corriente y caída del ahorro interno, los que junto al shock petrolero de 1973 formaron el mercado del eurodólar que inundó a Europa y a los países exportadores de petróleo. Además, se rompen los tratados de Bretton Woods y es el fin de la convertibilidad directa del dólar con respecto al oro, lo cual contribuyó al crecimiento explosivo de la liquidez internacional, la crisis inflacionaria de los años setenta y la posterior irrupción del neoliberalismo orientado hacia la recuperación de la rentabilidad capitalista. En tanto, la revolución de la informática tiende a reducir costos internacionales de producción y circulación, con especial relevancia en el ámbito financiero por la banda ancha de internet, los softwares y servidores que controlan las operaciones bancarias y financieras de forma continua las 24 horas del día en tiempo real, el big data permite el manejo de algoritmos de encriptación de las redes de seguridad financiera que realizan millones de operaciones por segundo, la Red Profunda o Deep Web y las cadenas blockchain que han posibilitado el surgimiento de las criptomonedas y el crédito virtual (Bouveret y Vikram, 2018). A nivel financiero, la respuesta neoliberal a la crisis inflacionaria de 1972-1981 fue la implantación de un nuevo sistema financiero desregulado y liberalizado (Correa, 1998) que a partir del angel capital (capitales de riesgo a altas tasas de interés), contribuyó al arranque de la actual revolución tecnológica y sobre todo, a la sobreinversión sectorial que condujo a la crisis bursátil punto. com de 2000-2001 y a la ingeniería financiera, al aumento de empresas offshore. Estos procesos con los nuevos instrumentos financieros: los derivados3, estallaron en la gran crisis de 2008-2009 y la aparición pública de instituciones formadas años atrás llamada la banca en la sombra (Sha- 2 Caídas estimadas de la tasa de ganancia en EUA del 8.3% en 1965, a 7.7% en 1966-67 y 5.5% en 1971-1973 (Nordhaus, 1974); y del 7% hacia 19982000, a 5.7% en 2008, al 2% en 2009, recuperación al 4.8% en 2014-2106 y nueva caída al 4% en 2019-2020 (Roberts, 2020). 3 Los derivados ya no son activos financieros (créditos), sino valores indirectos derivados de otros activos “subyacentes” de los cuales depende su precio, pueden ser acciones, títulos, índices bursátiles, precios futuros de commodities o dinero (divisas, tasas de interés, bonos) o seguros contra créditos –públicos o privados- no pagados (los más difundidos fueron los CDS, Credit Default Swaps), mediante actos de apuesta o especulación sobre cambios esperados del valor de referencia (Mex-Der, 2016). En EUA en la vorágine de la creciente especulación se titularizaron todo tipo de créditos: hipotecarios, tarjetas de crédito, automotrices, de construcción, maquinaria y equipo, hasta los préstamos estudiantiles y “otros créditos” (U. S. Government, 2011). 282 Transformaciones del sistema monetario… / Vargas y Hernández dow Banking System), por surgir al margen de las regulaciones financieras existentes. La sofisticación en los mercados corporativos de deuda siguió con los instrumentos estructurados (contratos cuya rentabilidad no se liga a tasas de interés, sino a algunas acciones específicas, la inflación o a algún índice bursátil), de flujo de caja o híbridos y los sintéticos (Criado y Van Rixtel, 2008), estableciendo contratos complejos que diluyen la naturaleza de las operaciones originales (Vink, 2007). Por otra parte, se institucionalizaron las valoraciones de calificadoras privadas The big three (Moody’s, Standard and Poors y Fitchs), que concentran casi la totalidad de valoraciones a nivel internacional, incluidas las calificaciones de las deudas soberanas de los gobiernos nacionales. Se constituye así, una descomunal masa de capital ficticio (Marx, 1979) que se desploma con la ruptura de la sofisticadísima y moderna cadena de pagos, algunas de cuyas partes están casi completamente desvinculadas de la producción y valorización del capital invertido en bienes tangibles para obtener ganancias medias o extraordinarias, pues, las grandes corporaciones prefieren inversiones en instrumentos financieros con ganancias financieras de corto plazo, que recuperaciones lentas con sucias y molestas actividades manufactureras con inversiones en capital fijo (la valorización del capital a interés o del capital dinero llevada a su máxima expresión D – D’ en la era digital). Se puede establecer, en términos generales, que el nuevo sistema financiero internacional conjuga seis grandes procesos históricos: 1. La actualización del viejo sistema internacional de crédito a las nuevas condiciones de la acumulación, modificado con las dos guerras mundiales y la Gran Depresión de 1929, cuyas resonancias contradictorias se prolongaron hasta la década de los sesenta y el intervencionismo estatal de primera generación que comenzó al inicio de la Primera Guerra Mundial. Esta actualización inicia con la ruptura de los acuerdos de Bretton Woods, la crisis de 1974, la aceleración de la acumulación por los grandes conglomerados, y, señoreaje monetario con predominio del dólar que desplaza a la libra esterlina. 2. La convergencia entre la globalización (integración productiva global del capital e incremento de las transacciones financieras a nivel mundial), el neoliberalismo (desregulación y liberalización) y la revolución tecnológica en curso (trasferencias financieras en tiempo real, ingeniería financiera), en la globalización financiera. 3. El combate a la inflación mediante la aplicación de políticas monetarias restrictivas, alza de tasas de interés y contracción de la base monetaria, que favorecieron la rentabilidad del capital financiero. 4. La sustitución de un sistema financiero basado en el crédito bancario, por otro basado en los títulos de deuda o titularización (Chapoy y Girón, 2008), con la desintermediación bancaria (sustitución de bancos regulados por fondos mutuales desregulados –hedge funds), a los instrumentos derivados y estructurados como agentes instrumentales de la especulación financiera a gran escala, así como paralelamente favorece las ventajas del costo del capital como una porción creciente en el señoreaje internacional4. 5. Los procesos de desregulación financiera iniciada por el Big Bang londinense en 1986 (Robertson, 2016) que internacionalizó las operaciones bursátiles en tiempo real, liberalizó las tasas de interés, reemplazó la ley Glass-Steagall que prohibía la fusión entre banca comercial y de inversión, por la Gramm-Leach-Bliley a fines de 1999, derogó la reglamentación Q de la Reserva Federal (FED) vigente desde 1933 que limitaba la salida de los capitales privados de EUA hacia el resto del mundo y la fijación de las tasas de interés que podían pagar los bancos a los depositantes derogada en 2011, abriendo los canales de las 3D, des-regulación, des-supervisión y des-penalización financiera (Fernández, 1990; Latimer, 2011). 6. Modelos computarizados de ingeniería financiera especulativa, fiscalmente evasivos, uso masivo de tarjetas de crédito o cajeros automáticos, titularización del crédito, instrumentos derivados y agencias privadas calificadoras de riesgo, etc. Este conjunto de elementos, a 4 Se trata de los ingresos que obtienen los bancos centrales de cada país por emitir moneda nacional. Este señoreaje se obtiene estableciendo la diferencia entre el valor facial que tiene el dinero, por ejemplo, un billete de 20 pesos, menos su costo de producción, supongamos, 2 pesos, entonces el ingreso por señoreaje será en este caso de 18 pesos. En la medida que los países tienen sus reservas internacionales en dólares, entonces, renuncian al derecho de señoreaje fortaleciendo con ello al dólar a escala internacional, pues generan el señoreaje para la economía norteamericana (Millán, 2010). 283 Revista Academia & Negocios Vol. 8 (2) 2022 pp. 279 - 306 su vez, posibilitaron enormes fugas de capital hacia paraísos fiscales para evadir impuestos, lavar dineros provenientes de operaciones criminales o, simplemente, para eludir el escrutinio público. Lo que en principio tendría que pasar por el sistema internacional de pagos centralizado del Banco de Pagos Internacionales (BPI) y el sistema de trasferencia monetarias del código SWIFT (Society for Worldwide Interbank Financial Telecommunication), ubicado en Bélgica, permite a EUA acceder a su conocimiento y a la posibilidad de bloquear aquellas transferencias que desapruebe, de origen o destino de un determinado país (Rosanovich y Converti, 2017). A nivel teórico, el nuevo sistema financiero expresa el cambio de concepción de la teoría económica convencional en cuanto a la relación ahorro-inversión. A diferencia del keynesianismo, se plantea que el ahorro determina la inversión. En consecuencia, a mayor tasa de interés, mayor ahorro y mayores posibilidades de inversión futura, sin considerar que un mayor ahorro puede generar también mayor especulación o atesoramiento privado, que es lo que efectivamente se ha incrementado colosalmente a nivel global. Esta concepción friedmaniana se aplicó en EUA a partir del gobierno de Reagan y según Rodríguez (2005), combate la inflación a través de una política monetaria draconiana a costa del empleo y los salarios. En los años ochenta estaba madura la teoría de las expectativas racionales5, que junto a la política monetaria de Friedman constituyen el nuevo consenso neoliberal, el cual prescribe menor intervención del Estado, fomenta la producción por medios monetarios, la diversificación y minimización del riesgo financiero mediante pronósticos econométricos y modelos probabilísticos sofisticados. Estos modelos y la desregulación financiera llevaron a la crisis financiera de 2008, pues se abandonaron rápidamente las tesis neoliberales para regresar a políticas monetarias de corte neokeynesiano, ahora de Flexibilidad Monetaria (Quantitative Easing), aumentando la oferta de dinero por los bancos centrales de países desarrollados mediante la compra de activos financieros, accio- nes, bonos privados y/o bonos del estado, desvalorizando en forma controlada las monedas locales en un entorno de baja inflación o deflación, tendiendo a reducir las tasas de interés a largo plazo en cero o negativas, como expresión de la enorme sobreacumulación de capital dinerario en circulación. Lo anterior, con la expectativa de reactivar la inversión productiva iniciada por Japón hacia el año 2000, aquejado por un estancamiento de tres décadas de crecimiento promedio nulo y seguida por EUA en sucesivas rondas a partir de 2008 y posteriormente por los bancos de Inglaterra y el Banco Central Europeo en 2014. El ascenso económico y social de EUA a nivel internacional produjo diversos aspectos progresivos, tales como el impulso a una nueva etapa de la globalización en el siglo XX, espacialmente de mayor alcance y profundidad que las precedentes, el aumento de los niveles de vida de segmentos importantes de población en los países avanzados, la revolución tecnológica a partir de la conformación de un nuevo sector electrónico informático y de telecomunicaciones SEI-T (Dabat y Ordóñez, 2009), alterno al complejo químico-metalmecánico-automotor. Es decir, un sistema financiero internacional en torno a instituciones reguladas, bancos centrales, privados y públicos. Además, la conformación de una nueva división internacional del trabajo a partir de la segmentación de los procesos productivos que posibilitaron extender los aprendizajes tecnológicos a nivel social en algunos países emergentes con políticas y Estados activos, que les permitieron generar tecnología, marcas propias y mejora de los niveles de vida de sus poblaciones y, al mismo tiempo, como toda realidad contradictoria, generaron aspectos regresivos como el uso militarista de las tecnologías informáticas, el sesgo hacia las actividades financieras crecientemente especulativas, sistemas contables de simulación, evasión fiscal y crecimiento de empresas fantasmas en paraísos fiscales vinculados a actividades delictivas internacionales. El auge y posterior declinación del nuevo sistema financiero será un aspecto fundamental tanto del ascenso de EUA y su hegemonía mundial, como de su descomposición posterior en el siglo XXI. 5 Esta corriente neoclásica (Lucas, 1972; Sargent, 1977) establece que los consumidores, trabajadores y empresas toman decisiones racionales en búsqueda de beneficios individuales con la información disponible generalmente correcta; las predicciones sobre el valor futuro de variables económicas hechas por los agentes no son sistemáticamente erróneas y que los errores son aleatorios (ruido blanco); Stiglitz (2002) destruye ese supuesto, demostrando las grandes asimetrías de información existentes. 284 Transformaciones del sistema monetario… / Vargas y Hernández Las teorías convencionales monetaristas de expectativas racionales y neokeynesianas, han mostrado sus limitaciones, quedando un sustrato pragmático de ensayo y error en las políticas públicas. Es importante hacer notar que junto a algunos premios Nobel de economía como Stiglitz6, Tirole (1988), Piketty, Romer7 u otros economistas de la talla de Sachs, han socavado la teoría neoclásica e intelectual de diversos ámbitos a nivel internacional como a los filósofos Patel, Lomborg, Kharas, o ingenieras como Winnie Byanyima de Oxfam Internacional. Se amplía la disputa al seno de los organismos internacionales (OCDE, FMI, Banco Mundial), en torno a políticas que buscan limitar, regular y sancionar a la banca en la sombra, los paraísos fiscales, las empresas offshore, las grandes corporaciones, y, en especial los gigantes tecnológicos estadounidenses que paguen impuestos adecuadamente en los países de origen como en los que se localicen fiscal o físicamente. La declinación relativa de la hegemonía estadounidense se debe al impulso revolucionario de las fuerzas productivas, la erosión de la rentabilidad, la revolución tecnológica actual, la globalización, aumento de la productividad laboral, nueva división internacional del trabajo y, en contraparte, a las relaciones de producción más progresivas que se generaron en los países emergentes del sudeste asiático, en los cuales los aspectos más negativos han sido acotados por sistemas de regulación pública y un ecosistema empresarial orientado a la rentabilidad y a la producción de valores de uso y mejoras paulatinas en los niveles de vida. Por su parte, Latinoamérica ha resentido los efectos negativos del neoliberalismo, y se explica que desde la década de los noventa intenta romper con el neoliberalismo en Argentina, Brasil, Venezuela, Ecuador, Bolivia, Uruguay, o recientemente Perú, Chile y Colombia, motivada por factores externos como la coyuntura alcista de precios de la materias primas entre 2011 y 2014 (Dabat y Leal, 2020). Como prueba de lo descrito, el 8 de octubre de 2021, 136 naciones establecieron un impuesto mínimo global de 15 por ciento a las multinacionales en cada una de los países firmantes donde generan beneficios8, los que servirán para ser redistribuidos a los sectores más vulnerables de la población (Ayuso, 2021). Latinoamérica, impulsada por la creciente demanda china y su política de inversiones de corte no imperialista, las tasas de interés a la baja, gobiernos progresistas que lograron resultados mayormente positivos, procesos que tuvieron reveses de los sectores conservadores locales apoyados por una contraofensiva estadounidense con golpes suaves (campañas de desprestigio, judicialización de la política, uso de iglesias de diferente signo, etc.) y vientos progresistas que llegan con dos décadas de rezago. En el plano monetario, solo tres economías relativamente pequeñas (Panamá en 1904, Ecuador en 2000 y El Salvador en 2001), adoptaron el dólar como moneda de curso interno, y aunque con condiciones históricas diferentes, tienen en común reducir sus márgenes gubernamentales de maniobra en política monetaria y fiscal, convirtiéndose la primera en un paraíso fiscal9. RASGOS DEL NUEVO SISTEMA MONETARIO Y FINANCIERO ESPECULATIVO INTERNACIONAL El centro de gravedad de la economía internacional ha girado desde el noroeste euro-estadounidense hacia el sudeste asiático, diseminándose a otros países que formalmente son aliados de EUA (Japón, Corea del Sur, India, Australia), y cuyo principal flujo comercial y financiero es la región Asia-Pacífico (Arrighi, 2007; Dabat, Leal y Romo, 2012, Maddison y van der Eng, 2013, Hernández, 2018). En el capitalismo son inexistentes los equilibrios pretendidos por las teorías convencionales, pues, lo que existe son procesos contradictorios de la competencia interna e internacional en el merca- 6 “El modelo de equilibrio competitivo (mediante el cual los productores maximizan las ganancias, los consumidores maximizan la utilidad y los precios se determinan en mercados competitivos que igualan la oferta y la demanda) que ha dominado el pensamiento de los economistas durante más de un siglo, no proporciona un buen resultado. Tenemos una economía plagada de poder de mercado y explotación” (Stiglitz, 2020). 7 Al criticar la aseveración de Lucas de 2003 respecto a que la teoría convencional había resuelto hace décadas la prevención de las depresiones, “si utilizamos la pérdida mundial de producción como medida, la crisis financiera de 2008-9 muestra que la predicción de Lucas es un fracaso mucho más grave que su predicción de que los modelos keynesianos estaban equivocados” (Romer, 2016). 8 Por la guerra de Ucrania, el FMI y la OCDE aplazaron la entrada en vigor de este acuerdo hasta 2024. 9 Los circuitos de dolarización en Venezuela, Argentina, Cuba, o con cierta recurrencia en otros países latinoamericanos requieren un tratamiento que no abordamos en este trabajo. 285 Revista Academia & Negocios Vol. 8 (2) 2022 pp. 279 - 306 do mundial, a partir de la capacidad productiva de cada nación y de la creciente esfera financiera que se viene fragmentando fruto de la propia declinación relativa de la economía estadounidense y el ascenso de otras regiones a nivel productivo (Asia, Europa, Medio Oriente, Rusia o América Latina) o financiero (Europa y en menor medida del Sudeste asiático), lo cual modifica el sistema financiero internacional que se ilustra en la Figura 1. Figura 1. Diagrama del actual sistema financiero internacional Notas: BPI: Banco de Pagos Internacionales, B. Regionales: por ej. BID (Banco Interamericano), etc.; Bolsas EUA: NYSE, Chicago, Filadelfia, Nasdaq, Dow Jones; ETN: Empresas Trasnacionales; PF: Paraísos Fiscales, Empresas Offshore; IP: Internet Profundo, Cripto-Monedas; BCE: Banco Central Europeo; IBAN: International Bank Account Number; OFI: Otros Organismos Financieros, BS: Banca en la Sombra; MUNFI: Universo de Intermediación Financiera No Bancaria (Pensiones, Seguros); BRICS: Brasil, Rusia, India, China, Sudáfrica; CIPS: Sistema Internacional de Pagos (BRICS); BDS: Sistema de Posicionamiento Beidou (alterno al GPS). Fuente: Elaboración propia. Sobre el actual sistema financiero internacional, China y el grupo de los Bric´s han construido una institucionalidad comercial y financiera internacional alterna a la Occidental, como parte de un entramado más amplio ligado a la moderna Ruta de la Seda por mar y por tierra (OBOR, One Belt One Road por sus siglas en inglés), con bancos regionales en África, Asia y América Latina. Las crecientes dificultades de las economías occidentales para competir con las nuevas zonas dinámicas acentúan cinco rasgos del sistema financiero internacional, algunos de los cuales acentúan rasgos especulativos y parasitarios que se agregan a un sexto elemento asociado a la predominancia monetaria internacional de las monedas fuertes, a saber: 1) procesos de incremento de la intermediación de flujos financieros como profundización financiera o financiarización; 2) caída de la inter286 mediación bancaria y crecimiento de un nuevo sector de intermediarios financieros no regulado denominado banca en la sombra, 3) la desvinculación entre economía real y banca de inversión, 4) el incremento del capital dinero inmovilizado en paraísos fiscales, 5) flujos de evasión y elusión fiscales deslocalizados en paraísos fiscales a través de empresas offshore o cascarones vacíos (Special Purpose Entities), que se mezclan con la delincuencia organizada o el terrorismo internacional, y 6) los privilegios del señoreaje internacional. La financiarización Los circuitos financieros omnipresentes desde el mercantilismo al capitalismo industrial y financiero, lograron institucionalizar estructuras Transformaciones del sistema monetario… / Vargas y Hernández de intermediación más profundas en los países avanzados que incorporan la capitalización en bolsa, la deuda pública, deuda privada extrabursátil, bonos de empresas financieras y no financieras, la creciente titularización, créditos no titularizados, que hacia 2010 presentaba el siguiente panorama (Figura 2). Figura 2. Estructura de intermediación financiera 2010 (% del PIB) y profundización financiera. Fuente: Roxburgh, Lund y Piotrowsky (2011) Desde una noción básica de profundización financiera, entendida como la relación entre la deuda patrimonial total y el PIB de una economía, se avanza a una más amplia que contempla los diferentes componentes de la intermediación financiera que varían entre países. Considerando los principales flujos intermediados por los sectores financieros, los mayores niveles de profundización financiera medidos en índices de una consultora internacional en 2010, indican que los mayores niveles se encuentran en EUA (462) y Japón (457), seguidos por un índice 15% menor, los países europeos (400), a continuación los países emergentes como China (280), India (209) alrededor de 40% por debajo de los líderes, América Latina se encuentra bastante lejos (148) con cerca de un tercio de EUA o Japón, mientras que las regiones de Asia y África son difíciles de evaluar por el tipo de agregación, pero aparecerían superando a América Latina. La categoría de otros desarrollados adolece de las clasificaciones de países por nivel de ingreso per cápita que incorpora a naciones que no son desarrolladas (Kuwait, Bahrein, Puerto Rico, etc.) Otra estimación de la profundidad financiera como proporción del PIB es una media de 103% en países desarrollados, 68% en Asia-Pacífico, 53.1% Medio Oriente-Norte de África y sólo 34.5% en América Latina y el Caribe (World Bank, 2014, Tabla A.1.5). Al analizar la composición particular de los dos líderes, en EUA destaca el peso creciente de la capitalización bursátil, bonos de instituciones financieras y titularización (derivados, estructurados), mientras que en Japón domina su abultada deuda pública y los créditos tradicionales no titulizados. En Europa Occidental la capitalización bursátil es la mitad de la estadounidense, destacando los bonos de instituciones financieras y los préstamos no titulizados a un nivel similar al japonés. De manera contrastante, en China destaca el crédito público que está abocado a financiar a las grandes empresas estatales, seguido de la participación bursátil y de los fondos de inversión internacionales que financian a las medianas y pequeñas empresas, mientras la India invierte esa relación. Se destaca que tienen una estructura más ligada a la inversión pública y privada no titularizada, menos dependiente de la especulación financiera internacional. 287 Revista Academia & Negocios Vol. 8 (2) 2022 pp. 279 - 306 Declinación de la intermediación crediticia bancaria frenada y la crisis de 1929-1933. Esta característica histórica se ha revertido desde la década de los ochenta, en detrimento de la banca en general y particular, y en favor de otros intermediarios financieros no regulados: la banca en la sombra que en 2018 se aproxima a aquellas en la magnitud de activos financieros, considerando las veintinueve economías más grandes a nivel global que representan más del 80% del producto mundial (Tabla 1). El advenimiento de la etapa del capitalismo monopolista clásico significó la centralidad de la importancia de los bancos y el sector financiero, como tendencia que culmina más de trecientos años de avance en esa dirección, cuando el capitalismo se fortaleció y posteriormente se auto-bloqueó fruto de sus contradicciones con las confrontaciones bélicas, la especulación desen- Activos financieros globales totales Bancos centrales Bancos Instituciones financieras públicas MUNFI * Compañías de seguros Fondos de pensiones Otras instituciones financieras Otros intermedia-rios financieros Tabla 1. Caída de la banca en el sistema financiero global (diciembre de 2018) 378.9 30.1 147.9 17.3 183.7 32.9 35.6 114.3 1.0 Tasa de activos financieros globales (%) 100 7.9 39.0 4.6 48.5 8.7 9.4 30.2 0.3 Crecimiento medio anual 2018 (%) 1.4 2.5 2.8 3.2 -0.1 0.2 0.4 -0.4 8.8 Crecimiento 2012-2017 anualizado (%) 5.9 8.5 3.4 4.7 7.8 5.5 6.3 9.0 8.8 Dimensión (billones** USD) Notas: * MUNFI: Universo de Intermediación Financiera No Bancaria (siglas en inglés); *Billones Sistema Métrico Decimal. Fuente: FSB (2020). La participación de la banca privada y pública en 2018 constituye 43.6% de los activos financieros globales, mientras que la de la banca en la sombra representa 30.5%, con un ritmo de crecimiento de esta última de más del doble que la banca regulada durante el período 2002-2017. Esta tendencia general tiene manifestaciones notoriamente diferenciadas entre cuatro distintos tipos de economías. En este punto se destaca que es más acusada en las economías neoliberales, un tanto menor en los emergentes de América Latina y Asia, significativamente menor en econo- mías como China, India, Rusia y Corea del Sur, las cuales mantienen la mayor participación relativa de la banca pública. Después de un aumento espectacular en EUA de 1980 al 2000, con ritmos de crecimiento por encima de los de la banca regulada, con la contracción de esta última en la crisis de 2008, ambos sectores prácticamente igualaron sus activos financieros totales en 13 billones de dólares hacia 2010, para 2017 a nivel global la banca en la sombra alcanza 5210 billones de dólares, continuando encabezada por EUA (Figura 3). 10 Existen ligeras discrepancias de medición y clasificación entre el FSB, agencia especializada creada exprofeso para darle seguimiento después de la crisis de 2008, y agencias calificadoras privadas como Fitch Ratings. 288 Transformaciones del sistema monetario… / Vargas y Hernández Figura 3. Banca en la sombra por país 2017 (billones de dólares y %). Fuente: Elaboración propia con base en Fitch Ratings (2019). La importancia en magnitud y funcionamiento de la banca en la sombra es cualitativa y funcionalmente muy diferente en EUA que en China. El primero es la cuna de los procesos de titularización con dominio abrumador de la banca privada, servicios de salud semiprivados ligados a los seguros, o el sistema universitario que acumula más de 1.6 billones de dólares en deudas en becas estudiantiles que estrangulan a numerosas familias, los cuales Bernie Sanders propone cancelar (Gil, 2020), o el sistema hipotecario en el que las entidades no bancarias en 2019 originan más de la mitad de todas las nuevas hipotecas en comparación con solo el 10% de 2007 (FSOC, 2019). En contraste, en China predomina el sistema de banca pública y los poderosos grupos financieros privados trabajan con fuertes regulaciones, compatibles con el objetivo general de ser un país plenamente desarrollado en 2049 (Sachs, 2020). La magnitud de la banca en la sombra en china y su proporción con relación al PIB vienen declinando desde su aparente cima en 2017, según diversas estimaciones (Fitch Ratings, 2019; Yu, 2020). Es pertinente destacar en la distribución de la banca en la sombra por país, además del continente, que deben considerarse las islas y territorios que son paraísos fiscales con jurisdicción o área de influencia de EUA (Samoa, Samoa Americana, Guam, Islas Vírgenes, Islas Marshall, Barbados, Belice, Trinidad y Tobago) o del Reino Unido (Bermudas, Fiji, Vanuatu, Dominica, Anguila, Islas Vírgenes Británicas, Islas Caimán, Islas Cook, Saint Kitts y Nevis, Santa Lucía, Islas Mauricio), o los Países Bajos que incluyen Aruba, Curazao y San Martín, sub-registrando significativamente sus dimensiones. Por otra parte, la distribución funcional de los 52 billones de dólares en activos financieros de la banca en la sombra en 2017 es de poco más del 70% en fondos mutuales (37 billones), seguidos por las titularizaciones con 10% (5 billones) y acuerdos con corredores 8% (4.2 billones) (Fitch Ratings, 2019) (Figura 4). Desvinculación entre economía real y banca de inversión (Bolsas de valores) Las dificultades para mantener o elevar la tasa media de rentabilidad por las grandes corporaciones globales (Nota 4) lleva a dispersar los riesgos en diferentes economías nacionales, aprovechando las ventajas relativas en cada una de ellas. En sus países de origen los niveles de rentabilidad han verificado que las empresas financieras disfrutan de un margen de rentabilidad mayor que las no financieras, tras el Big Bang de 1986 (Kuvshinov y Zimmermann, 2018), lo cual dibuja un divorcio entre la economía real y el comportamiento de las bolsas de valores (Stiglitz y Weiss, 1981; Binswanger, 2004; De Loecker y Eeckhout, 2017). 289 Revista Academia & Negocios Vol. 8 (2) 2022 pp. 279 - 306 Figura 4. Capitalización bursátil en EUA 1780-2020 (% del PIB). Fuente: Taylor (2018). Es pertinente observar el comportamiento de largo plazo de los mercados de valores de EUA, destacando la relación entre capitalización de mercado y el PIB, y más de cincuenta mil tipos de valores negociados en el largo período de 225 años, precios de acciones, dividendos y acciones corporativas que la etapa clásica del imperialismo multiplicó por tres, de 15% a 50% en relación al PIB entre 1880 y 1920, y se duplicó en forma espectacular al 100% en menos de una década, alcanzando su cenit en 1929. Este nivel se recupera nuevamente setenta años después hasta la década de los dos mil, con una nueva cumbre cercana al 200% en la crisis de 2008, y a partir de entonces ronda un 150% con respecto al PIB estadounidense, que en los últimos treinta años ha crecido a un ritmo promedio en torno al 2%. Titularización y liquidez Aún con la tendencia a la disminución de la participación de la banca en los activos globales, en 2020 a un PIB global de 80 billones de dólares correspondería una liquidez global 62.5% más grande, al ubicarse en 130 billones de dólares de acuerdo a un especialista (Roberts, 2020) (Figura 5). Figura 5. Pirámide de liquidez con relación al PIB mundial (%). Fuente: Roberts (2020). La distribución de la liquidez favorece tanto a la titularización en instrumentos derivados como a los bonos, y en menor medida a los créditos bancarios con la Troika (FMI, Banco Central Europeo y Banco Mundial), además del Club de París a la 290 cabeza y al gran dinero organizado11 en las diversificadas y complejas corporaciones financieras de fondos de inversión y fondos soberanos que controlan las inversiones en bonos, derivados y otros instrumentos financieros de diseño. Transformaciones del sistema monetario… / Vargas y Hernández Atesoramiento en paraísos fiscales tirse en tesoros completamente improductivos que conducen a conductas parasitarias en localizaciones como Países Bajos, Luxemburgo, Irlanda, islas sin desarrollo industrial o financiero endógeno, Reino Unido o EUA que muestran ciertas características de los paraísos fiscales, tendiendo este último a desplazar a Suiza en la captación de grandes fortunas12. Esa ruptura de la cadena de inversión con vistas a la obtención de ganancias financieras de corto plazo, ha propiciado la conversión de numerosas economías en refugios de capitales y fortunas que se depositan sacándolas de los circuitos de acumulación productiva o financiera, para conver- Tabla 2. Clasificación de economías seleccionadas conforme sus activos financieros 2006 y 2018 (porcentajes) E 2006 2018 2006 2018 2006 2018 2006 2018 China 69 60 20 10 8 10 4 6 India 60 55 17 14 4 2 10 Rusia 59 54 38 20 4 2 Corea Sur 58 42 7 5 nd nd 2006 A+B+C 2018 F Fondos Pensiones Instituciones Fin. Públicas Bancos D Otros Interm. Financieros C Aseguradoras B Bancos Centrales A 2018 2006 2018 nd nd 2 20 80 11 nd nd 10 20 71 1 1 nd nd 0 20 76 10 18 0 3 22 30 47 17 15 7 7 80 Economías mixtas social productivas Emergentes Argentina 43 40 30 40 nd nd 4 4 Brasil 42 40 15 19 4 4 1 1 11 11 25 26 63 México 40 31 15 15 15 15 4 4 15 15 16 20 61 Indonesia 59 61 25 18 3 5 3 5 3 3 6 9 84 Turquía 70 70 18 16 2 4 2 2 nd nd 6 6 90 Alemania 70 47 3 15 1 1 10 13 3 4 10 19 63 Francia 65 59 3 7 1 1 19 19 nd nd 8 18 67 Japón 44 44 5 16 18 8 13 13 4 3 13 14 68 Reino Unido 58 45 1 3 1 1 10 8 5 8 23 36 49 EUA 20 22 2 4 10 9 10 10 20 22 28 25 35 1 nd nd 46 90 8 Neoliberales Paraísos fiscales Islas Caimán 42 8 nd nd nd nd 1 Luxemburgo 18 4 nd nd 1 1 2 2 nd nd 80 92 4 Irlanda 42 10 1 2 2 2 4 4 nd nd 45 79 14 Países Bajos 37 21 1 3 1 1 4 3 12 16 44 59 25 Nota: Incluye sociedades que captan depósitos, instituciones financieras cautivas, prestamistas y auxiliares financieros. Fuente: Elaboración con base en FSB (2020), Anexo 2. 11 Se hace referencia a la expresión usada por el presidente estadounidense Roosevelt durante el New Deal. “Estar gobernados por el dinero organizado es tan peligroso como estarlo por el crimen organizado”. 12 Según un reporte, la reglamentación 2014 de la OCDE es más severa con los evasores fiscales, medidas que fueron ratificadas por 97 países con las excepciones de tres pequeños países (Bahrein, Nauru y Vanuatu) y uno gigante (EUA). El Departamento del Tesoro de EUA ni siquiera se disculpó por no sumarse al marco de estándares elaborado por la OCDE, porque “representa un importante factor de crecimiento de nuestro negocio”. Las fortunas se trasladan de Bahamas o Islas Vírgenes Británicas a Nevada, Wyoming o Dakota del Sur (Mega Ricos, 2019; Bloomberg, 2016). 291 Revista Academia & Negocios Vol. 8 (2) 2022 pp. 279 - 306 La clasificación de economías inicia con las denominadas mixtas social productivistas, en las cuales las orientaciones de rentabilidad en los mercados son atendidas en forma regulada por los Estados que priorizan ganancias sociales y desarrollo científico-técnico a nivel social amplio. Se diferencian de los países neoliberales que anteponen los criterios de rentabilidad a corto plazo, incluso por encima de la vida de los ciudadanos como ha quedado de manifiesto con la pandemia de Covid-19 (Dabat y Hernández, 2020), constituyendo los extremos de dos vías de desarrollo notoriamente contrastantes dentro de los marcos del capitalismo informático global (CIG). Como se observa en la Tabla 2, las economías social productivistas tienen la mayor proporción de participación en el agregado de Banca Central, Bancos privados y públicos (Columna A+B+C) en China 80%, Rusia 76% e India 71%, con la notable excepción de Corea del Sur (47%), porque no se encuentra disponible información de su Banca pública, además de la menor escala de su economía. Mientras que dentro de los países neoliberales se distinguen dos grupos muy diferentes: Japón (68%) (rasgo cultural e histórico común con el primer tipo de economías), Francia (68%) y Alemania (63%) de la suma de Bancas centrales, públicas y privadas; y, por otro lado, Reino Unido (49%) y EUA (35%), que los aproxima en este rubro a los paraísos fiscales, pero con economías maduras diversificadas concentradoras de importantes corporaciones con ingresos tecnológicos y de propiedad intelectual. Las economías emergentes más grandes de América Latina y Asia seleccionadas (Indonesia, Turquía), se encuentran en este indicador bancario en un rango intermedio entre las economías social productivistas y las neoliberales, aunque sus dinámicas de acumulación y de comportamiento financiero las vuelve más próximas y, al mismo tiempo, vulnerables a la gravitación occidental (Figura 6). Figura 6. Estimación de atesoramiento en paraísos fiscales en 2007 (% del PIB) Notas: Incluye todos los países con PIB superior a USD 200,000 millones en 2007. Rusia es una estimación más gruesa, porque incorpora la acumulación de errores y omisiones netos (EON) en la balanza de pagos. Fuente: Alstadsæter y cols. (2017). Las economías que prácticamente han abandonado el sector Bancario son los paraísos fiscales, con diferencias significativas pues las economías europeas (Irlanda, Países Bajos, Luxemburgo)13 deben cumplir estándares de funcionamiento de la Unión Europea (UE), en tanto las islas sin desarrollo propio se refugian en este triste rol para poder subsistir, que les imponen los países económica y políticamente más poderosos. 13 Luxemburgo con apenas algunos enclaves industriales, 600 mil habitantes, según estadísticas oficiales tiene 4 billones de inversión extranjera directa, tanta como EUA y mucho más que China, en conjunto con los Países Bajos, albergan casi la mitad de la inversión “fantasma” a nivel global (Damgaard, y cols., 2019). 292 Transformaciones del sistema monetario… / Vargas y Hernández De acuerdo con la clasificación de la UE, cada año se reclasifican de la lista negra los paraísos fiscales que en 2019 se redujeron de 15 a 5, en tanto que la lista gris descendió de 65 a 34 países (Pérez-Díaz, 2019), buena parte de los cuales en ambas categorías son islas ligadas al Reino Unido y a EUA, o a dominios semicoloniales como Panamá y otros en diferentes continentes. Las estimaciones de la suma de recursos financieros en paraísos fiscales en las empresas offshore, en los cascarones vacíos que usan las grandes empresas trasnacionales globales y el lavado de dinero en complicidad con el gran crimen organizado internacional que vienen quedando al descubierto con las filtraciones al periodismo de investigación (Panama Papers en 2016, Paradise Papers en 2017, Mauritius Leaks en 2019; FinCen en 2019-2020, Pandora Papers en 2021), difieren ampliamente de un monto global de 2 a 5% del PIB mundial (OCDE, 2016) que representarían de 1.6 a 4 billones de dólares, o la de Altstadsaeter, Niels y Zucman (2017) que la ubican en 10% del PIB mundial o alrededor de 10 billones de dólares. De cualquier manera, constituyen enormes montos de recursos atesorados que benefician al 0.01% de la población mundial, y que podrían financiar el mejoramiento de los sistemas de salud severamente castigados, la transición energética hacia energías limpias en menor tiempo, o la limpieza de los mares, ríos; la mejora de la diversidad biológica con mayores y mejores niveles de sustentabilidad, o cumplir los objetivos del milenio de Naciones Unidas. La presión fiscal a los Estados nacionales El cambio de un sistema financiero con predominio de instituciones financieras reguladas a otro desregulado, ha contado con una creciente presión del capital financiero e industrial por reducir las contribuciones tributarias, supuestamente con el propósito de incrementar los niveles de inversión y, por ende, de empleo y bienestar económico que en realidad trajeron mayor concentración y centralización de capitales y reducción de las masas salariales en EUA, en la Comunidad Europea y en los países más ligados al neoliberalismo (Harvey, 2007; Piketty, 2014; Milanovic, 2014; Wright y Zucman, 2018). Además, se erosionaron las bases tributarias generales con la reducción de las tasas impositivas que cayeron a la mitad entre 1980 y 2013 (Crivelli, De Mooij y Keen, 2015) (Figura 7). Figura 7. Tasas de impuesto sobre la renta, 1980-2013. Fuente: Crivelli, De Mooij y Keen (2015). 293 Revista Academia & Negocios Vol. 8 (2) 2022 pp. 279 - 306 Con una pérdida de ingresos acumulada estimada de 95 billones de dólares en las economías de la OCDE y 28 billones en las economías No-OCDE, equivalentes al 1% y 1.3% de los PIB globales de esos grupos respectivamente, las pérdidas son de mayor impacto en los países No-OCDE porque sus bases gravables son menores debido a que la media de ingresos fiscales al PIB en estos países es de alrededor del 15%, en comparación con cerca del 35% en la OCDE. Estas pérdidas son más importantes en países de América Latina y el Caribe, África Sub-Sahariana y el Sudeste Asiático (Cobham y Janský, 2018). Las pérdidas serían del orden de 500 a 650 mil millones de dólares anuales (Crivelli, De Mooij y Keen, 2015; Cobham y Janský, 2018), de las cuales 200 mil corresponden a las economías de ingresos bajos, que son incluso mayores a los 150 mil millones que reciben como ayuda financiera para el desarrollo (AOD) (Shaxson, 2019). Es decir, lo que ingresa por una ventana sale aumentado por la puerta, pero afectando las bases tributarias de los países con menor madurez de sus sistemas fiscales, presionados en la competencia por capturar mayores montos de inversión y reproducida al interior de los países entre provincias o estados que compiten por los fondos financieros14. La evasión y elusión fiscales a nivel internacional incidiría en la disminución global del crecimiento económico, que, inicialmente favorecería a los países receptores de inversión directa o de cartera hasta llegar a un punto donde sería decreciente el efecto inicial y se tornaría regresivo para la economía internacional en conjunto y con pendiente negativa en los países prestatarios (Sahay, Čihák, N’Diaye y Barajas, 2015) (Tabla 3). El motor del sistema de evasión fiscal internacional en combinación con empresas offshore o cascarones vacíos (Entidades de Propósito Especial), es la competencia entre Estados para propor- cionar las mejores formas de evadir impuestos, divulgación y regulación financiera, es decir, una forma de macro-corrupción estructural generada por la concurrencia en el capitalismo electrónico informático (CIG) atribuida a los Estados a los que se responsabiliza de la misma, cuando en realidad son las grandes corporaciones privadas las que producen y reproducen la evasión impositiva (reducción de tasas, ingeniería financiera, contabilidad creativa, empresas offshore, cascarones vacíos), generando ganancias financieras de corto plazo y creación de paraísos fiscales15. Múltiples iniciativas impulsadas por organismos financieros internacionales y Estados como Reino Unido en 2010, con una ley de alivio de la deuda de los países pobres muy endeudados (HIPC, por sus siglas en inglés), Bélgica que aprobó en 2015 la Ley contra los fondos buitre16 (holdouts) (Bulow, Reinhart, Rogoff y Trebesch, 2020), las iniciativas de la OCDE de reportes financieros estandarizados que pretende eliminar la evasión tributaria y los paraísos fiscales (Common Reporting Standard, CRS) o sobre la erosión de bases y traspaso de beneficios (Base Erosion and Profit Shifting, BEPS) desde 2015 hasta 2020, se han considerado un relativo fracaso. Lo anterior un fracaso, especialmente, para la economía digitalizada al posponer para después de la pandemia de Covid-19, gravar a los gigantes tecnológicos estadounidenses o las iniciativas de la ONG red de Justicia Fiscal (Tax Justice Network, TJN) o la liderada por el colombiano Ocampo con el FMI para que las empresas trasnacionales paguen correctamente impuestos en su matriz o en los países que radiquen fiscalmente sus empresas (Independent Commission for the Reform of International Corporate Taxation, ICRICT), que son letra muerta frente al poder de las grandes corporaciones. . 14 Uno entre innumerables ejemplos de ventajas fiscales, las otorgadas a Audi por una fábrica con inversión por 2 mil millones de dólares en Puebla, México, vendiéndole el terreno a precio por m2 inferior al costo de expropiación a campesinos, exención de impuesto predial por diez años, reembolso de impuesto sobre nómina por 12 años a Audi y por 10 años a VW, pago de 53.6 millones de dólares desde pago de exámenes psicométricos, transporte aéreo en viajes de capacitación, construcción de Centro de Capacitación, transporte al personal y publicidad para exhibir el cumplimiento de normas ecológicas por la empresa alemana (libro de Sergio Mastretta comentado por Camarena (2019). 15 Estas prácticas contribuyen a segmentar aún más a las clases trabajadoras, pues los sectores contables, legales, de bienes raíces, financieros, en los países de origen de las empresas trasnacionales y en los paraísos fiscales, tienen salarios y prestaciones muy por encima del promedio del resto de los trabajadores, que algunos autores han denominado la “maldición financiera” análogo a la de las materias primas (Shaxson, 2019; así como Christensen ex asesor económico del paraíso británico de Jersey). 16 Compran a bajo precio en el mercado deuda de Estados y empresas al borde de la quiebra o default y luego proceden judicialmente para el cobro total de los bonos más los intereses por los años adeudados. Un 2 o 5% de bonistas manejados en empresas de países acreedores imponen largas renegociaciones y buscan no sólo ganancias financieras, sino humillar a los deudores degradando su calificación crediticia posterior (ver caso argentino con juez Griesa de 2001 a 2016, (Guzmán y Stiglitz, 2016). 294 Transformaciones del sistema monetario… / Vargas y Hernández Tabla 3. Estimaciones de pérdidas (+) o ganancias (-) fiscales en 2013, países seleccionados (billones de do4lares y porcentajes del PIB) FMI Fiscal Gobiernos $ % PIB $ % PIB Economias mixtas social productivas China 77.13 0.86 66.81 0.75 India 47.53 2.70 41.17 2.34 Malasia 2.70 0.86 2.33 0.75 Corea Sur 1.64 0.14 1.12 0.09 MEDIANA 25.12 0.86 21.75 0.75 Emergentes Argentina 24.71 5.10 21.41 4.42 Brasil -25.19 -1.15 -21.82 -1.00 México ND Indonesia 7.48 0.86 6.48 0.75 Turquía -0.77 -0.09 -0.52 -0.06 Sudáfrica 6.73 1.90 5.83 1.65 MEDIANA 6.73 0.86 5.83 0.75 Neoliberales Alemania 22.09 0.61 15.02 0.42 Francia 29.08 1.06 19.78 0.72 Japón 68.79 1.37 46.79 0.93 Reino Unido 1.56 0.06 1.03 0.04 EUA 277.61 1.66 188.83 1.13 MEDIANA 48.94 1.22 33.29 0.83 Paraísos fiscales Islas Caimán ND Panamá 0.36 0.90 0.32 0.78 Luxemburgo 0.33 0.55 0.23 0.37 Irlanda -0.66 -0.30 -0.45 -0.20 Países Bajos 1.53 0.19 1.04 0.13 Suiza -0.26 -0.04 -0.18 -0.03 MEDIANA 0.04 0.08 0.03 0.05 Fuente: Cobham y Janský (2018), Tabla A2. Visto en profundidad, este estudio aun cuando es un avance junto a otras estimaciones (Grubert, 2012; Curcuru, Thomas y Warnock, 2013; Wright y Zucman, 2018), denota que las investigaciones disponibles subestiman las pérdidas fiscales en las economías neoliberales o las ganancias en los paraísos fiscales por tres tipos de razones: a) en la que hay consenso entre todos los autores de no contar con información fidedigna de las empresas trasnacionales17 de punto de partida, lo cual 17 Solo las empresas estadounidenses Fortune 500 tenían un estimado de 2.6 billones en el extranjero en 2017, las ganancias corporativas que trasladan a paraísos fiscales han aumentado de un estimado de 5 a 10% de las ganancias brutas en la década de 1990 a alrededor de 25 a 30% en la actualidad (Fortune, 2017; Shaxson, 2019). 295 Revista Academia & Negocios Vol. 8 (2) 2022 pp. 279 - 306 bitante el privilegio fiscal de las grandes corporaciones trasnacionales, además de los aportes de Gourinchas, Rey y Govillot (2010). sesga significativamente la información; b) la no incorporación de los territorios de jurisdicción de EUA, Reino Unido o los Países Bajos, pues como se observa hay una gran subestimación en estos tres casos, pero es escandalosa en Reino Unido y en los paraísos fiscales (pues Panamá, Luxemburgo y los Países bajos tendrían pérdidas fiscales); y c) no se consideran los diferentes tipos de economías y relaciones sociales de producción más generales, pues, por ejemplo, de las 500 empresas más grandes a nivel global en 2019, las de propiedad pública (State-Owned Enterprises, SOE, por sus siglas en inglés) son 132, de las cuales Asia tiene 100 (China 90, India 4, Japón 2, Resto Asia 4) más Rusia 3; Europa 17 (Francia 9, Alemania e Italia 3 cada una y Resto de Europa 2); en tanto el Resto del mundo cuenta con 12 (Brasil 4 18, EUA 3, México 2 y Medio Oriente 3) (OECD, 2020, Cuadro 6.1). Señoreaje y el (otro) “privilegio exorbitante” Los sueños de los viejos alquimistas de encontrar-inventar el flogisto, el sistema capitalista los ha vuelto realidad de una manera menos extravagante y regida objetivamente por la ley del valor (Marx, 1867/1979), en la configuración del sistema financiero internacional con monedas fiduciarias. El exministro de finanzas francés Giscard D’Estaing, a la postre presidente francés, calificó a la dominancia avasalladora del dólar como un privilegio exorbitante, a lo cual respondieron con arrogancia las autoridades del Tesoro estadounidense que el dólar es nuestra moneda, pero es su problema, refiriéndose a Europa y al resto del mundo (Tabla 4). De manera positiva y propositiva en lo que existe también amplio consenso, es en considerar exor- Estados Unidos Señoreaje 11 11 Participación SUMA 2010 2008 2006 2004 2002 2000 1998 1996 1994 1992 Promedio Ventaja costo de capital Suma 1990 1960-89 Tabla 4. Beneficios internacionales de divisas dominantes (MMD), 1960-2010 87% 12 9 15 16 16 22 13 13 25 14 12 178 33.0% 9 10 11 15 19 22 29 40 55 72 80 362 67.0% 21 19 26 31 35 44 42 53 80 86 92 540 Euro Área* 13% Señoreaje 3 3 4 5 8 3 26 32.5% Ventaja costo de capital 4 5 8 9 14 14 54 67.5% Suma 7 8 12 14 22 17 80 Gran total 620 100% Fuente: Para el periodo 1960-1989, Neumann (1992), para 1990-2010, World Bank (2011), Box 3.2. 18 Brasil pertenecería a la categoría de “social productivista” por su estructura social, además de integrante de los BRIC´S, pese a su gobierno neoliberal (Dabat y Hernández, 2020). 296 Transformaciones del sistema monetario… / Vargas y Hernández En las diversas épocas históricas ha existido una moneda dominante, lo fue la libra esterlina en los siglos XIX y el primer tercio del XX, momento a partir del cual el dólar se convirtió en la divisa dominante (Eichengreen y Flandreau, 2008), acompañada en el último tramo por el euro. Las monedas dominantes progresivamente cumplen las funciones de medida de los valores, medio de circulación, atesoramiento, medio de pago y dinero mundial, compartiendo esos privilegios con la moneda dominante antecesora (libra esterlina inglesa) o sucesora (el euro). Conforme a los estudios disponibles, en un primer momento, los factores del señoreaje se deben a estas funciones, y, en un segundo momento a lo que se denomina ventajas de costo capital, que vienen a ser una actualización de esas mismas funciones en un sistema financiero desregulado, con operaciones financieras en tiempo real y con la búsqueda de refugios alternativos a las monedas dominantes y al oro, las criptomonedas ligadas al internet profundo o deep web. Los beneficios de las monedas dominantes durante cincuenta años sumarían 178 mil millones de dólares (MMD) para el señoreaje monetario o restringido en el caso del dólar, que representa un tercio del señoreaje total y 362 MMD para el señoreaje por ventajas de costo de capital o amplio que significan los otros dos tercios del señoreaje total por 540 MMD que ascienden al 87% del total de los 620 MMD del señoreaje del dólar y el euro. Por su parte, el euro accede como socio minoritario en los privilegios funcionales de dinero mundial desde el año 2000 con una participación del 13%, estimando que durante una década el señoreaje monetario o restringido es de 26 MMD, equivalentes también a una tercera parte del total, y a 54 MMD en el señoreaje con costos de capital, que son las dos terceras partes de los 80 MMD de dólares que corresponden a la divisa comunitaria europea19. La preminencia de este duopolio monetario y financiero inhibe la competencia de posibles rivales como las criptomonedas de los gigantes tecnológicos estadounidenses, que tienen diversos grados de avance y que han sido pospuestas en diversas ocasiones. La que buscaba ser una seria competencia al Bitcoin es Libra de la red social Facebook, que al parecer debido a las fuertes críticas al comportamiento general de la compañía, el giro del gobierno estadounidense de investigar fiscal y operativamente a los GAFA (Google, Amazon, Facebook, Apple) por considerar que han abusado de su poder monopólico (Andiotis y Rudegeair, 2019; Romm, y cols., 2020; Kang, McCabe y Wakabayashi, 2020), pasará por una larga disputa en tribunales, por lo cual se mantiene inalterado el duopolio del dólar y el euro. Libra parece que se transforma de desafío al Bitcoin a una alternativa a Paypal ligada a las monedas ya establecidas. El intento de regulación a los GAFA apenas inicia, por lo cual se vislumbra que el turno de las criptomonedas llegaría posteriormente, pues el internet profundo es terreno de todos y de nadie, donde conviven las buenas intenciones de preservar un espacio fuera de las intromisiones gubernamentales y las calculadas diversificaciones del crimen organizado. De igual manera, las criptomonedas de China requerirán pasar por amplios acuerdos internacionales (Eichengreen, 2020; Stiglitz, 2020) que puedan agregar una alternativa de moneda de reserva junto a los derechos especiales de giro del FMI (DEG) o al oro. Hoy día, una empresa multinacional puede utilizar la ingeniería financiera para transferir grandes sumas de dinero en todo el mundo, reubicar fácilmente activos intangibles altamente rentables o vender servicios digitales desde paraísos fiscales sin tener una presencia física. Estos fenómenos pueden impactar enormemente las estadísticas macroeconómicas tradicionales, como inflar las cifras del PIB y la inversión extranjera directa en los paraísos fiscales, por ejemplo, el caso del crecimiento del PIB irlandés del 26% en 2015, cuya base es la reducción de su tasa impositiva del 50% en la década de los ochenta al 12.5% actual que hacen aparecer expresiones como doble irlandés con sándwich holandés, que implica transferencias de beneficios entre subsidiarias en Irlanda y los Países Bajos con paraísos fiscales en el Caribe como destino final (Damgaard, Elkjaer y Johannesen, 2019). Y la empresa originadora de todo ello, Apple, sigue disfrutando de prestigio internacional y la más alta capitalización global de mercado. 19 Estos privilegios pueden aparecer ocultos en las cuentas de capital o subregistrados en las cuentas nacionales, como ajustes o bien bajo el rubro “errores u omisiones” que ayudan a financiar los déficits en cuenta corriente o los gastos diversos de los Estados que controlan esas divisas. 297 Revista Academia & Negocios Vol. 8 (2) 2022 pp. 279 - 306 LOS REQUERIMIENTOS ESTRUCTURALES DE LA NUEVA FASE DE DESARROLLO Y LAS LIMITACIONES DEL SISTEMA MONETARIO Y FINANCIERO INTERNACIONAL Para Leal (2015), al abrirse una nueva etapa del desarrollo del capitalismo en el mundo, cambia la base tecno-productiva de las economías nacionales, así como otros ámbitos del sistema, como el surgimiento de un nuevo espacio económico que corresponde a esa nueva estructura productiva, un nuevo método de producción de plusvalor, una nueva división social e internacional del trabajo, una nueva institucionalidad jurídico-política, un nuevo sistema financiero nacional e internacional, etc. Eso fue lo que sucedió a mediados de los años ochenta, después de que el capitalismo mundial superó parcialmente la crisis de la etapa fordista-keynesiana en los años setenta y ochenta, etapa que se caracterizó por el predominio del complejo tecnológico automotriz, acerero y petrolero. Ahora, la nueva etapa abierta hacia mediados de los años ochenta (Dabat, Rivera y Suárez, 2004), está comandada por el complejo tecnológico de la industria electrónica, donde las ramas de la información y las telecomunicaciones se transformaron en las más dinámicas, que al articularse con la fuerza de trabajo altamente calificada posibilita la creación de diseños que pueden ser transformados en productos y en nuevas ramas industriales. Asimismo, es la base para la creación de marcas, patentes, franquicias, etc., que son nuevas formas de propiedad del capital (Kaplinsky, 1995) y que dinamizan a la acumulación en los tiempos modernos. Es decir, el trabajo altamente calificado al articularse con las nuevas tecnologías de la información y las telecomunicaciones (TIC) se ha transformado en la base de la valorización capitalista y del crecimiento económico de las naciones industrializadas y emergentes (Rivera, 2005). Al mismo tiempo, las nuevas TIC han irradiado a todos los sectores de las economías nacionales, posibilitando la modernización productiva de las grandes empresas y la reforma económica de los Estados. Sin embargo, la emergencia de la nueva fase de desarrollo económico en el mundo, coincidió con la entrada del neoliberalismo (Dabat, 2010; Harvey, 2007), que inicialmente permitió la intro298 ducción de estas nuevas tecnologías en las actividades de las grandes empresas y los servicios educativos y gubernamentales desde principios de los años setenta del siglo pasado. Luego, se transformó en una traba para la difusión de las nuevas tecnologías y la consolidación de la nueva fase de desarrollo, porque estimuló la conformación de un sistema financiero desregulado y liberalizado que: Alienta legalmente la especulación en detrimento del crédito productivo, que a su vez, incide directamente en la producción, provocando un crecimiento muy bajo en las economías neoliberales. Asimismo, anula el papel promotor e interventor de los Estados nacionales, que en lugar de abocarse a la creación de las instituciones que requiere esta nueva etapa de desarrollo, se ha dedicado a reforzar el papel dominante de los monopolios y oligopolios en el comando de la acumulación capitalista (Vargas, 2014, p. 50). Cada nueva etapa del capitalismo requiere solucionar el conflicto social que implica el tránsito de la vieja etapa a la nueva, donde la clase dominante necesita desarrollar elementos socio-políticos culturales ajenos a ella, pero necesarios para preservar su hegemonía. Se trata de que sea únicamente la burguesía la que logre desarrollar todas sus potencialidades de acción, para no permitir ser superada históricamente por las clases dominadas (Ordóñez, 1996). La solución a esta conflictividad implica desarrollar las instituciones económicas, políticas, sociales y culturales que estén en consonancia con los requerimientos estructurales de la nueva etapa. En este caso, se habla de los requerimientos estructurales del capitalismo contemporáneo, que transita por la etapa que se denomina indistintamente como capitalismo informático global (Dabat, 2002) o economía del conocimiento (Ordóñez y Bouchain, 2011). Los requerimientos estructurales tienen que ver con las exigencias de la nueva base tecno-productiva conformada por las TIC, que para poder extenderse al conjunto de las ramas económicas de las naciones requieren del apoyo estatal y del financiamiento en todas sus modalidades. En oposición a estas exigencias, los regímenes neoliberales desregularon y liberalizaron las actividades financieras para que fueran las instituciones financieras “las que determinaran las tasas de interés y los plazos asociados a las operaciones activas y pasivas, la eliminación de los cajones se- Transformaciones del sistema monetario… / Vargas y Hernández lectivos de crédito” (Vargas, 2013, p. 71). Al tiempo que otorgaron autonomía a los bancos centrales para que ya no financiaran a los Estados nacionales, con lo cual pusieron a disposición de los grandes monopolios y oligopolios toda la política monetaria y el sistema de crédito, con cuyas acciones le quitaron a los Estados la capacidad e influir en la economía y de utilizar al sistema financiero como palanca del crecimiento económico. En contraste, las economías de orientación productivista como Corea del Sur, Tailandia, Malasia, Singapur, China, la India, Rusia, etc., siguieron un camino distinto en sus políticas de industrialización y mantuvieron a sus sistemas financieros vinculados estrechamente a las actividades productivas. Mientras las economías industrializadas como EUA, Alemania, Francia, Inglaterra, Canadá e Italia han sufrido los estragos de las políticas neoliberales y sufren de un estancamiento secular. En el contexto descrito, el sistema monetario y financiero que requiere la etapa actual de industrialización en las economías nacionales es un sistema financiero público que garantice el financiamiento a las actividades productivas. Al mismo tiempo, se requiere que los Estados nacionales establezcan requisitos de desempeño a las instituciones financieras privadas para que canalicen prioritariamente el crédito a la producción y no a la especulación como sucede en las economías neoliberales. Las acciones anteriores, tendrían el propósito de desarrollar la infraestructura física que corresponde a la actual fase de desarrollo del capitalismo, consistente en: Promover la introducción de la banda 5G, inversión en investigación y desarrollo, dotar de internet a todos los territorios de los Estados nacionales, internet en todos los sitios públicos, digitalización de todos los servicios que ofrecen los gobiernos del mundo, impulso a las redes de investigación científica, tecnológica y la educación, promover el desarrollo y consolidación de la ciencia abierta, consistente en la socialización de los conocimientos que son elaborados por investigaciones independientes para que sean difundidas a través de redes y plataformas (libro electrónico, revistas, etc.). impulso a las investigaciones relativas a la biotecnología y nanotecnología, impulso al desarrollo de las tecnologías verdes, aumento a la inversión destinada a la educación en todas las modalidades y niveles, para promover el aprendi- zaje tecnológico y la innovación, etc. Todo lo anterior, sin dejar de invertir en las áreas relativas a la infraestructura keynesiana (transportes y comunicaciones, infraestructura escolar, hospitalaria, etc., y el mantenimiento de esa infraestructura que se encuentra avejentada en todas las naciones del mundo (Vargas, 2019, p. 13-14). En términos internacionales y en materia de pagos y financiamiento, los gobiernos deben impulsar una transformación radical de las instituciones financieras para ponerlas en consonancia de las necesidades que requieren las empresas y los gobiernos para promover la difusión de la actual revolución tecnológica. Aunque en la praxis, el conjunto de naciones neoliberales está en disputa para construir la nueva institucionalidad de regulación del capitalismo en el mundo. Por una parte, los países neoliberales tienen el control de las instituciones emanadas después de la Segunda Guerra Mundial, que están al servicio de los intereses de EUA y sus aliados, mientras en las naciones social-productivista, están construyendo una nueva institucionalidad que se corresponde con las exigencias estructurales de la nueva etapa de desarrollo y que solo falta extender este modelo al resto del mundo. En el plano interno, las exigencias que impone la dinámica del sistema en cuanto al papel especulativo del dinero y el crédito proponen lo siguiente: Que no se posponga el cobro del 15% del impuesto a las trasnacionales acordado en octubre de 2021 por 136 países, para que estos recursos sean utilizados por los países donde operan esas compañías. Que el G-7 cumpla con los compromisos que ha asumido de destinar cuando menos el 1% del total de los recursos líquidos que se mueven en los paraísos fiscales y sean utilizados para renovar la avejentada infraestructura creada en el marco de la etapa fordista-keynesiana o para financiar la transición energética con energías limpias, así como para el combate a la pobreza en las naciones emergentes y en desarrollo. Para que sea efectivo el control de los flujos de capital que deambulan por el mundo es necesario que cada país decida la forma de establecer controles sobre estos movimientos en coordinación con las autoridades financieras internacionales y los gobiernos de los países centrales. 299 Revista Academia & Negocios Vol. 8 (2) 2022 pp. 279 - 306 CONCLUSIONES Desde la ruptura de los acuerdos monetarios de Bretton Woods en 1971, emergió un nuevo orden monetario y financiero internacional en el que los Estados nacionales perdieron el control sobre las monedas y después por la presión de los grandes grupos económico-financieros sobre los gobiernos para que se abriera y desregularan los sistemas financieros nacionales, conformando un ámbito abocado a la especulación, en detrimento del crédito productivo. Como expresión de esta institucionalización, el sistema financiero dejó de ser factor de promoción del desarrollo económico y se transformó en un obstáculo para ese propósito que en conjunción con la autonomización de los bancos centrales se dejó al Estado sin un instrumento para impulsar el desarrollo económico en las naciones, donde el neoliberalismo se transformó en una visión de Estado que privilegia el papel del mercado y deja el comando de la acumulación de capital en manos de los grandes monopolios y oligopolios nacionales e internacionales. A partir de la institucionalización de la desregulación y liberalización de los sistemas financieros nacionales y de la existencia de los tipos de cambio flotantes, se conformó un sistema monetario y financiero internacional que dio pie a la emergencia de procesos y mercados financieros que limitan el papel promotor del crédito nacional e internacional, a favor de la acumulación productiva. Además, posibilitó el despliegue de procesos perniciosos contra la estructura productiva de los países como la desintermediación bancaria, privilegiando el espacio bursátil que posibilita la desvinculación entre la economía real y crediticia, al tiempo que alienta la especulación sin que se le imponga a este espacio ningún requisito de desempeño o de carácter fiscal en la mayoría de las naciones neoliberales. Asimismo, se desplegaron otros procesos como la financiarización. Se generaron las condiciones para que el resto del mundo continuara financiando a los EUA mediante el señoreaje, que representan cuantiosos ingresos para la Reserva Federal de esa nación y sin ningún beneficio económico para los países receptores de los dólares que son emitidos en EUA. 300 Por otra parte, ha alentado la evasión y la elusión fiscales por parte de las grandes trasnacionales que con facilidad trasladan su dinero a los paraísos fiscales, donde reciben exenciones de impuestos y facilidades para evadir cualquier restricción que limite la movilidad del capital. Esto también se ha traducido en una presión fiscal para los Estados donde arriban esos capitales, porque tienen que desembolsar recursos para la atracción de estos. Este sistema monetario y financiero internacional también se ha convertido en un obstáculo para el crecimiento económico, porque la libertad que gozan en cada nación los mercados monetarios y financieros, les permite abocarse a la especulación en detrimento del crédito productivo. En síntesis, este mercado no es el que corresponde a los requerimientos de la nueva etapa de desarrollo capitalista en el mundo, que necesita de sistemas financieros públicos y privados que tengan la exigencia de cumplir con requisitos de desempeño impuestos por los Estados nacionales para que garanticen los recursos para desarrollar la infraestructura física que necesita la estructura productiva y el despliegue pleno de la actual revolución tecnológica. En términos de propuesta de transformación de los organismos financieros internacionales y de los entes políticos que sirven de base para las negociaciones y acuerdos supranacionales entre sus miembros, no existen aún condiciones para visualizar el rumbo que tomará el orden mundial en disputa, por lo tanto, no se puede hablar de una nueva arquitectura monetaria y financiera internacional que corresponderá a la nueva fase de desarrollo vigente. FINANCIAMIENTO Este artículo forma parte del proyecto de investigación DGAPA-PAPIIT con clave IN304019, “El siglo XXI en perspectiva actual”, adscrito al Instituto de Investigaciones Económicas, UNAM, que fue coordinado por el Dr. Alejandro Ulises Dabat Latrubesse, durante 2019-2021, siendo el Dr. Vargas corresponsable del proyecto. 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Financial Times, 5 de Agosto. https://www.ft.com/content/04a0a603-9f08-419b-a54f-1cfb0922acc8 305 Revista Academia & Negocios Vol. 8 (2) 2022 pp. 279 - 306 306
https://openalex.org/W3085306507	https://ajas.uoanbar.edu.iq/article_29191_a6e44e0577b85a31cd46212bc0d5feb1.pdf	Chinese	null	Effect of Tobacco and Sulpur powders for the control of lesser date moth Batrachedra amydraula. Myer. Cosmopterygidae: Lepidoptera on the Khastawi cultiva	Maǧallaẗ al-Anbār li-l-ʿulūm al-zirāʿiyyaẗ/Maǧallaẗ al-anbār li-l-ʻulūm al-zirāʻiyyaẗ	2,010	cc-by	2,639	ﺔﺻﻼﺧ اﻟ ﺗﻌد ﺣﺷرة اﻟﺣﻣﯾرة Batrachedra amydraula . Myer. ﻣن أﻓﺎت اﺷﺟﺎر اﻟﻧﺧﯾل اﻟرﺋﯾﺳﯾﺔ ﻓﻲ ﻋدد ﻣن اﻟﻣﻧﺎطق ﻓﻲ اﻟﻌﺎﻟم واﻟﻌراق، ﺗﺳﺑب ﻫذﻩ اﻵﻓﺔ ﺧﺳﺎﺋر اﻗﺗﺻﺎدﯾﺔ ﻛﺑﯾرة ﺧﺎﺻﺔ ﻓﻲ اﻟﺻﻧف ﺧﺳﺗﺎوي . ﻧﻔذت دراﺳﺔ ﺣﻘﻠﯾﺔ ﻟﺗﻘوﯾم ﻓﻌﺎﻟﯾﺔ ﻣﺳﺣوق اﻟﺗﺑﻎ وﻣﺳﺣوق اﻟﻛﺑرﯾت و ﺧﻠﯾط ﯾﻬﻣﺎ ﻟﻣﻛﺎﻓﺣﺔ ﺣﺷرة اﻟﺣﻣﯾرة ﻋﻠﻰ ﺻﻧف اﻟﻧﺧﯾل اﻟﺧﺳﺗﺎوي ﻓﻲ ﻣﻧطﻘﺔ اﻟﺻﻘﻼوﯾﺔ ﺧﻼل ﻋﺎم 2008 . ﺑﯾﻧت اﻟﻧﺗﺎﺋﺞ ﺗﻔوق ﻣﺳﺣوق اﻟﺗﺑﻎ وﻣﺳﺣوق اﻟﺧﻠﯾط ﻋﻠﻰ ﻣﺳﺣوق اﻟﻛﺑرﯾت ﺗﻌﻔﯾرا ﻋﻧد إﺟراء اﻟﻣﻌﺎﻣﻠﺔ ﻓﻲ ﻣرﺣﻠﺔ ﺑداﯾﺔ ظﻬور اﻹﺻﺎﺑﺔ ﻋﻧدﻣﺎ ﻛﺎﻧت اﻟﺛﻣﺎر ﻓﻲ ﻣرﺣﻠﺔ اﻟﺣﺑﺎﺑوك ﺣﯾث أدى إﻟﻰ ﺧﻔض ﻧﺳﺑﺔ اﻹﺻﺎﺑﺔ اذ ﺑﻠﻎ اﻟﻣﻌدل اﻟﻌﺎم ﻟﻼﺻﺎﺑﺔ ﺑﻌد اﺳﺑوع ، اﺳﺑوﻋﺎن وﺛﻼث اﺳﺎﺑﯾﻊ ﻣن اﻟﻣﻌﺎﻣﻠﺔ 11.7 ، 12.7 , 24.7 % ﻋﻠﻰ اﻟﺗواﻟﻲ ﻗﯾﺎﺳ ًﺎ ﻣﻊ وﻣﻌﺎﻣﻠﺔ اﻟﻣﻘﺎرﻧﺔ اﻟﺗﻲ ﺑﻠﻐت )21.1 % (ﺑﻣﺎ ان ﻛﻼ اﻟﻣﺎدﺗﯾن اﻣﻧﺔ ﻧﺳﺑﯾﺎ ﻟذا ﯾﻣﻛن ادﺧﺎﻟﻬﻣﺎ ﺿﻣن ﺑراﻣﺞ اﻟﻣﻛﺎﻓﺣﺔ ﻛﺎﺣد اﻟﺑداﺋل اﻟﻣﻣﻛﻧﺔ ﺗﺟﺎﻩ اﻻﻓﺔ . Batrachedra amydraula. Myer. Cosmopterygidae: Lepidoptera ﻋﻠﻰ ﺻﻧف اﻟﻧﺧﯾل ﺧﺳﺗﺎوي Batrachedra amydraula. Myer. Cosmopterygidae: Lepidoptera ﻋﻠﻰ ﺻﻧف اﻟﻧﺧﯾل ﺧﺳﺗﺎوي ط ﺎرق ﻣﺣﻣد ﻋﺑد اﻟﻔﻬداوي و ﺧﻣﯾس ﻋﺑود ﻋﻠﯾوي ﻗﺳم وﻗﺎﯾﺔ اﻟﻧﺑﺎت-ﻛﻠﯾﺔ اﻟزراﻋﺔ / ﺟﺎﻣﻌﺔ اﻻﻧﺑﺎر Tarik M. Al-Fahdawi and Khamees A. Aliwey Plant protection depart.\ College of Agriculture\ University of Al-Anbar Tarik M. Al-Fahdawi and Khamees A. Aliwey Plant protection depart.\ College of Agriculture\ University of Al-Anbar Effect of Tobacco and Sulpur powders for the control of lesser date moth Batrachedra amydraula. Myer. Cosmopterygidae: Lepidoptera on the Khastawi cultiva Tarik M. Al-Fahdawi and Khamees A. Aliwey Plant protection depart.\ College of Agriculture\ University of Al-Anbar اﻟﻣﻘدﻣﺔ ﺗﻌد ﻧﺧﻠﺔ اﻟﺗﻣر phoenix dectylifera L.ﻣن أﺷﺟﺎر اﻟﻔﺎﻛﻬﺔ اﻟ ﺗﻲ ﺗﻧﺗﺷر ﻓﻲ اﻟ ﻣﻧﺎطق اﻻﺳﺗواﺋﯾﺔ )1 (اذ ﯾﻘدر ﻋدد أﺷﺟﺎر اﻟﻧﺧﯾل ﻓﻲ اﻟﻌﺎﻟم ﺑﻧﺣو )90 ( ﻣﻠﯾون ﻧﺧﻠﺔ ﻣﻧﻬﺎ ﺣواﻟﻲ62 ﻣﻠﯾون ﻧﺧﻠﺔ ﻓﻲ اﻟوطن اﻟﻌرﺑﻲ . وﯾﻌد اﻟﻌراق ﻣرﻛزا ﻣﻬﻣﺎً ﻻﻧﺗﺷﺎر اﻟﻧﺧﯾل ﻓﻲ اﻟﻌﺎﻟم إذ ﯾﺿم ﺑﺣ دود 10ﻣﻠﯾون ﻧﺧﻠﺔ وﺑﺣدود اﻛﺛر ﻣن8 ﻣﻠﯾون ﻧﺧﻠﺔ ﻣﻧﺗﺟﺔ ﻣﻧﺗﺷرة ﻓﻲ اﻧﺣﺎء اﻟﻌراق وﯾﻘدر اﻻﻧﺗﺎج ﺑﻧﺣوة اﻛﺛر ﻣن 432 اﻟف طن ﺳﻧوﯾﺎ ﺣﺳب اﺣﺻﺎﺋﺎت وزارة اﻟﺗﺧطﯾط واﻟﺗﻌﺎون اﻻﻧﻣﺎﺋﻲ / ﺟﻬﺎز اﻟﻣرﻛزي ﻟﻼﺣﺻﺎء / ﻣدﯾرﯾﺔ اﻻﺣﺻﺎء اﻟزراﻋﻲ ﻟﻌﺎم 2006 ) 2 ( ﺗﺻﺎب أﺷﺟﺎر اﻟﻧﺧﯾل وﺛﻣﺎرﻫﺎ ﺑﻌدد ﻣن اﻵ ﻓﺎت اﻟ ﺣﺷرﯾﺔ و اﻟ ﻣرﺿﯾﺔ ﻋ دﯾدة وﺗﻌد ﺣﺷرة ﺣﻣﯾرة اﻟﻧﺧﯾلBatrachedra amydraula ﻣن اﻫم اﻵﻓﺎت اﻟﺗﻲ ﺗؤﺛر ﻓﻲ اﻹﻧﺗﺎج ﻛﻣﺎ . اذ ﺗﺻﯾب اﻟﺛﻣﺎر وﻫﻲ ﻋﻠﻰ اﻟﻧﺧﻠﺔ وﺗﺗﻐذى ﯾرﻗﺎﺗﻬﺎ ﻋﻠﻰ اﻟﺛﻣﺎر ﻣﻧذ ﺑداﯾﺔ اﻟﻌﻘد وﺣﺗﻰ طور اﻟﺧﻼل ﻓﺗﺎﺗﻲ ﻋﻠﻰ ﻣﻌظم ﻣﺣﺗوﯾﺎت اﻟﺛﻣرة وﻣن ﺛم ﺗﺗﺳﺎﻗط ﻧﺳﺑﺔ ﻛﺑﯾ رة ﻣﻧﻬﺎ ﺗﺻل اﻟﻰ 100 % ﻓﻲ ﺣﺎﻟﺔ اﻹﺻﺎﺑﺔ اﻟﺷدﯾدة ﻣﺣدﺛﺔ اﺿرار اﻗﺗﺻﺎدﯾﺔ ﻛﺑﯾرة ﻟﻠﺑﻠدان اﻟﻣﻧﺗﺟﺔ ﻟﻬذﻩ اﻟﺗﻣور )3 ( وﺗﺗﺎﺛر ﻧﺳﺑﺔ اﻻﺻﺎﺑﺔ ﺗﺑﻌﺎ ﻟﻠﺻﻧف اﻟﻣزروع واﻟظروف اﻟﺑﯾﺋﯾﺔ اﻟﺳﺎﺋدﻩ )4 ، 5 ، 6 ، 7 ( ﻧﻔذت ﻓﻲ اﻟﻌراق اﻟﻌدﯾد ﻣن اﻟدراﺳﺎت اﻟﻣﺗﻌﻠﻘﺔ ﺑﺣﯾﺎة وﺑﯾﺋﺔ اﻟﺣﺷرة و و ﺳﺎﺋل ﻣﻛﺎﻓﺣ )ﺎ ﻬﺗ8 ، 9 ، 10 ( ﻧﺗﯾﺟﺔ ﻟﻠﺧﺳﺎرة اﻟﺗﻲ ﯾﺳﺑﺑﻬﺎ ﻫذﻩ اﻟﺣﺷرة وﻗﻠﺔ اﻻﺑﺣﺎث اﻟﻌﻠﻣﯾﺔ ﻓﻲ ﻣﺟﺎل اﺳﺗﺧدام اﻟﻣﺳﺎﺣﯾق اﻟﻧﺑﺎﺗﯾﺔ ﻟﻣﻛﺎﻓﺣﺗﻬﺎ ﻓﻘد ﺗرﻛزت اﻷﻫداف اﻷﺳﺎﺳﯾﺔ ﻟﻬذ ا اﻟﺑﺣث ﻋﻠﻰ ﻣدى ﺗﺎﺛﯾر ﻣﺳﺣوق اﻟﺗﺑﻎ وﻣﺳﺣوق اﻟﻛﺑرﯾت وﺧﻠ ﯾ طﻬﻣﺎ ﻓﻲ اﻟ ﻧﺳﺑﺔ اﻟ ﺋو ﻣ ﯾﺔ ﻟﻺﺻﺎﺑﺔ ﺑ ﻬذﻩ اﻟﺣﺷ ةر ﻋﻧد ظروف اﻟﺣﻘل . Abstract The lesser date moth Batrachedra amydraula .Meyr. is considered a major pest of date palm in many places of the world including Iraq causing significant economic losses. A field study was conducted to evaluate the effect of Tobacco powder, Sulphur powder and their mixture on the lesser date moth infesting khastawi cultivar close to saglawia region in Al-anbar province during the spring season of 2008 . Results revealed that a better control was obtained with the Tobacco powder and it,s mixture with Sulphur, at one week, two weeks, and three weeks after treatment. The general means were (11.7, 12.7, 24.7%) for the three treatments respectively compared to (21.1%) in the control treatment. 271 ﺗﺣﺿﯾر ﻣﺳﺣوق اﻟﺗﺑﻎ واﻟﻛﺑرﯾت ﻎ ﺗم ﺷراء اوراق اﻟﺗﺑﻎ ﻣن اﻻﺳواق اﻟﻣﺣﻠﯾﺔ وﺟﻔﻔت ﻓﻲ اﻟظل ﺗﺣت ﻣروﺣﺔ ﺳﻘﻔﯾﺔ ﺛم طﺣﻧت ﺑواﺳطﺔ ﻫﺎون ﯾدوي ﺑﻌدﻫﺎ ﻏرﺑل اﻟﻣﺳﺣوق ﺑواﺳطﺔ ﻣﻧﺧل ﻗﯾﺎس )40 - 50 (ﻣش . ﺣﻔظ ﻫذا اﻟﻣﺳﺣوق ﻓﻲ ﻣﻛﺎن ﻣﻌزول، اﻣﺎ ﺑﺎﻟﻧﺳﺑﺔ ﻟﻣﺳﺣوق اﻟﻛﺑرﯾت اﻟﻣﺎ ﯾﻛروﻧﻲ ﺣﺻل ﻋﻠﯾﻪ ﻣن اﻻﺳواق اﻟﻣﺣﻠﯾﺔ وﻛﺎن ﻣطﺣون ﺟﯾدا و ﺟﺎﻫزا ﻟﻼﺳﺗﻌﻣﺎل . ﻋﻧد اﻟﻣﻌﺎﻣﻠﺔ ﺟرى اﻟﺗﻌﻔﯾر ﺑﺎﺳﺗﺧدام ﻋﻠﺑﺔ ﻣﻌدﻧﯾﺔ زﻧﺔ 1 / 2 ﻛﻐم ﻣﺛﻘﺑﺔ ﻣن اﻻﺳﻔل ﻋدة ﺛﻘوب ﺑواﺳطﺔ ﻣﺳﻣﺎر ﻣﻌدﻧﻲ ووﺿﻌت اﻟﻛﻣﯾﺔ اﻟﻣطﻠوﺑﺔ داﺧل اﻟﻌﻠﺑﺔ وﻋﻔرت اﻟﻌذوق ﺑﻣﻌدل 30ﻏم ﻣن اﻟﻣﺎدة / ﻋذق ﻓﻲ ﻛل ﻣﻛرر ﻓﻲ ﻣرﺣﻠﺔ اﻟﺣﺑﺎﺑوك ﻣن ﻧﻣو اﻟﺛﻣﺎر . وزﻋت اﻟﻣﻌﺎﻣﻼت ﻋﻠﻰ اﻟﻧﺣو اﻟﺗﺎﻟﻲ 1 -ﻣﻌﺎﻣﻠﺔ ﻣﺳﺣوق اﻟﺗﺑﻎ ﺗﻌﻔﯾر اﻟﻌذوق ﺑﻛﻣﯾﺔ 30/ ﻏم ﻋذق . 2 - ﻣﻌﺎﻣﻠﺔ ﻣﺳﺣوق اﻟﻛﺑرﯾت ﺗﻌﻔﯾراﻟﻌذوق30/ﻏم ﻋذق . 3 - ﻣﻌﺎﻣﻠﺔ اﻟﺧﻠﯾط ) ﻣﺳﺣوق اﻟﺗﺑﻎ + ﻣﺳﺣوق اﻟﻛﺑرﯾت اﻟﻌذوق30/ ﻏم ﻋذق (. 4 - اﻟﻣﻘﺎرﻧﺔ ﺑدون ﺗﻌﻔﯾر . ﻧﻔذت اﻟﺗﺟرﺑﺔ ﻓﻲ اﺣد ﺑﺳﺎﺗﯾن اﻟﻧﺧﯾل ﻓﻲ ﻧﺎﺣﯾﺔ اﻟﺻﻘﻼوﯾﺔ / ﻣﺣﺎﻓظﺔ اﻻﻧﺑﺎر ﻟﻣوﺳم ﻋﺎم 2008 .اذﺗ م ﺗﺣدﯾد اﻟﺻﻧف اﻟﺧﺳﺗﺎوي ﻓﻘط ﻟﻛوﻧﻪ اﻛﺛر اﺻﻧﺎف ﺣﺳﺎﺳﯾﺔ ﻣن ا ﻻ ﺻﻧﺎف اﻟﻧﺧﯾل اﻻﺧرى )8 ، 9 .( ﺣددت ارﺑﻊ ﻣﻌﺎﻣﻼت و ﺛﻼث ﻣﻛررات ) ﻓﻲ ﻛل ﻣﻛرر ﺛﻼث اﺷﺟﺎر ﻣن ﺻﻧف اﻟﺧﺳﺗﺎوي ، ( وزﻋت اﻟﻣﻌﺎﻣﻼت وﻣﻛرراﺗﻬﺎ ﺑﺷﻛل ﻋﺷواﺋﻲ ﺗﺑﻌﺎ ﻟﺗﺻﻣﯾم اﻟﻌﺷواﺋﻲ اﻟﻛﺎﻣل )C.R.D ( ) 11 ( . ﻧﻔذت اﻟﺗﺟرﺑﺔ ﻋﻧدﻣﺎ ظﻬرت اﻻﺻﺎﺑﺔ ﺑﺣﺷرة ﺣﻣﯾرة اﻟﻧﺧﯾل . ﺣﺳﺑت اﻟﻧﺳﺑﺔ اﻟ ﻣ ﺋوﯾﺔ ﻟﻼﺻﺎﺑﺔ ﻓﻲ اﻟﻣﻌﺎﻣﻼت اﻟﻣﺧﺗﻠﻔﺔ . ﻛﺎن ﻣوﻋد ﺗم ﺷراء اوراق اﻟﺗﺑﻎ ﻣن اﻻﺳواق اﻟﻣﺣﻠﯾﺔ وﺟﻔﻔت ﻓﻲ اﻟظل ﺗﺣت ﻣروﺣﺔ ﺳﻘﻔﯾﺔ ﺛم طﺣﻧت ﺑواﺳطﺔ ﻫﺎون ﯾدوي ﺑﻌدﻫﺎ ﻏرﺑل اﻟﻣﺳﺣوق ﺑواﺳطﺔ ﻣﻧﺧل ﻗﯾﺎس )40 - 50 (ﻣش . ﺣﻔظ ﻫذا اﻟﻣﺳﺣوق ﻓﻲ ﻣﻛﺎن ﻣﻌزول، اﻣﺎ ﺑﺎﻟﻧﺳﺑﺔ ﻟﻣﺳﺣوق اﻟﻛﺑرﯾت اﻟﻣﺎ ﯾﻛروﻧﻲ ﺣﺻل ﻋﻠﯾﻪ ﻣن اﻻﺳواق اﻟﻣﺣﻠﯾﺔ وﻛﺎن ﻣطﺣون ﺟﯾدا و ﺟﺎﻫزا ﻟﻼﺳﺗﻌﻣﺎل . ﻋﻧد اﻟﻣﻌﺎﻣﻠﺔ ﺟرى اﻟﺗﻌﻔﯾر ﺑﺎﺳﺗﺧدام ﻋﻠﺑﺔ ﻣﻌدﻧﯾﺔ زﻧﺔ 1 / 2 ﻛﻐم ﻣﺛﻘﺑﺔ ﻣن اﻻﺳﻔل ﻋدة ﺛﻘوب ﺑواﺳطﺔ ﻣﺳﻣﺎر ﻣﻌدﻧﻲ ووﺿﻌت اﻟﻛﻣﯾﺔ اﻟﻣطﻠوﺑﺔ داﺧل اﻟﻌﻠﺑﺔ وﻋﻔرت اﻟﻌذوق ﺑﻣﻌدل 30ﻏم ﻣن اﻟﻣﺎدة / ﻋذق ﻓﻲ ﻛل ﻣﻛرر ﻓﻲ ﻣرﺣﻠﺔ اﻟﺣﺑﺎﺑوك ﻣن ﻧﻣو اﻟﺛﻣﺎر . وزﻋت اﻟﻣﻌﺎﻣﻼت ﻋﻠﻰ اﻟﻧﺣو اﻟﺗﺎﻟﻲ 1 -ﻣﻌﺎﻣﻠﺔ ﻣﺳﺣوق اﻟﺗﺑﻎ ﺗﻌﻔﯾر اﻟﻌذوق ﺑﻛﻣﯾﺔ 30/ ﻏم ﻋذق . 2 - ﻣﻌﺎﻣﻠﺔ ﻣﺳﺣوق اﻟﻛﺑرﯾت ﺗﻌﻔﯾراﻟﻌذوق30/ﻏم ﻋذق . 3 - ﻣﻌﺎﻣﻠﺔ اﻟﺧﻠﯾط ) ﻣﺳﺣوق اﻟﺗﺑﻎ + ﻣﺳﺣوق اﻟﻛﺑرﯾت اﻟﻌذوق30/ ﻏم ﻋذق (. ﺗﺣﺿﯾر ﻣﺳﺣوق اﻟﺗﺑﻎ واﻟﻛﺑرﯾت 4اﻟﻘﺎﻧﺔ د نﺗﻔ ﻧﻔذت اﻟﺗﺟرﺑﺔ ﻓﻲ اﺣد ﺑﺳﺎﺗﯾن اﻟﻧﺧﯾل ﻓﻲ ﻧﺎﺣﯾﺔ اﻟﺻﻘﻼوﯾﺔ / ﻣﺣﺎﻓظﺔ اﻻﻧﺑﺎر ﻟﻣوﺳم ﻋﺎم 2008 .اذﺗ م ﺗﺣدﯾد اﻟﺻﻧف اﻟﺧﺳﺗﺎوي ﻓﻘط ﻟﻛوﻧﻪ اﻛﺛر اﺻﻧﺎف ﺣﺳﺎﺳﯾﺔ ﻣن ا ﻻ ﺻﻧﺎف اﻟﻧﺧﯾل اﻻﺧرى )8 ، 9 .( ﻧﻔذت اﻟﺗﺟرﺑﺔ ﻓﻲ اﺣد ﺑﺳﺎﺗﯾن اﻟﻧﺧﯾل ﻓﻲ ﻧﺎﺣﯾﺔ اﻟﺻﻘﻼوﯾﺔ / ﻣﺣﺎﻓظﺔ اﻻﻧﺑﺎر ﻟﻣوﺳم ﻋﺎم 2008 .اذﺗ م ﺗﺣدﯾد اﻟﺻﻧف اﻟﺧﺳﺗﺎوي ﻓﻘط ﻟﻛوﻧﻪ اﻛﺛر اﺻﻧﺎف ﺣﺳﺎﺳﯾﺔ ﻣن ا ﻻ ﺻﻧﺎف اﻟﻧﺧﯾل اﻻﺧرى )8 ، 9 .( ﺣددت ارﺑﻊ ﻣﻌﺎﻣﻼت و ﺛﻼث ﻣﻛررات ) ﻓﻲ ﻛل ﻣﻛرر ﺛﻼث اﺷﺟﺎر ﻣن ﺻﻧف اﻟﺧﺳﺗﺎوي ، ( وزﻋت اﻟﻣﻌﺎﻣﻼت وﻣﻛرراﺗﻬﺎ ﺑﺷﻛل ﻋﺷواﺋﻲ ﺗﺑﻌﺎ ﻟﺗﺻﻣﯾم اﻟﻌﺷواﺋﻲ اﻟﻛﺎﻣل )C.R.D ( ) 11 ( . ﻧﻔذت اﻟﺗﺟرﺑﺔ ﻋﻧدﻣﺎ ظﻬرت اﻻﺻﺎﺑﺔ ﺑﺣﺷرة ﺣﻣﯾرة اﻟﻧﺧﯾل . ﺣﺳﺑت اﻟﻧﺳﺑﺔ اﻟ ﻣ ﺋوﯾﺔ ﻟﻼﺻﺎﺑﺔ ﻓﻲ اﻟﻣﻌﺎﻣﻼت اﻟﻣﺧﺗﻠﻔﺔ . ﻛﺎن ﻣوﻋد 272 اﻟﻣﻛﺎﻓﺣﺔ12 / 6 .ﺟرت ﻋﻣﻠﯾﺔ اﻟﻔﺣص ﺑﻌد اﺳﺑوع، و اﺳﺑوﻋﯾن و ﺛﻼث اﺳﺎﺑﯾﻊ ﻣن اﻟﻣﻌﺎﻣﻠﺔ ﻋﻠﻰ اﻟﺗواﻟﻲ ، ﺣﺳﺑت اﻟﻧﺳﺑﺔ اﻟ ﻣ ﺋوﯾﺔ ﻟﻼﺻﺎﺑﺔ ﻓﻲ اﻟﺛﻣﺎر اﻟﻣﺗﺳﺎﻗطﺔ ﺣﯾث اﺧذت100 ﺛﻣرة ﻣن ﻛل ﻣﻛرر ) ﻧﺧﻠﺔ ( وﻓﺣﺻت ﺟﯾدا ﻟﺣﺳﺎب ﻋدد اﻟﺛﻣﺎر اﻟﻣﺻﺎﺑﺔ ﺑﺣﺷرة اﻟﺣﻣﯾرة وﻋدد اﻟﺛﻣﺎر اﻟﺳﺎﻗطﺔ ﻻﺳﺑﺎب اﺧرى، وﺑﻌد ذﻟك ﻧظ ف ﺗﺣت اﺷﺟﺎر اﻟﻧﺧﯾل وأزﯾﻠت ﺟﻣﯾﻊ اﻟﺛﻣﺎر اﻟﺳﺎﻗطﺔ ﻟﺗﻼﻓﻲ ﺣدوث ﺧﻠط ﺑﯾن اﻟﻘراءات . ﺟرى ﺣﺳﺎب ﻣﻌدﻻت اﻟﻧﺳب اﻟﻣؤوﯾﺔ ﻟﻼﺻﺎﺑﺔ ﺑﻌد اﺳﺑوع واﺳﺑوﻋﺎن وﺛﻼث اﺳﺎﺑﯾﻊ ﻣن اﻟﻣﻌﺎﻣﻠﺔ وﺣﺳﺑت اﻟﻣﺗوﺳطﺎت ﻟﻠﻣﻌﺎﻣﻼت اﻻرﺑﻌﺔ ﺣﻠﻠت ا اﻟﻧﺗﺎﺋﺞ اﺣﺻﺎﺋﯾﺎ وﻓق اﺧﺗﺑﺎر ﺗﺣﻠﯾل اﻟﺗﺑﺎﯾن وﻗورﻧت اﻟﻔروﻗﺎت ﺗﺑﻌﺎ ﻻﺧﺗﺑﺎر اﺻﻐر ﻓرق ﻣﻌﻧوي L.S.D) ( ﻋﻧد ﻣﺳﺗوى اﺣﺗﻣﺎل 0.5 % ) 11 (. اﻟﻧﺗﺎﺋﺞ واﻟﻣﻧﺎﻗﺷﺔ اظﻬرت اﻟﻧﺗﺎﺋﺞ ان اﺳﺗﻌﻣﺎل ﻣﺳﺣوق اﻟﺗﺑﻎ وﻣﺳﺣوق اﻟﺧﻠﯾط ) اﻟﺗﺑﻎ +اﻟﻛﺑرﯾت ( ﻛﺎن اﻛﺛر ﻛﻔﺎﺋﻪ ﻓﻲ اﻟﺣد ﻣن اﻻﺻﺎﺑﺔ ﻟﻣوﻋد اﻟﻘراءة اﻻول ﺑﻌد اﻟﻣﻌﺎﻣﻠﺔ اﻟﺗﻲ ﺣﺳﺑت ﻓﻲ 12 / 6 / 2008 .اﻣﺎ اﻟﻘراءة اﻟ ﺛﺎﻧﯾﺔ اﻟﺗﻲ ﺗم اﺟراءﻫﺎ ﺑﺗﺎرﯾﺦ 26 / 6ﻟ وﺣظ ﻓﻲ اﻟﻣﻌ ﺎ ﻣﻼت اﻟﻣﺧﺗﻠﻔﺔ ﺗﺎﺛﯾر ﻣ ﺗﺑﺎﯾﻧﺎً ﻓﻲ ﺧﻔض اﻟﻧﺳﺑﺔ اﻟﻣﺋوﯾﺔ ﻟﻼﺻﺎﺑﺔ اذ ﻛﺎﻧت ﻧﺳﺑﺔ اﻻﺻﺎﺑﺔ ﻓﻲ ﻣﺳﺣوق اﻟﻛﺑرﯾت واﻟﻣﻘﺎرﻧﺔ 24.7 % ، 38.3 % ﻋﻠﻰ اﻟﺗواﻟﻲ وﻟم ﺗﻼﺣظ ﻓروق ﻣﻌﻧوﯾﺔ ﻋﻧد ﻣﺳﺗوى اﺣﺗﻣﺎل 5 % ﺑﯾن ﻣﻌﺎﻣﻠﺔ اﻟﺧﻠﯾط وﻣﻌﺎﻣﻠﺔ اﻟﺗﺑﻎ ﻟﻛن وﺟدت ﻓروق ﻣﻌﻧوﯾﺔ ﺑﯾن ﻛل ﻣن ﻣﻌﺎﻣﻠﺔ اﻟﺧﻠﯾط واﻟﺗﺑﻎ ﻣﻘﺎرﻧﺔ ﺑﻣﻌﺎﻣﻠﺔ اﻟﻛﺑرﯾت وﻣﻌﺎﻣﻠﺔ اﻟﻣﻘﺎرﻧﺔ . إﻣﺎ اﻟﻘراءة اﻟﺛﺎﻟﺛﺔ اﻟﺗﻲ اﺟر ﯾت ﺑ ﻌد ﺛﻼث اﺳﺎﺑﯾﻊ ﻣن اﻟﻣﻌﺎﻣﻠﺔ ﺑﺗﺎرﯾﺦ )3 / 7 ﻟم ﺗﻼﺣظ ﻓرق ﻣﻌﻧوﯾ ﺔ ﺑﯾن ﻣﻌ ﺎﻣﻠﺔ اﻟﺧﻠﯾط وﻣﻌﺎﻣﻠﺔ ﻣﺳﺣوق اﻟﺗﺑﻎ ﻓﻲ ﺣﯾن ﻛﺎن ﻫﻧﺎك ﻓروق ﻣﻌﻧوﯾ ﺔ ﺑﯾن ﻣﻌﺎﻣﻠﺔ اﻟﺧﻠﯾط وﻣﻌﺎﻣﻠﺔ ﻣﺳﺣوق اﻟﺗﺑﻎ و ﻣﻌﺎﻣﻠﺔ اﻟﻛﺑرﯾت و ﻣﻌﺎﻣﻠﺔ اﻟﻣﻘﺎرﻧﺔ أظﻬرت ﻧﺗﺎﺋﺞ اﻟﺗﺣﻠﯾل اﻹﺣﺻﺎﺋﻲ وﺟود اﺧﺗﻼﻓﺎت ﻓﻲ ﻛل ﻣن ﻣﻌﺎﻣﻠﺔ اﻟﺗﺑﻎ وﻣﻌﺎﻣﻠﺔ اﻟﺧﻠﯾط وﻣﻌﺎﻣﻠﺔ اﻟﻛﺑرﯾت واﯾﺿﺎ وﺟود اﺧﺗﻼﻓﺎت ﻣﻌﻧوﯾ ﺔ ﺑﯾن ﻣﻌﺎﻣﻠﺔ ﻣﺳﺣوق اﻟﺗﺑﻎ وﻣﻌ ﺎ ﻣﻠﺔ اﻟﻛﺑرﯾت . ﻫذﻩ اﻟﻧﺗ ﺋﺞﺎ ﺗﺷﯾر اﻟﻰ ان ﻣﻌﺎﻣﻠﺔ اﻟﺧﻠﯾط وﻣﻌﺎﻣﻠﺔ اﻟﺗﺑﻎ ﺗﻔوﻗ ﺎ ﻋﻠﻰ ﻣﻌﺎﻣﻠ ﺔ ﻣﺳﺣوق اﻟﻛﺑرﯾت وﻣﻌﺎﻣﻠﺔ اﻟﻣﻘﺎرﻧﺔ . ﯾﻼﺣظ ان ﻣﺗوﺳطﺎت ﻣﻌﺎﻣﻠﺔ اﻟﺧﻠﯾط وﻣﻌﺎﻣل اﻟﺗﺑﻎ وﻣﻌﺎﻣﻠﺔ ﻣﺳﺣوق اﻟﻛﺑرﯾت وﻣﻌﺎﻣﻠﺔ اﻟﻣﻘﺎرﻧﺔ وﺻﻠت اﻟﻰ )12.7 ، 11.7 24.7 21.1 (ﻋﻠﻰ اﻟﺗواﻟﻲ ﻣن اﻟﻧﺗﺎﺋﺞ ﺗﺑﯾن ان ﻣﺳﺣوق اﻟﺗﺑﻎ ﻛﺎن اﻻﻓﺿل ﻓﻲ اﻟﺛﺎﺛﯾر ﻓﻲ ﺣﺷرة اﻟﺣﻣﯾرة ﺑﺳﺑب وﺟود ﻣﺎدة اﻟﻧﯾﻛوﺗﯾ ن اﻟﻘﺎﺗﻠﺔ ﻟﻠﺣﺷرات ﺑﯾﻧﻣﺎ ﻟم ﯾﺧﺗﻠف اﻟﻛﺑرﯾت ﻋن اﻟﻣﻘﺎرﻧﺔ ﻓﻲ ﻧﺳﺑﺔ اﻻﺻﺎﺑﺔ وان اﻟزﯾﺎدة اﻟظﺎﻫرﯾﺔ ﻓﻲ ﻫذﻩ اﻟﻣﻌﺎﻣﻠﺔ ﻗد ﯾﻌزى ﺳﺑﺑﻬﺎ اﻟﻰ ﻗﺗل ﺑﻌض اﻻ ﻋداء اﻟﺣﯾﺎﺗﯾﺔ اﻟﺗﻲ رﺑﻣﺎ ﺗﺗﻐذى ﻋﻠﻰ ادوار اﻟﺣﻣﯾرة ﻣن اﻟﻧﺗﺎﺋﺞ اﻟﺗﻲ ﺣﺻل ﻋﻠﯾﻬﺎ ﯾﺑدو واﺿﺣﺎ ان ﻣﺳﺣوق اﻟﺗﺑﻎ وﺧﻠﯾط ﻪ ﻣﻊ اﻟﻛﺑرﯾت اظ ﻬرا ﻛﻔﺎءﻩ ﺟﯾدة ﻓﻲ ﻣﻛﺎﻓﺣﺔ ﺣﺷرة اﻟﺣﻣﯾرة ﻋﻠﻰ ﺻﻧ ف اﻟﻧﺧﯾل ﺧ ﺳﺗﺎوي وﺗ ﻛون ﻫذﻩ اﻟﻣواد اﻣﻧﺔ ﻧﺳﺑﯾﺎ ﻋﻠﻰ اﻟﺑﯾﺋﺔ ﻣﺗوﻓرﻩ ﻣﺣﻠﯾﺎ ﻟذﻟك ﯾﻣﻛن اﻟﺗوﺳﻊ ﻓﻲ اﻟﺗﺟرﺑﺔ اﻟﺣﺎﻟﯾﺔ وﺗطﺑﯾﻘﻬﺎ ﻓﻲ ﻣﻧﺎطق اﺧرى ﻣن اﺟل اﻟﺗوﺻل اﻟﻰ ﺗﺻور ﻛﺎﻣل ﻋن اﻟﻔﺎﺋدﻩ اﻟﻣﺗو ﻗ ﻌﺔ ﻣن اﻋﺗﻣﺎد ﻫذﻩ اﻟﻣواد ﻛﺎﺣد اﻟﻣواد اﻟﻣ ﻣﻛﻧﺔ ﻓﻲ ﻣﻛﺎﻓﺣﺔ اﻓﻪ اﻗﺗﺻﺎدﯾﺔ ﻣﻬﻣﺔ ﺗﺻﯾب ﻧﺧﯾل اﻟﺗﻣور ﻓﻲ اﻟﻌراق . اﻟﻧﺗﺎﺋﺞ واﻟﻣﻧﺎﻗﺷﺔ 3 -ﺑﻣﺎ ان اﻟﻣواد اﻟﻣﺳﺗﺧدﻣﺔ طﺑﯾﻌﯾﺔ وﻏﯾر ﻣؤﺛرة ﻧﺳﺑﯾﺔ ﻓﻲ اﻟﺑﯾﺋﺔ ﻟذﻟك ﯾﻣﻛن ان ﻧوﺳﻊ ﻫذﻩ اﻟﺗﺟرﺑﺔ ا ﻟﻰ ﻣﻧﺎطق اﺧرى ﻣن اﺟل اﻟﺗوﺻل إﻟﻰ اﻻﺳﺗﻧﺗﺎج اﻟﺳﻠﯾم ﺣول اﻋﺗﻣﺎدﻫﺎ ﻛﺄﺣد اﻟﺑداﺋل اﻟﻔﻌﺎﻟﺔ ﻓﻲ ﻣﻛﺎﻓﺣﺔ آﻻﻓﺔ اﻻﻗﺗﺻﺎدﯾﺔ اﻟﻣﻬﻣﺔ اﻟﺗﻲ ﺗﺻﯾب ﻧﺧﯾل اﻟﺗﻣر ﻓﻲ اﻟﻌراق . اﻻﺳﺗﻧﺗﺎﺟﺎت اﻟﺗوﺻﯾﺎت 1 -أن ﺣﺷرة ﺣﻣﯾرة اﻟﻧﺧﯾل ﺗﺳﺑب ﺧﺳﺎ ﺋ ر اﻗﺗﺻﺎدﯾﺔ ﻣﻬﻣﺔ ﻋﻠﻰ ﺻﻧف اﻟﺧﺳﺗﺎوي . 2 -أن أﻓﺿل اﻟﻣﻌﺎﻣﻼت ﻫﻲ ﻣﻌﺎﻣﻠﺔ اﻟﺗﺑﻎ وﻣﻌﺎﻣﻠﺔ ﻣﺳﺣوق ﺧﻠﯾط اﻟﺗﺑﻎ ﻣﻊ اﻟﻛﺑرﯾت وان ﻣﺳﺣوق اﻟﻛﺑرﯾت ﻟﯾس ﻟﻪ ﺗﺄﺛﯾر ﻋﻠﻰ ﺣﺷرة اﻟﺣﻣﯾرة . 3 -ﺑﻣﺎ ان اﻟﻣواد اﻟﻣﺳﺗﺧدﻣﺔ طﺑﯾﻌﯾﺔ وﻏﯾر ﻣؤﺛرة ﻧﺳﺑﯾﺔ ﻓﻲ اﻟﺑﯾﺋﺔ ﻟذﻟك ﯾﻣﻛن ان ﻧوﺳﻊ ﻫذﻩ اﻟﺗﺟرﺑﺔ ا ﻟﻰ ﻣﻧﺎطق اﺧرى ﻣن اﺟل اﻟﺗوﺻل إﻟﻰ اﻻﺳﺗﻧﺗﺎج اﻟﺳﻠﯾم ﺣول اﻋﺗﻣﺎدﻫﺎ ﻛﺄﺣد اﻟﺑداﺋل اﻟﻔﻌﺎﻟﺔ ﻓﻲ ﻣﻛﺎﻓﺣﺔ آﻻﻓﺔ اﻟﻌراق ﺗﺻﯾبﻧﺧﯾل اﻟﺗﻣرﻓ اﻻﻗﺗﺻﺎدﯾﺔ اﻟﻣﻬﻣﺔ اﻟﺗ اﻻﺳﺗﻧﺗﺎﺟﺎت اﻟﺗوﺻﯾﺎت 1 -أن ﺣﺷرة ﺣﻣﯾرة اﻟﻧﺧﯾل ﺗﺳﺑب ﺧﺳﺎ ﺋ ر اﻗﺗﺻﺎدﯾﺔ ﻣﻬﻣﺔ ﻋﻠﻰ ﺻﻧف اﻟﺧﺳﺗﺎوي . 2 -أن أﻓﺿل اﻟﻣﻌﺎﻣﻼت ﻫﻲ ﻣﻌﺎﻣﻠﺔ اﻟﺗﺑﻎ وﻣﻌﺎﻣﻠﺔ ﻣﺳﺣوق ﺧﻠﯾط اﻟﺗﺑﻎ ﻣﻊ اﻟﻛﺑرﯾت وان ﻣﺳﺣوق اﻟﻛﺑرﯾت ﻟﯾس ﻟﻪ ﺗﺄﺛﯾر ﻋﻠﻰ ﺣﺷرة اﻟﺣﻣﯾرة . 3 -ﺑﻣﺎ ان اﻟﻣواد اﻟﻣﺳﺗﺧدﻣﺔ طﺑﯾﻌﯾﺔ وﻏﯾر ﻣؤﺛرة ﻧﺳﺑﯾﺔ ﻓﻲ اﻟﺑﯾﺋﺔ ﻟذﻟك ﯾﻣﻛن ان ﻧوﺳﻊ ﻫذﻩ اﻟﺗﺟرﺑﺔ ا ﻟﻰ ﻣﻧﺎطق اﺧرى ﻣن اﺟل اﻟﺗوﺻل إﻟﻰ اﻻﺳﺗﻧﺗﺎج اﻟﺳﻠﯾم ﺣول اﻋﺗﻣﺎدﻫﺎ ﻛﺄﺣد اﻟﺑداﺋل اﻟﻔﻌﺎﻟﺔ ﻓﻲ ﻣﻛﺎﻓﺣﺔ آﻻﻓﺔ اﻻﻗﺗﺻﺎدﯾﺔ اﻟﻣﻬﻣﺔ اﻟﺗﻲ ﺗﺻﯾب ﻧﺧﯾل اﻟﺗﻣر ﻓﻲ اﻟﻌراق . اﻟﻧﺗﺎﺋﺞ واﻟﻣﻧﺎﻗﺷﺔ إﻣﺎ اﻟﻘراءة اﻟﺛﺎﻟﺛﺔ اﻟﺗﻲ اﺟر ﯾت ﺑ ﻌد ﺛﻼث اﺳﺎﺑﯾﻊ ﻣن اﻟﻣﻌﺎﻣﻠﺔ ﺑﺗﺎرﯾﺦ )3 / 7 ﻟم ﺗﻼﺣظ ﻓرق ﻣﻌﻧوﯾ ﺔ ﺑﯾن ﻣﻌ ﺎﻣﻠﺔ اﻟﺧﻠﯾط وﻣﻌﺎﻣﻠﺔ ﻣﺳﺣوق اﻟﺗﺑﻎ ﻓﻲ ﺣﯾن ﻛﺎن ﻫﻧﺎك ﻓروق ﻣﻌﻧوﯾ ﺔ ﺑﯾن ﻣﻌﺎﻣﻠﺔ اﻟﺧﻠﯾط وﻣﻌﺎﻣﻠﺔ ﻣﺳﺣوق اﻟﺗﺑﻎ و ﻣﻌﺎﻣﻠﺔ اﻟﻛﺑرﯾت و ﻣﻌﺎﻣﻠﺔ اﻟﻣﻘﺎرﻧﺔ أظﻬرت ﻧﺗﺎﺋﺞ اﻟﺗﺣﻠﯾل اﻹﺣﺻﺎﺋﻲ وﺟود اﺧﺗﻼﻓﺎت ﻓﻲ ﻛل ﻣن ﻣﻌﺎﻣﻠﺔ اﻟﺗﺑﻎ وﻣﻌﺎﻣﻠﺔ اﻟﺧﻠﯾط وﻣﻌﺎﻣﻠﺔ اﻟﻛﺑرﯾت واﯾﺿﺎ وﺟود اﺧﺗﻼﻓﺎت ﻣﻌﻧوﯾ ﺔ ﺑﯾن ﻣﻌﺎﻣﻠﺔ ﻣﺳﺣوق اﻟﺗﺑﻎ وﻣﻌ ﺎ ﻣﻠﺔ اﻟﻛﺑرﯾت . ﻫذﻩ اﻟﻧﺗ ﺋﺞﺎ ﺗﺷﯾر اﻟﻰ ان ﻣﻌﺎﻣﻠﺔ اﻟﺧﻠﯾط وﻣﻌﺎﻣﻠﺔ اﻟﺗﺑﻎ ﺗﻔوﻗ ﺎ ﻋﻠﻰ ﻣﻌﺎﻣﻠ ﺔ ﻣﺳﺣوق اﻟﻛﺑرﯾت وﻣﻌﺎﻣﻠﺔ اﻟﻣﻘﺎرﻧﺔ . ﯾﻼﺣظ ان ﻣﺗوﺳطﺎت ﻣﻌﺎﻣﻠﺔ اﻟﺧﻠﯾط وﻣﻌﺎﻣل اﻟﺗﺑﻎ وﻣﻌﺎﻣﻠﺔ ﻣﺳﺣوق اﻟﻛﺑرﯾت وﻣﻌﺎﻣﻠﺔ اﻟﻣﻘﺎرﻧﺔ وﺻﻠت اﻟﻰ )12.7 ، 11.7 24.7 21.1 (ﻋﻠﻰ اﻟﺗواﻟﻲ ﻣن اﻟﻧﺗﺎﺋﺞ ﺗﺑﯾن ان ﻣﺳﺣوق اﻟﺗﺑﻎ ﻛﺎن اﻻﻓﺿل ﻓﻲ اﻟﺛﺎﺛﯾر ﻓﻲ ﺣﺷرة اﻟﺣﻣﯾرة ﺑﺳﺑب وﺟود ﻣﺎدة اﻟﻧﯾﻛوﺗﯾ ن اﻟﻘﺎﺗﻠﺔ ﻟﻠﺣﺷرات ﺑﯾﻧﻣﺎ ﻟم ﯾﺧﺗﻠف اﻟﻛﺑرﯾت ﻋن اﻟﻣﻘﺎرﻧﺔ ﻓﻲ ﻧﺳﺑﺔ اﻻﺻﺎﺑﺔ وان اﻟزﯾﺎدة اﻟظﺎﻫرﯾﺔ ﻓﻲ ﻫذﻩ اﻟﻣﻌﺎﻣﻠﺔ ﻗد ﯾﻌزى ﺳﺑﺑﻬﺎ اﻟﻰ ﻗﺗل ﺑﻌض اﻻ ﻋداء اﻟﺣﯾﺎﺗﯾﺔ اﻟﺗﻲ رﺑﻣﺎ ﺗﺗﻐذى ﻋﻠﻰ ادوار اﻟﺣﻣﯾرة ﻣن اﻟﻧﺗﺎﺋﺞ اﻟﺗﻲ ﺣﺻل ﻋﻠﯾﻬﺎ ﯾﺑدو واﺿﺣﺎ ان ﻣﺳﺣوق اﻟﺗﺑﻎ وﺧﻠﯾط ﻪ ﻣﻊ اﻟﻛﺑرﯾت اظ ﻬرا ﻛﻔﺎءﻩ ﺟﯾدة ﻓﻲ ﻣﻛﺎﻓﺣﺔ ﺣﺷرة اﻟﺣﻣﯾرة ﻋﻠﻰ ﺻﻧ ف اﻟﻧﺧﯾل ﺧ ﺳﺗﺎوي وﺗ ﻛون ﻫذﻩ اﻟﻣواد اﻣﻧﺔ ﻧﺳﺑﯾﺎ ﻋﻠﻰ اﻟﺑﯾﺋﺔ ﻣﺗوﻓرﻩ ﻣﺣﻠﯾﺎ ﻟذﻟك ﯾﻣﻛن اﻟﺗوﺳﻊ ﻓﻲ اﻟﺗﺟرﺑﺔ اﻟﺣﺎﻟﯾﺔ وﺗطﺑﯾﻘﻬﺎ ﻓﻲ ﻣﻧﺎطق اﺧرى ﻣن اﺟل اﻟﺗوﺻل اﻟﻰ ﺗﺻور ﻛﺎﻣل ﻋن اﻟﻔﺎﺋدﻩ اﻟﻣﺗو ﻗ ﻌﺔ ﻣن اﻋﺗﻣﺎد ﻫذﻩ اﻟﻣواد ﻛﺎﺣد اﻟﻣواد اﻟﻣ ﻣﻛﻧﺔ ﻓﻲ ﻣﻛﺎﻓﺣﺔ اﻓﻪ اﻗﺗﺻﺎدﯾﺔ ﻣﻬﻣﺔ ﺗﺻﯾب ﻧﺧﯾل اﻟﺗﻣور ﻓﻲ اﻟﻌراق . 273 ﺟدول )1 (ﺗﺎﺛﯾر ﻣﺳﺎﺣﯾق اﻟﺗﺑﻎ واﻟﻛﺑرﯾت وﺧﻠﯾط ﻬﻣﺎ ﻓﻲ اﻟﻧﺳﺑﺔ اﻟﻣﺋوﯾﺔ ﻟﻼﺻﺎﺑﺔ ﺑﺣﺷرة اﻟﺣﻣﯾرة اﻟﻧﺧﯾل ﻋﻠﻰ اﻟﺻﻧف ﺧﺳﺗﺎوي ﺧﻼل رﺑﯾﻊ2008 ﻧوع اﻟﻣﻌﺎﻣﻠﺔ ﻣﻌدل اﻟﻧﺳﺑﺔ اﻟﻣﺋوﯾﺔ ﻟﻼﺻﺎﺑﺔ ﻗﺑل اﻟﻣﻌﺎﻣﻠﺔ ﻓﻲ اﻟﺛﻣﺎر اﻟﺳﺎﻗطﺔ ﻣﻌدل اﻟﻧﺳﺑﺔ اﻟﻣﺋوﯾﺔ ﻟﻼﺻﺎﺑﺔ ﺑﻌد7اﯾﺎم ﻣﻌدل اﻟﻧﺳﺑﺔ اﻟﻣﺋوﯾﺔ ﻟﻼﺻﺎﺑﺔ ﺑﻌد14اﯾﺎم ﻣﻌدل اﻟﻧﺳﺑﺔ اﻟﻣﺋوﯾﺔ ﻟﻼﺻﺎﺑﺔ ﺑﻌد 21ﯾوم اﻟﻣﺗوﺳط ﻣﺳﺣوق اﻟﺧﻠﯾط )ﻣﺳﺣوق اﻟﺗﺑﻎ + ﻣﺳﺣوق اﻟﻛﺑرﯾت ( 46.3 10.7 22 5.3 12.7 ﻣﺳﺣوق اﻟﺗﺑﻎ 55.3 6 23.7 5.3 11.7 ﻣﺳﺣوق اﻟﻛﺑرﯾت 50.3 17 42.7 14 24.7 اﻟﻣﻘﺎرﻧﺔ Control 62.6 16.7 38.3 8.3 21.1 L.S.D > 0.05 8.3 10.18 1.88 اﻟﺗﺎرﯾﺦ 12 / 6 / 2008 19 / 6 26 / 6 3 / 7 اﻻﺳﺗﻧﺗﺎﺟﺎت اﻟﺗوﺻﯾﺎت 1 -أن ﺣﺷرة ﺣﻣﯾرة اﻟﻧﺧﯾل ﺗﺳﺑب ﺧﺳﺎ ﺋ ر اﻗﺗﺻﺎدﯾﺔ ﻣﻬﻣﺔ ﻋﻠﻰ ﺻﻧف اﻟﺧﺳﺗﺎوي . 2 -أن أﻓﺿل اﻟﻣﻌﺎﻣﻼت ﻫﻲ ﻣﻌﺎﻣﻠﺔ اﻟﺗﺑﻎ وﻣﻌﺎﻣﻠﺔ ﻣﺳﺣوق ﺧﻠﯾط اﻟﺗﺑﻎ ﻣﻊ اﻟﻛﺑرﯾت وان ﻣﺳﺣوق اﻟﻛﺑرﯾت ﻟﯾس ﻟﻪ ﺗﺄﺛﯾر ﻋﻠﻰ ﺣﺷرة اﻟﺣﻣﯾرة . ا ﻟﻣﺻﺎدر 1 - ، اﻟﺑﻛر ، ﻋﺑد اﻟﺟﺑﺎر1972 . ﻧﺧﻠﺔ اﻟﺗﻣر ﻣﺎﺿﯾﻬﺎ وﺣﺎﺿرﻫﺎ واﻟﺟدﯾد ﻓﻲ زراﻋﺗﻬﺎ وﺻﻧﺎﻋﺗﻬﺎ وﺗﺟﺎر ﺗ ﻬﺎ ، ﻣ طﺑﻌﺔ اﻟﻌﺎﻧﻲ _ ﺑﻐداد . 2 -ﺟﻬﺎز اﻟﻣرﻛزي ﻟﻼﺣﺻﺎء ، 2006 ، ﻣدﯾرﯾﺔ اﻻﺣﺻﺎء اﻟزراﻋﻲ . 3 - اﻟﺣﯾدري ، ﺣﯾدر و اﻟﺣﻔﯾظ ، ﻋﻣﺎد .1986 اﻓﺎق اﻟﻧﺧﯾل و اﻟﺗﻣور اﻟﻔﺻﻠﯾﺔ ﻓﻲ اﻟﺷرق اﻻدﻧﻰ وﺷﻣﺎل اﻓرﯾﻘﯾﺎ . ﻣطﺑﻌﺔ اﻟوطن . 126 ﺻﻔﺣﺔ . 3 - اﻟﺣﯾدري ، ﺣﯾدر و اﻟﺣﻔﯾظ ، ﻋﻣﺎد .1986اﻓﺎق اﻟﻧﺧﯾل و اﻟﺗﻣور اﻟﻔﺻﻠﯾﺔ ﻓﻲ اﻟﺷرق اﻻدﻧﻰ وﺷﻣﺎل اﻓرﯾﻘﯾ . ﻣطﺑﻌﺔ اﻟوطن . 126 ﺻﻔﺣﺔ . 4 -اﻟراوي ، ﻣﺣﻣد ﻋﻣﺎر وﻓوزﯾﺔ ﻣﺣﻣد ﻋزﯾز 2002 .ﺗﺄﺛﯾر اﻟﺗرﻛﯾب اﻟﻛﯾﻣﯾﺎ و ي ﻟﺳﻧﺔ اﺻﻧﺎف ﻣن ﻧﺧﻠﺔ اﻟﺗﻣر ﻓﻲ اﻻداء اﻟﺣﯾﺎﺗﻲ ﻟﺣﺷرة اﻟﺣﻣﯾرة Batrachedra sp اﻟﻣﺟﻠﺔ اﻟطرﻓﯾﺔ ﻟﻠﻌﻠوم . 43 ب )1 ( 17 - 31 . 5 - اﻟﻌﻠﻲ ، ﺣﺳﯾن ﻋﺑﺎس : 2000 ﻣﻘﺎوﻣﺔ اﻟﺗﻣر ﺿد اﻻﺻﺎﺑﺔ ﺑﺎﻟﺣﻣﯾرة ﺑﺗﻠﻘﯾﺢ اﻟﻧﺧﯾل ﺑﺎﺻﻧﺎف ﺟﯾد ﻣن طﻠﻊ ذﻛور اﻟﻧﺧﯾل . ﻣﺟﻠﺔ وﻗﺎﯾﺔ اﻟﻧﺑ ﺎت اﻟﻌرﺑﯾﺔ 18 ) 2 ( 91 - 95 . 274 6- Ahmed , T.R.and H.F. Al.Rubaiee . 2000. Thermal Threshold and degree-day required for development of Batrachedra amydraula Iraqi .J. Agric .5.(1) 120-123. 7. Ahmed , T.R.and H.F. Al.Rubaiee .1996 Bionomic of two species Batrachedra (Lepidoptera : Momphidae )and susceptibility of different varieties of dates to the species amydraula. ipa. Jgric .res .3:6 . 8 -ﻋزﯾز، ﻓوزﯾﺔ ﻣﺣﻣد ،1990 . ﺣﺳﺎﺳﯾﺔ ﺑﻌض اﺻﻧﺎف اﻟﻧﺧﺑل ﻟﻼﺻﺎﺑﺔ ﺑﺣﺷرة اﻟﺣﻣﯾرة اﻟﻧﺧﯾل Batrachedra amydraula .Meyr.رﺳﺎﻟﺔ ﻣﺎﺟﺳ ﺗﯾر – ﻛﻠﯾﺔ اﻟﻌﻠوم / ﺟﺎﻣﻌﺔ ﺑﻐداد . 9 -اﻟدﻟﯾﻣﻲ، ﺧﻣﯾس ،2004 . دراﺳﺎت اﻗﺗﺻﺎدﯾﺔ وﺑﯾﺋﯾﺔ ﻋﻠﻰ ﺣﺷرة ﺣﻣﯾرة اﻟﻧﺧﯾلBatrachedra amydraula .Meyr. ﻓﻲ وﺳط اﻟﻌراق وﺑﻌض طراﺋق ﻣﻛﺎﻓﺣﺗﻬﺎ . رﺳﺎﻟﺔ ﻣﺎﺟﺳﺗﯾر – ﻛﻠﯾﺔ اﻟزراﻋﺔ / ﺟﺎﻣﻌﺔ ﺑﻐداد 10 – ، ﻋزﯾز ﻓوزﯾﺔ ﻣﺣﻣد ،2005 , دراﺳﺎت ﺣﯾﺎﺗﯾﺔ و ﺑﯾﺋﯾﺔ ﻋﻠﻰ ﺣﺷرة ﺣﻣﯾرة اﻟﻧﺧﯾل (Lepidoptera: Cosmopterygidae) Batrachedra sp واﻟﺗﻧﺑؤ ﺑﻣوﻋد ظﻬورﻫﺎ واﺻﺎﺑﺗﻬﺎ ﻟﻠﻧﺧﯾل ﻓﻲ اول اﻟرﺑﯾﻊ . اطروﺣﺔ دﻛﺗوراة / ﻛﻠﯾﺔ اﻟﻌﻠوم / ﺟﺎﻣﻌﺔ ﺑﻐداد . 99ﺻﻔﺣﺔ . 11 - اﻟﺳﺎﻫوﻛﻲ ، ﻣدﺣت ، وﻛرﯾﻣﺔ ﻣﺣﻣد وﻫﯾب . 1990 . ﺗطﺑﯾﻘﺎت ﻓﻲ ﺗﺣﻠﯾل اﻟﺗﺟﺎر ب ، ﺟﺎﻣﻌﺔ ﺑﻐداد / ﺑﯾت اﻟﺣﻛﻣﺔ . 10 – ، ﻋزﯾز ﻓوزﯾﺔ ﻣﺣﻣد ،2005 , دراﺳﺎت ﺣﯾﺎﺗﯾﺔ و ﺑﯾﺋﯾﺔ ﻋﻠﻰ ﺣﺷرة ﺣﻣﯾرة اﻟﻧﺧﯾل (Lepidoptera: Cosmopterygidae) Batrachedra sp واﻟﺗﻧﺑؤ ﺑﻣوﻋد ظﻬورﻫﺎ واﺻﺎﺑﺗﻬﺎ ﻟﻠﻧﺧﯾل ﻓﻲ اول اﻟرﺑﯾﻊ . اطروﺣﺔ دﻛﺗوراة / ﻛﻠﯾﺔ اﻟﻌﻠوم / ﺟﺎﻣﻌﺔ ﺑﻐداد . 99ﺻﻔﺣﺔ . 11 - اﻟﺳﺎﻫوﻛﻲ ، ﻣدﺣت ، وﻛرﯾﻣﺔ ﻣﺣﻣد وﻫﯾب . 1990 . ا ﻟﻣﺻﺎدر ﺗطﺑﯾﻘﺎت ﻓﻲ ﺗﺣﻠﯾل اﻟﺗﺟﺎر ب ، ﺟﺎﻣﻌﺔ ﺑﻐداد / ﺑﯾت اﻟﺣﻛﻣﺔ . 10 – ، ﻋزﯾز ﻓوزﯾﺔ ﻣﺣﻣد ،2005 , دراﺳﺎت ﺣﯾﺎﺗﯾﺔ و ﺑﯾﺋﯾﺔ ﻋﻠﻰ ﺣﺷرة ﺣﻣﯾرة اﻟﻧﺧﯾلLepidoptera: Cosmopterygidae) Batrachedra sp واﻟﺗﻧﺑؤ ﺑﻣوﻋد ظﻬورﻫﺎ واﺻﺎﺑﺗﻬﺎ ﻟﻠﻧﺧﯾل ﻓﻲ اول اﻟرﺑﯾﻊ . اطروﺣﺔ دﻛﺗوراة / ﻛﻠﯾﺔ اﻟﻌﻠوم / ﺟﺎﻣﻌﺔ ﺑﻐداد . 99ﺻﻔﺣﺔ . رﯾﻊ ور رو / وم ﯾ / 11 - اﻟﺳﺎﻫوﻛﻲ ، ﻣدﺣت ، وﻛرﯾﻣﺔ ﻣﺣﻣد وﻫﯾب . 1990 . ﺗطﺑﯾﻘﺎت ﻓﻲ ﺗﺣﻠﯾل اﻟﺗﺟﺎر ب ، ﺟﺎﻣﻌﺔ ﺑﻐداد / ﺑﯾت اﻟﺣﻛﻣﺔ . 275 275
https://openalex.org/W1872121276	https://researchonline.lshtm.ac.uk/id/eprint/2228470/1/The%20Impact%20of%20a%20Community%20Awareness%20Strategy_GOLD%20VoR.PDF	English	null	The Impact of a Community Awareness Strategy on Caregiver Treatment Seeking Behaviour and Use of Artemether-Lumefantrine for Febrile Children in Rural Kenya	PloS one	2,015	cc-by	12,207	RESEARCH ARTICLE Data Availability Statement: All relevant data are within the paper. Pre- and post-intervention cross-sectional household surveys were used to evaluate the impact of the intervention on prompt and correct use of AL for febrile children below five years of age. The primary outcome was the proportion of children below five years of age with fever in the last 14 days accessing AL within 48 hours of fever onset. Funding: This study was principally funded by Pfizer Inc’s Mobilize against Malaria initiative (Contract number 88A732140). RWS is a Wellcome Trust Principal Fellow (# 079080 & # 103602). EAO, RWS and CJ acknowledge the support provided by the Wellcome Trust Major Overseas Programme grant to the KEMRI/Wellcome Trust Research Programme (number 092654) BW, EAO and RWS also acknowledge support from the Kenya Medical Background Access to prompt and effective treatment is the cornerstone for malaria control. Population Services International in collaboration with the Ministry of Health launched a malaria behav- iour change communication intervention in Nyanza province, Kenya. The initiative aimed to improve: symptom recognition and prompt access to government health facilities for febrile children; effective treatment with the recommended first-line drug artemether-lumefantrine (AL) in public health facilities and adherence to the AL regimen. Copyright: © 2015 Wasunna 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. Beatrice Wasunna1, Emelda A. Okiro2,3, Jayne Webster4, Jim Todd5, Robert W. Snow2,3, Caroline Jones3,4,6 Beatrice Wasunna1, Emelda A. Okiro2,3, Jayne Webster4, Jim Todd5, Robert W. Snow2,3, Caroline Jones3,4,6 1 Eastern and Southern Africa Centre of International Parasite Control (ESACIPAC), Kenya Medical Research Institute, P.O. Box 54840–00200, Nairobi, Kenya, 2 Department of Public Health Research, Kenya Medical Research Institute/Wellcome Trust Research Programme, Centre for Geographic Medicine Research-Coast (CGMRC), P.O. Box 43640–00100 GPO, Nairobi, Kenya, 3 Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, CCVTM, Oxford, United Kingdom, 4 Disease Control Department, London School of Hygiene and Tropical Medicine (LSHTM), London, Keppel Street, WCIE 7HT, London, United Kingdom, 5 Department of Population Health, London School of Hygiene and Tropical Medicine, London, United Kingdom, Keppel Street, WCIE 7HT, London, United Kingdom, 6 Health Systems and Social Science Research, Kenya Medical Research Institute/Wellcome Trust Research Programme, Centre for Geographic Medicine Research-Coast (CGMR-C), P.O. Box 230, Kilifi, Kenya OPEN ACCESS Citation: Wasunna B, Okiro EA, Webster J, Todd J, Snow RW, Jones C (2015) The Impact of a Community Awareness Strategy on Caregiver Treatment Seeking Behaviour and Use of Artemether-Lumefantrine for Febrile Children in Rural Kenya. PLoS ONE 10(7): e0130305. doi:10.1371/ journal.pone.0130305 Editor: Grace C. John-Stewart, University of Washington, UNITED STATES Received: May 8, 2014 Accepted: May 18, 2015 Published: July 2, 2015 Copyright: © 2015 Wasunna 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. Citation: Wasunna B, Okiro EA, Webster J, Todd J, Snow RW, Jones C (2015) The Impact of a Community Awareness Strategy on Caregiver Treatment Seeking Behaviour and Use of Artemether-Lumefantrine for Febrile Children in Rural Kenya. PLoS ONE 10(7): e0130305. doi:10.1371/ journal.pone.0130305 * anyangob@gmail.com * anyangob@gmail.com The Impact of a Community Awareness Strategy on Caregiver Treatment Seeking Behaviour and Use of Artemether- Lumefantrine for Febrile Children in Rural Kenya Beatrice Wasunna1, Emelda A. Okiro2,3, Jayne Webster4, Jim Todd5, Robert W. Snow2,3, Caroline Jones3,4,6 Introduction In September, 2008, the Roll Back Malaria (RBM) partnership launched the Global Malaria Action Plan (GMAP) to provide a framework for action around which stakeholders in malaria endemic nations could coordinate their malaria control efforts [1]. As part of this strategy pre- elimination countries were expected to make sure that at least 80% of febrile children in malaria endemic regions accessed effective antimalarial treatment early in their illness by 2015 [1]. By 2006 every country in sub-Saharan Africa had abandoned failing monotherapies in favor of artemisinin-based combination therapy (ACT) as recommended first-line treatment for uncomplicated malaria [2], but, despite these policy changes, recent data suggest that there was a large variation in the proportion of children under five with a fever treated with an anti- malarial who received an ACT ranging from under 5% in Chad to over 90% in Rwanda [3]. g g [ ] In Kenya, the first-line antimalarial drug policy was changed officially in 2004 from sulpha- doxine-pyrimethamine (SP) to artmether-lumefantrine (AL) but it wasn’t until late 2006 that AL was procured and distributed to all levels of the government and mission health sectors alongside revised standard treatment guidelines and in-service training for health care provid- ers [4]. Despite a few pilot interventions targeting retail-sector provision, AL remained a pre- scription-only medicine and largely confined to formal public health services across Kenya [5] through to the end of 2010. Cross-sectional health facility surveys undertaken in Kenya, four to six months after AL policy roll out, reported that only 28% of children meeting the national guidelines definition of uncomplicated malaria were prescribed AL, despite AL being in stock on the day of the survey [6]. Even fewer children were receiving the effective antimalarial early in their illness with studies undertaken at various sites in Kenya between 2006 and 2007, reporting that only 11% of febrile children below 5 years received AL within 48 hours of fever onset [7]. In an attempt to address the large gap between the GMAP targets and the proportion of febrile children under five accessing AL from public sector services early in their illness in Kenya, Population Services International (PSI) in collaboration with the Ministry of Public Health and Sanitation (MoPHS) developed and implemented an intensive malaria case man- agement behavioral change initiative. Conclusion The findings of this evaluation demonstrate that interventions that target only one sector may have a limited impact on improvements in prompt and effective treatment where multi- ple sources of treatments are sought for febrile illness. Additionally, the context in which an intervention is implemented is likely to influence the process and outcomes. Behaviour Change Communication and Fever Treatment Seeking Behaviour or next day) for their febrile children. However, there was a decrease in the use of govern- ment health facilities in the post-intervention period. There was a small increase in the proportion of children accessing AL within 48 hours of fever onset [18.4% vs 23.5% (0.1– 10.0)]. Research Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Pfizer Mobilize against Malaria Initiative (MAM) provided funding for this study but was not involved in study design, data collection, and interpretation of the results or decisions to submit the final manuscript. RWS has participated in the Novartis National Malaria Control Programme Best Practice Workshops in Africa and has received consultative fees for participation. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials. Results There was an increase from 62.8% pre-intervention to 79.4% post-intervention (95% CI: 11.1, 22.1) in caregivers who reported seeking formal treatment promptly (on the same day, 1 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0130305 July 2, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0130305 July 2, 2015 Study district Bondo district was selected as the evaluation district from the twelve districts located in Nyanza Province. Bondo is on the shores of Lake Victoria with intense, perennial malaria transmission with a predicted 2009 parasite prevalence in children in excess of 40% [10], where hospital admissions have not declined since 1999 [11], where previous studies have suggested that access to prompt treatment with AL was poor following the change in drug policy [7] and per- formance of health workers in the government sector sub-optimal with respect to managing febrile children [6,12]. The district has four main administrative divisions namely: Bondo, Usigu, Madiany and Rarieda. Forty one government and mission health facilities serve the dis- trict population of over 280,000 inhabitants and are staffed by over 100 health workers. Introduction The initiative was undertaken during the previous malaria treatment policy recommendations of presumptive treatment of all fevers in malaria endemic regions of Kenya. This initiative began in February 2009 with financial support from the Pfizer Mobilize against Malaria program [8] and identified two principal target popula- tions: prescribers of anti-malarials operating from government health facilities and caretakers of young children residing in the community. The impact of the intervention targeting PLOS ONE \| DOI:10.1371/journal.pone.0130305 July 2, 2015 2 / 19 Behaviour Change Communication and Fever Treatment Seeking Behaviour prescribers (enhanced health worker training) has been reported elsewhere [9]. In this paper we focus on the impact of the second aspect of the intervention, a community based behaviour change communication campaign designed to improve the numbers of febrile children attend- ing public health facilities promptly and increase the proportion completing the AL treatment regime. The core theme of the behavioural change initiative was prompt action for all fevers in children branded under the phrase haraka upesi (English = prompt action). A professional advertising agency based in Nairobi was employed to work on communication messages, chan- nels and materials following a communications brief provided by PSI that sought to reinforce three key behaviours: 1) the importance of prompt symptom recognition specifically, getting febrile children into clinics promptly, 2) effective treatment with the recommended first-line dug (AL) at a public health facility and 3) adherence to the AL regimen. The channels of communication included mass media (including radio), posters, distribution of messaging materials, message dissemination through road-shows and established community-based orga- nisations. Here we report on the evaluation of the impact of the intervention on the appropriate treatment (time/drug/dose/duration) for children under five who had suffered from a fever in the past two weeks. Community level intervention activities were undertaken between Novem- ber 2009 and September 2010. Radio messaging was undertaken between October 2009 and September 2010. PLOS ONE \| DOI:10.1371/journal.pone.0130305 July 2, 2015 Study design The study was designed as a pre-post intervention cluster sample survey [13]. A GIS platform was developed including enumeration areas (EA) and associated population data was created based on EAs used during the 1999 national census [14], roads, rivers, schools, health facilities, market centres, administration offices and major landmarks created from national digital data sources and updated through detailed mapping in July 2010. All data were stored in ArcView GIS 3.2 (ESRI Inc., New York, USA) for survey work and estimation of travel distances to health facilities, market centre’s and proximity to posters and CBO small group sessions. Sample size estimates for each of the household surveys were calculated for the proportion of children who access effective malaria treatment within 48 hours of fever [15]. We assumed an initial proportion of 11% of febrile children access AL within 48 hours on onset of symp- toms (primary outcome) as described in 2007 in Bondo district [7] with a 4% minimum point increase post-intervention. Included in the sample size estimation was an estimate of the pro- portion of children aged less than five years, a 10% non-response rate and a design effect of 2 to adjust for clustering. The anticipated sample size was therefore computed as 600 households located in 30 EA clusters. Two samples were selected, first for the pre-intervention survey and 3 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0130305 July 2, 2015 Behaviour Change Communication and Fever Treatment Seeking Behaviour Fig 1. The distribution of randomly selected pre- (Green) and Post-intervention (Pink) enumeration area clusters for the household surveys. Blue clusters indicate where ten clusters were randomly selected in both pre- and post-intervention selection Fig 1. The distribution of randomly selected pre- (Green) and Post-intervention (Pink) enumeration area clusters for the household surveys. Blue clusters indicate where ten clusters were randomly selected in both pre- and post-intervention selection doi:10.1371/journal.pone.0130305.g001 separately for the post-intervention survey. The second sample selection was increased to accommodate an unexpectedly higher AL use at baseline and thus included 700 households located in 35 clusters. Sample EAs were selected using the spatial randomization function in ArcView 3.2 to ensure all areas across the district were equally represented. The distribution of survey clusters is shown in Fig 1. Within each cluster, household were randomly selected using the World Health Organization Expanded Programme of Immunization random walk [16,17]. separately for the post-intervention survey. Study design The second sample selection was increased to accommodate an unexpectedly higher AL use at baseline and thus included 700 households located in 35 clusters. Sample EAs were selected using the spatial randomization function in ArcView 3.2 to ensure all areas across the district were equally represented. The distribution of survey clusters is shown in Fig 1. Within each cluster, household were randomly selected using the World Health Organization Expanded Programme of Immunization random walk [16,17]. PLOS ONE \| DOI:10.1371/journal.pone.0130305 July 2, 2015 Household surveys Evaluation of community level intervention activities was undertaken through two cross-sec- tional household surveys. Pre-intervention (baseline) household surveys were undertaken between June and July 2009 and post-intervention surveys undertaken between July and August 2010. Post-intervention surveys were delayed due to inadequate AL stocks in public health facilities in Bondo between May and July 2010. All mothers or caretakers of children aged under-five years were asked whether their child had experienced a fever in the last 14 days and whether the child was febrile on the day of the survey. For each fever the sources, types and timing of treatment actions were documented. A photo-illustrated guide was used to assist recognition of proprietary forms of anti-malarials likely to be purchased from the retail sector and those available in the public sector. Questions were also asked on numbers of tablets taken and mothers were asked to show packaging if available to confirm completed doses. Finally mother’s and caretakers were interviewed about their knowledge of malaria, AL and sources of treatment and whether they had been exposed to any public messages on prompt fever treat- ment and the source of these messages. A second photo-illustrated guide of all PSI community PLOS ONE \| DOI:10.1371/journal.pone.0130305 July 2, 2015 4 / 19 Behaviour Change Communication and Fever Treatment Seeking Behaviour intervention materials was developed to enable mothers identify IEC materials distributed as part of the intervention. The primary outcome was ‘the proportion of children under five years of age with fever in the last 14 days accessing AL within 48 hours of fever onset’. Adherence was examined among all fevers where treatment started four or more days pre- ceding the survey day. A participant was defined as adherent if the caregivers presented an empty AL blister pack and/or stated that all medicines had been administered. Non-adherents were those who had leftover tablet (s) in a blister pack where treatment was started four days and/or reported to have not completed the dose that they had been given. Caregivers who used AL already existing in the household were excluded from the adherence analysis. To establish whether caregivers adhered to AL treatment, questions were asked in the household surveys on the total number of tablets given, those given in a single dose, number of times per day a drug was given, number of days and whether all tablets were given. Household surveys The number of left over tablets was assessed by requesting to see incomplete AL blister packs (blister packs with left over tablets) and, if not available, by calculating the total number of tablets adminis- tered against the total number of tablets received. Data were captured directly on data screens designed using Pendragon version 5.1 (Pendra- gon Software Corporation, Libertyville, Illinois) with internal consistency checks, using per- sonal digital assistants (model-HP Ipaq 114 classic handheld, Hewlett-Packard Company, Palo Alto, California) and downloaded into Microsoft Office Excel (2007) comma separated value (CSV) files. Assessment of AL availability at government and mission health facilities Data on drug availability was necessary to interpret the results from the pre- and post-interven- tion community surveys. Inadequate AL supply at the point of care will inevitably result in severely compromised malaria case management. Telephone interviews and physical audits were undertaken with health facility in-charges in the universe of 36 government and five mis- sion health facilities between January 2009 and June 2010 to assess AL availability. PLOS ONE \| DOI:10.1371/journal.pone.0130305 July 2, 2015 Analysis All analyses were performed using STATA, version 11 (StataCorp, College Station, Texas). A household wealth index was constructed from household characteristics and asset data using principal component analysis (PCA) [18,19]. Weights were derived from the first principal component and used to construct the wealth index that classified households into wealth quin- tiles, based on whether the household had electricity, radio, bicycle, mobile phone, household head occupation status and level of education, household ownership of cows, main source of drinking water, type of toilet facility, source of fuel for household cooking, household wall con- struction material, and household floor material. A descriptive analysis of fever treatment actions was first undertaken to obtain all frequency distributions of relevant variables/indicators for pre-and post-intervention surveys. The analy- sis accounted for the survey design by adjusting for cluster selection using svy command in STATA. The survey design was self-weighting. The sample size and initial hypothesis was based on the proportion of children with fever accessing AL within 48 hours, but the analysis also assessed many different outcomes to assess the impact of the sensitization program. Differ- ences in proportions were compared for significance using the chi-squared test, notably: 1) changes in fever treatment seeking behaviour through a pre- versus post-intervention compari- son; 2) access to antimalarial medicines by socio-economic quintile pre and post-intervention; 3) changes in the proportions of febrile children accessing AL at any time within 14 days, and a subset within 48 hours of fever onset pre- and post-intervention; and 4) the changes in the 5 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0130305 July 2, 2015 Behaviour Change Communication and Fever Treatment Seeking Behaviour proportions of febrile children accessing AL from public sector facilities within 48 (same day/ next day) of fever onset pre- and post-intervention. Logistic regression was used to determine factors associated with prompt (within 48 hours) treatment of fever with AL among children < 5 years of age in each of the surveys. The factors assessed included age of child, child’s gender, caregiver’s age, caregivers’ level of education, number of children below five years of age in the household, socio-economic status and distance to the nearest public health facility. Associations were assessed using adjusted Wald test on the three outcomes. Variables were screened using univariable analysis and those that gave a p value 0.05 were considered potential predictors of the treatment outcome. Ethical Approval Ethical approval for this study was obtained from the KEMRI National Ethical Review Com- mittee (KEMRI SSC number: 1375) and the London School of Hygiene and Tropical Medicine Ethics Committee (Ethical approval number: 5313). Analysis Univariate analysis of factors was performed using logistic regression to take account of clustering. To decide which predictive factors to include in the regression models the likelihood ratio test was used, and assessment of con- founding and effect modification was considered only in factors that were associated with the outcome (prompt treatment of fever). The test showed strong evidence (p<0.001) of variability between clusters and between caregivers. Univariable and multivariable logistic regression analysis with the child as the unit of analysis was undertaken using the random intercept model (xtmelogit command) in STATA accounting for clustering at EA and caregiver levels. Children with fever Of the 958 children under five years of age sampled in 2009, 506 (52.8%) were reported to have experienced a fever event in the last 14 days. This was a higher proportion than in 2010 when of the 1,123 children under five, 515 (45.9%) were reported to have had fever in the last 14 days (p = 0.006) (Table 1). Of the children with reported fever, 81.8% and 73.2% in 2009 and 2010 respectively were not febrile on the day of the survey. PLOS ONE \| DOI:10.1371/journal.pone.0130305 July 2, 2015 Fever treatment seeking behaviour pre- (2009) post- (2010) intervention Table 2 shows the proportion of febrile children for whom any treatment was sought (includ- ing home-medication with ‘western medicines’ but not including prayers, or use of herbal med- icines) and those for whom any treatment was sought within 48 hours across the two surveys. In both surveys most caregivers of febrile children reported seeking some form of treatment. However, there was a increase from 87.3% in 2009 to 91.8% in 2010, in the proportion of febrile children who sought any treatment (p = 0.02; Table 2). There was also an increase from 62.8% in 2009 to 79.4% in 2010 (p<0.001; Table 2) in caregivers who reported seeking formal treat- ment promptly (on the same day, or next day) for their febrile children. Sources of treatment for fever were similar between observation periods, but the proportion of fevers where first treatment actions included the formal public sector declined from 39.5% in 2009 to 22.3% in 2010 following the intervention (p = <0.001, Table 2). A decrease from 23.1% to 16.5% was also observed in the proportion of febrile children accessing treatment from the public sector within 48 hours (p = 0.04, Table 2) between the 2009 and 2010 surveys. There was little varia- tion pre- and post-intervention in the proportions of caregivers first seeking treatment from the retail sector (29.4%, in 2009 and 31.4% in 2010 or from private clinics (3.0%) in 2009 and 4.0% in 2010 (Table 2). There were, however, changes in the proportion of childhood fevers PLOS ONE \| DOI:10.1371/journal.pone.0130305 July 2, 2015 6 / 19 Behaviour Change Communication and Fever Treatment Seeking Behaviour Table 1. Characteristics of surveyed communities, households, children and caregivers pre- and post-intervention. Proportions are cluster adjusted. Fever treatment seeking behaviour pre- (2009) post- (2010) intervention Pre-intervention (June- July 2009) Post-intervention (July- August 2010) Number of clusters 30 35 Number of households 600 700 Number of children under ﬁve 958 1, 123 Number (%) children who were female 483 (50.4%) 558 (49.7%) Number of children aged <1 year 204 (21.3%) 232 (20.7%) Number of children aged 1 year 208 (21.7%) 202 (18.0) Number of children aged 2 years 546 (57.0%) 689 (61.3) Number of households with more than one child < 5 years 293 (48.8) 355 (50.7) Mean Euclidian distance (km) of household to nearest public health facility (+/- SD) 2.6km (1.3) 2.3km (1.2) Key household variables used for wealth assets Household head employed in wage economy 99 (16.5%) 88 (12.6%) Household owner occupied 486 (81.0%) 629 (89.9%) Source of drinking water—piped water 123 (20.5%) 138 (19.7%) Source of drinking water—lake 224 (37.3%) 187 (26.7%) Toilet = pit latrine 364 (60.7%) 417 (59.6%) Household owns at least one bicycle 400 (66.7%) 483 (69.0%) Household owns a radio 475 (79.2%) 553 (79.0%) Household owns television 51 (8.5%) 86 (12.3%) Household owns at least one mobile phone 384 (64.0%) 536 (76.6%) Household connected to electricity 13 (2.2%) 5 (0.7%) Household uses Firewood for cooking fuel 501 (83.5%) 621 (88.7%) Household owns > = 10 cows 21 (3.5%) 34 (4.9%) Household walls made from brick/cement 135 (22.5%) 115 (16.4%) Household walls made from clay/mud 458 (76.3%) 579 (82.7%) Household ﬂoor made from cement 145 (24.2%) 146 (20.9%) Household ﬂoor made of earth 454 (75.7%) 553 (79.0%) Household head education Primary complete 407 (67.8%) 508 (72.6%) Secondary or higher 168 (28.0%) Number of caregivers interviewed 628 717 Refers to households that own the structure/house in which they live in. Table 1. Characteristics of surveyed communities, households, children and caregivers pre- and post-intervention. Proportions are cluster adjusted. managed with drugs available within the household: from 12.4% in 2009 to 31.1% in 2010 (p = <0.001, Table 2). Community health workers were rarely accessed as the first source of fever treatment in either 2009 or 2010. managed with drugs available within the household: from 12.4% in 2009 to 31.1% in 2010 (p = <0.001, Table 2). Community health workers were rarely accessed as the first source of fever treatment in either 2009 or 2010. Behaviour Change Communication and Fever Treatment Seeking Behaviour Table 2. Caregivers first source of treatment seeking and antimalarials received for febrile children < 5 years of age. Pre-intervention (June-July 2009) Post-intervention (July-August 2010) n (%) n (%) Difference in proportions (95% CI) P- value*** Fevers and source of treatment (First action) Total Number of children <5 years of age 958 1,123 Proportion of children< 5 years reporting fever in last 14 days 506 (52.8) 515 (45.9) -6.9 (-11.1, -2.6) 0.006 Proportion of children< 5 years reporting fever in last 14 days but not febrile on day of survey 414 (81.8) 377 (73.2) -8.6 (13.7, -3.5) 0.001 Number of children< 5 years reporting fever in last 14 days but not febrile on day of survey N = 506 N = 515 Proportion of all febrile children seeking any treatment (not including prayers) 442 (87.3) 473 (91.8) 4.5 (0.7, 8.2) 0.02 Proportion of all febrile children seeking any treatment (not including prayers) within 48 hours 318 (62.8) 409 (79.4) 16.6 (11.1, 22.1) <0.001 Proportion febrile children accessing treatment from formal government of Kenya (GoK)/mission health facilities 200 (39.5) 115 (22.3) -17.2 (-22.8,-11.6) <0.001 Proportion febrile children accessing treatment from formal government of Kenya (GoK)/mission health facilities within 48 hours 117 (23.1) 85 (16.5) -6.6 (-11.5, -1.7) 0.04 Proportion febrile children accessing treatment from formal private sector 14 (3.0) 20 (4.0) 1.0 (-1.2, 3.2) 0.43 Proportion of febrile children accessing treatment from formal retail sector 149 (29.4) 162 (31.4) 2.0 (-3.6, 7.6) 0.63 Proportion of febrile children accessing treatment from drugs available in household 63 (12.4) 160 (31.1) 18.7 (13.8, 23.6) <0.001 Proportion of febrile children accessing treatment from community health workers (CHWs) 16 (3.2) 16 (3.1) -0.1 (-2.2, 2.0) 1.00 Proportion of all febrile children not seeking any treatment 64 (12.7) 42 (8.2) -4.5 (-8.2, -0.7) 0.02 Drugs used to treat malaria Proportion of febrile children accessing any anti-malarial drug in last 14 days 224 (44.3) 202 (39.2) -5.1 (-11.1, 0.9) 0.12 Proportion of febrile children accessing AL at any time in last 14 days 157 (31.0)* 165 (32.0)** 1.0 (-4.7, 6.7) 0.76 Proportion of febrile children treated with quinine 33 (6.5) 27 (5.2) -1.3 (-4.2, 1.6) 0.44 Proportion of febrile children treated with sulfadoxine- pyrimethamine (SP) 5 (1.0) 6 (1.3) 0.3 (-1.0, 1.6) 0.76 Proportion of febrile children treated with amodiaquine 38 (7.5)* 9 (1.7) -5.8 (-8.3, -3.2) <0.001 Proportion of febrile children treated with chloroquine 3 (0.6) 1 (0.2) -0.4 (-1.2, 0.4) 0.30 Proportion of febrile children treated with dihydroartemisinin + piperaquine 1 (0.2) 0 -0.2 (-0.6, 0.2) - rce of treatment seeking and antimalarials received for febrile children < 5 years of age. Antimalarial treatments for febrile-children pre-post intervention Of the 506 febrile children in the 2009 survey, the proportion accessing any antimalarial drug in the last 14 days was 44.3%, similar to the 39.2% of the 515 febrile children who were reported to have accessed antimalarial drugs during the 2010 survey (p = 0.13, Table 2). In addition, there were no changes in the proportion of febrile children accessing AL at any time in the last 14 days between the two observation periods, 31.0% (2009) versus 32.0% (2010) (Table 2). 7 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0130305 July 2, 2015 doi:10.1371/journal.pone.0130305.t002 AL treatment, timing and adherence For the primary indicator, ‘the proportion of children under five years of age with fever in the last 14 days accessing AL within 48 hours of fever onset’ there was a 5.1 percentage point increase from baseline 18.4% to 23.5% post-intervention (p = 0.06, Table 3). However, there were no increases in the proportion of febrile children accessing AL from a public health facil- ity within 48 hours of the onset of fever (Table 3). There was an increase in the numbers of febrile children treated using AL available from within the household from 1.6% to 5.4% between 2009 and 2010 (Table 3: 95% CI: 1.6, 6.0) but, since no data were collected on the orig- inal source of the drugs which were used ‘from within the household’, it is not possible to iden- tify where these drugs originally came from. Information on the source of drugs from outside the household was collected and, although there were no increases in the proportion of febrile children accessing the retail sector as the first source of fever treatment, there was a significant increase in the proportion of febrile children whose fever treatment included AL obtained from the retail sector. The proportion of febrile children who received treatment with AL that had been sourced from the retail sector rose from 1.4% in 2009 to 4.0% in 2010) (p = 0.01, Table 3), Table 3. Comparison of AL treatment, timing and adherence between pre- (2009) and post-intervention (2010). All proportions are cluster adjusted Pre-intervention (June July 2009) Post-intervention (July August 2010) Fevers and source of treatment (First action) There was, however, a decline in the proportion of febrile children accessing amodiaquine from 7.5% in 2009 to 1.7% in 2010 (p = <0.001, Table 2). Six percent (33) of febrile children in 8 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0130305 July 2, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0130305 July 2, 2015 Behaviour Change Communication and Fever Treatment Seeking Behaviour 2009 and 5.2% (27) in 2010 received quinine while a few of the febrile children received ineffective and/or non-recommended monotherapies ineffective and/or non-recommended monotherapies (Table 2). Across both surveys, only one child received dihydroartemisinin- piperaquine, launched as the recommended second-line antimalarial in August 2010 (Table 2). ineffective and/or non-recommended monotherapies ineffective and/or non-recommended monotherapies (Table 2). Across both surveys, only one child received dihydroartemisinin- piperaquine, launched as the recommended second-line antimalarial in August 2010 (Table 2). AL treatment, timing and adherence For the primary indicator, ‘the proportion of children under five years of age with fever in the last 14 days accessing AL within 48 hours of fever onset’ there was a 5.1 percentage point increase from baseline 18.4% to 23.5% post-intervention (p = 0.06, Table 3). However, there were no increases in the proportion of febrile children accessing AL from a public health facil- ity within 48 hours of the onset of fever (Table 3). There was an increase in the numbers of febrile children treated using AL available from within the household from 1.6% to 5.4% between 2009 and 2010 (Table 3: 95% CI: 1.6, 6.0) but, since no data were collected on the orig- inal source of the drugs which were used ‘from within the household’, it is not possible to iden- tify where these drugs originally came from. Information on the source of drugs from outside the household was collected and, although there were no increases in the proportion of febrile children accessing the retail sector as the first source of fever treatment, there was a significant increase in the proportion of febrile children whose fever treatment included AL obtained from the retail sector. The proportion of febrile children who received treatment with AL that had been sourced from the retail sector rose from 1.4% in 2009 to 4.0% in 2010) (p = 0.01, Table 3), Table 3. Comparison of AL treatment, timing and adherence between pre- (2009) and post-intervention (2010). Behaviour Change Communication and Fever Treatment Seeking Behaviour suggesting that AL was becoming more available in this sector and that caregivers were more aware of AL and asked for it from the retail shops. In 2009, 147 (93.6%) and in 2010, 152 (92.1%) of all AL treatments were started four or more days prior to the survey day (Table 3). Of these, there was an increase in the proportion who reported adhering to the complete course of AL treatment, from 68.7% in 2009 to 83.5% in 2010 (p = 0.01, Table 3). Initiating treatment promptly was not associated with the likeli- hood of dosage completion. Of all AL treatments that had been started within 48 hours of fever onset and where the initial dose had been taken four or more days prior to the survey there was no significant difference in the proportion of febrile children who were reported to have received a complete course of treatment: 60.2% in 2009 and 64.5% in 2010 (p = 0.58, Table 3). It is possible that the lack of change in adherence rates with increased promptness of treatment was influenced by the higher rate of treatments sourced from within the home in 2010 since these treatments were associated with high non-completion rates. Of the 28 febrile children in the 2010 survey who were reported to have been given AL that was obtained from within the household only nine (32.1%) reported that a complete course of drugs had been available at the start of the treatment and that the full dose had been taken. When the analysis was restricted to those where treatment was sought promptly at a public health facility, there was a significant increase in adherence from 67.5% in 2009 to 81.4% in 2010 (p = 0.05, Table 3). Predictors of fever treatment seeking behaviour and prompt access to artemether-lumefantrine In 2009, none of the variables were associated with prompt access to AL (Table 4). In 2010, uni- variable logistic regression showed that children aged two years and above were four times (p = 0.03, Table 5) more likely to access prompt fever treatment with AL compared with chil- dren aged one year and below. Febrile children in less poor (quintile 4) households were three times more likely to access AL within 48 hours of fever onset compared to those in poorer quintiles (p = 0.05, Table 6). In the final regression model in 2010 only child’s age (p = 0.03, Table 6) remained associated with prompt access to AL. AL availability at government and mission health facilities Over 70% of health facilities had any AL blister packs in stock across the two survey periods. Between April and May 2010, less than half of health facilities had AL paediatric packs (Table 7). Over 80% of health facilities had the adult AL blister packs (AL 24 tablets) in stock during the audit period. At no point during the 10 months did all health facilities have all AL blister packs in stock (Table 7). Less than half of health facilities had all AL blister packs in stock two months prior to the pre- and post-intervention surveys. Furthermore, only 41.5% and 19.5% of health facilities had all AL blister packs in April and May 2010. The study was predicated on measuring access and use of AL from the public sector. Prior to each household sample survey efforts were made to ensure adequate stocks of pediatric pack sizes were avail- able at least four weeks before the survey began and during the survey period to ensure univer- sal availability of AL at public health facilities. Two months prior to the post-intervention survey, less than half of all facilities had AL paediatric packs in stock; however, we ensured that AL was delivered to all health facilities in Bondo two weeks prior to the survey. AL treatment, timing and adherence All proportions are cluster adjusted Pre-intervention (June-July 2009) Post-intervention (July-August 2010) n/N (%) n/N (%) Difference in proportion (95% CI) P- value Source of AL treatment Public sector 137/506 (27.1) 108/515 (21.0) -6.1 (-11.3, -0.9) 0.07 Formal private sector 5/506 (1.0) 10/515 (1.9) 0.9 (-0.6, 2.4) 0.30 Retail commercial sector 7/506 (1.4) 19/515 (4.0) 2.6 (0.6, 4.6) 0.01 Drugs available in household 8/506 (1.6) 28/515 (5.4) 3.8 (1.6, 6.0) <0.001 AL treatments where treatment started 4 days ago 147/157 (93.6) 152/165 (92.1) -1.5 (-7.1, 1.4) 0.60 Of all AL treatments where treatment started 4 days ago number who completed dose 101/147 (68.7) 127/152 (83.5) 14.8 (5.3, 24.3) 0.01 Timing and adherence of AL treatment Proportion of febrile children accessing AL within 48 hours 93/506 (18.4)* 121/515 (23.5)* 5.1 (0.1, 10.0) 0.06 Proportion of fevers children AL within 48 hours from a public health facility 80/506 (15.8) 70/515 (13.6)** -2.2 (-6.5, 2.1) 0.39 Of all AL treatments within 48 hours and where treatment started > = 4 days ago number who completed dose 56/93 (60.2) 78/121 (64.5) 4.3 (-8.8, 17.4) 0.58 Of all AL treatments within 48 hours from a public health facility and where treatment started 4 days ago number who completed dose 54/80 (67.5) 57/70 (81.4) 13.9 (0.2, 27.6) 0.05 Proportion of caregivers who sourced AL from public sector who had dose explained 128/137 (93.4) 101/108 (93.5) 0.1 (-6.2, 6.3) 0.98 Range of proportion across clusters in 2009 (0–33.9%); 2010 (0–52.9%) P-value from chi-squared test adjusted for clustering doi:10 1371/journal pone 0130305 t003 Table 3. Comparison of AL treatment, timing and adherence between pre- (2009) and post-intervention (2010). All proportions are cluster adjusted ng and adherence between pre- (2009) and post-intervention (2010). All proportions are cluster adjusted Table 3. Comparison of AL treatment, timing and adherence between pre- (2009) and post-intervention (2010). All proportions are cluster adjusted PLOS ONE \| DOI:10.1371/journal.pone.0130305 July 2, 2015 9 / 19 Behaviour Change Communication and Fever Treatment Seeking Behaviour Table 4. Univariable and multivariable logistic regression analysis of factors predicting prompt (within 48 hours) treatment of fever with AL among children < 5 years of age in 2009. Univariable Multivariable Variable No. of febrile children N (%) febrile children with outcome OR (95% CI) P value OR (95% CI) P value Child age <1 yeara 90 18 (20.0) 1.0 1.0 1 year 118 17 (14.4) 0.5 (0.1, 1.6) 0.23 0.3 (0.1, 1.3) 0.11 2 years 298 58 (19.5) 0.9 (0.3, 2.4) 0.83 0.8 (0.2, 2.5) 0.71 Child gender Malea 246 39 (15.8) 1.0 1.0 Female 260 54 (20.8) 2.0 (0.9, 4.4) 0.10 2.5 (1.0, 6.5) 0.06 Caregiver level of education No educationa 22 2 (9.1) 1.0 1.0 Complete primary school 383 64 (16.7) 1.3 (0.2, 9.9) 0.81 2.2 (0.1, 32.7) 0.58 Complete secondary or higher 101 27 (6.7) 5.2 (0.6, 47.1) 0.14 5.3 (0.3, 94.2) 0.26 Caregiver age <20 yearsa 42 6 (14.3) 1.0 1.0 20–30 years 303 57 (18.8) 1.7 (0.4, 8.2) 0.47 1.2 (0.2, 8.1) 0.83 31–40 years 109 23 (21.1) 1.8 (0.3, 9.7) 0.49 1.0 (0.1, 7.7) 0.98 41–50 years 41 6 (14.6) 1.0 (0.1, 7.7) 0.99 0.5 (0.04, 6.7) 0.62 >50 years 11 1 (9.1) 0.5 (0.1, 14.1) 0.66 0.2 (0.002, 13.4) 0.43 Number of children under ﬁve years in household One childa 222 43 (19.4) 1.0 1.0 two children 284 50 (17.6) 0.8 (0.4, 1.8) 0.67 0.6 (0.2, 1.7) 0.34 Socio-economic category of household Quintile 1 (most poor)a 112 17 (15.2) 1.0 1.0 Quintile 2 (very poor) 86 21 (24.4) 3.4 (0.8, 13.6) 0.08 4.0 (0.8, 19.6) 0.08 Quintile 3 (poor) 106 24 (22.6) 2.9 (0.8, 10.9) 0.11 4.0 (0.9, 18.5) 0.08 Quintile 4 (less poor) 99 16 (16.2) 1.3 (0.3, 4.9) 0.70 1.0 (0.2, 5.1) 0.94 Quintile 5 (least poor) 103 15 (14.6) 1.1 (0.3, 4.0) 0.91 0.7 (0.1, 3.5) 0.68 Distance to nearest public health facility <1 kma 72 15 (20.8) 1.0 1.0 1-<2km 96 15 (15.6) 0.6 (0.1, 2.6) 0.53 0.6 (0.1, 3.1) 0.54 2-<3km 144 26 (18.1) 0.8 (0.2, 3.0) 0.78 0.7 (0.1, 3.1) 0.63 Table 4. Univariable and multivariable logistic regression analysis of factors predicting prompt (within 48 hours) treatment of fever with AL among children < 5 years of age in 2009. Discussion The haraka upesi behaviour change communication intervention to improve prompt and effec- tive treatment for children under five with a febrile illness targeted three steps on the fever 10 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0130305 July 2, 2015 Variable treatment seeking pathway: 1) prompt fever treatment, 2) fever treatment at a public health facility and 3) adherence to the AL treatment regimen. Overall, there were improvements in two key steps: prompt fever treatment and prompt access to AL, with a 5.1 percentage point increase observed in the primary outcome. In Bondo district, the majority of caregivers of children under five years of age with a fever sought treat- ment for their child both before the implementation of the intervention in 2009 (87.3%) as well as post intervention in 2010 (91.8%). There was a significant increase in prompt (within 48 hours) treatment seeking between 2009 and 2010 but this did not translate into increased access to anti-malarial treatment, with only approximately 40% of fevers (44.3% in 2009 and 39.2% in 2010) receiving any anti-malarial. However, the proportion of febrile children access- ing AL (32%) and accessing AL promptly (23%) in the post intervention period appears to be relatively high when compared to reports from earlier surveys on treatment seeking behaviour undertaken in Bondo district and in Kenya. The study by Gitonga and colleagues conducted in Bondo district between 2006 and 2007 found that only 11% of caregivers with a febrile child under 5 had accessed AL within 48 hours of fever onset [7]. It is possible that these differences are related to the short time that had elapsed between the implementation of the AL policy and the conduct of these surveys. Data from the most recent World Malaria Report shows that the proportion of febrile chil- dren accessing antimalarials in SSA ranges from 5.7% to 70% [20] and in most countries fewer than 20% of febrile children access ACTs promptly [21]. Despite interventions aimed at increasing prompt AL treatment in Bondo considerable efforts are required, as elsewhere in Africa, to reach the the RBM target of 80%. In Bondo, the proportion of febrile children whose caregivers reported using the govern- ment sector as the first source for fever treatment declined despite the focus of the communica- tion initiative being around prompt fever treatment at a public health facility. A recent multicountry study found that only 13.4% of febrile children in Benin, 16% in Uganda and the Democratic Republic of Congo (DRC), and 17.6% in Nigeria had been taken for treatment in a public health facility [22]. ariable logistic regression analysis of factors predicting prompt (within 48 hours) treatment of fever with AL in 2009. PLOS ONE \| DOI:10.1371/journal.pone.0130305 July 2, 2015 11 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0130305 July 2, 2015 Behaviour Change Communication and Fever Treatment Seeking Behaviour treatment seeking pathway: 1) prompt fever treatment, 2) fever treatment at a public health facility and 3) adherence to the AL treatment regimen. Overall, there were improvements in two key steps: prompt fever treatment and prompt access to AL, with a 5.1 percentage point increase observed in the primary outcome. In Bondo district, the majority of caregivers of children under five years of age with a fever sought treat- ment for their child both before the implementation of the intervention in 2009 (87.3%) as we as post intervention in 2010 (91.8%). There was a significant increase in prompt (within 48 hours) treatment seeking between 2009 and 2010 but this did not translate into increased access to anti-malarial treatment, with only approximately 40% of fevers (44.3% in 2009 and 39.2% in 2010) receiving any anti-malarial. However, the proportion of febrile children access ing AL (32%) and accessing AL promptly (23%) in the post intervention period appears to be relatively high when compared to reports from earlier surveys on treatment seeking behaviou undertaken in Bondo district and in Kenya. The study by Gitonga and colleagues conducted i Bondo district between 2006 and 2007 found that only 11% of caregivers with a febrile child under 5 had accessed AL within 48 hours of fever onset [7]. It is possible that these differences are related to the short time that had elapsed between the implementation of the AL policy an the conduct of these surveys. Data from the most recent World Malaria Report shows that the proportion of febrile chil- dren accessing antimalarials in SSA ranges from 5.7% to 70% [20] and in most countries fewe than 20% of febrile children access ACTs promptly [21]. Despite interventions aimed at increasing prompt AL treatment in Bondo considerable efforts are required, as elsewhere in Africa, to reach the the RBM target of 80%. In Bondo, the proportion of febrile children whose caregivers reported using the govern- ment sector as the first source for fever treatment declined despite the focus of the communic tion initiative being around prompt fever treatment at a public health facility. A recent multicountry study found that only 13.4% of febrile children in Benin, 16% in Uganda and th Democratic Republic of Congo (DRC), and 17.6% in Nigeria had been taken for treatment in public health facility [22]. Low use of government health facilities is not universal, however, and a study undertaken in Zambia reported almost half (49.8%) of febrile children accessed a public health facility for fever treatment [22], while public health facility utilization of 50% for fever treatment has been reported in Tanzania, Namibia, Mozambique, Djibouti, Sudan and Liberia [23]. A study undertaken in Tanzania following the roll out an intervention simila Table 4. (Continued) Univariable Multivariable Variable No. of febrile children N (%) febrile children with outcome OR (95% CI) P value OR (95% CI) P value 3-<4km 116 25 (21.5) 1.2 (0.3, 4.4) 0.81 1.4 (0.3, 6.9) 0.65 4-<5km 63 9 (14.3) 0.4 (0.1, 2.3) 0.34 0.4 (0.1, 2.7) 0.35 5km 15 3 (20.0) 1.3 (0.1, 15.6) 0.84 2.8 (0.1, 54.4) 0.50 a Reference group doi:10.1371/journal.pone.0130305.t004 Table 4. (Continued) Univariable Multivariable Variable No. of febrile children N (%) febrile children with outcome OR (95% CI) P value OR (95% CI) P value 3-<4km 116 25 (21.5) 1.2 (0.3, 4.4) 0.81 1.4 (0.3, 6.9) 0.65 4-<5km 63 9 (14.3) 0.4 (0.1, 2.3) 0.34 0.4 (0.1, 2.7) 0.35 5km 15 3 (20.0) 1.3 (0.1, 15.6) 0.84 2.8 (0.1, 54.4) 0.50 a Reference group doi:10 1371/journal pone 0130305 t004 Table 4. (Continued) Table 4. (Continued) Behaviour Change Communication and Fever Treatment Seeking Behaviour Table 5. Univariable and multivariable logistic regression analysis of factors predicting prompt (within 48 hours) treatment of fever with AL among children < 5 years of age in 2010. Univariable Multivariable Variable No. of febrile children N (%) febrile children with outcome OR (95% CI) p- value OR (95% CI) p- value Child age <1 yeara 90 15 (17.0) 1.0 1.0 1 year 105 19 (18.5) 1.4 (0.3, 5.7) 0.63 1.5 (0.3, 7.0) 0.56 2 years 320 87 (27.2) 4.5 (1.2, 17.4) 0.03 5.5 (1.3, 22.9) 0.02 Child gender Malea 257 61 (23.7) 1.0 1.0 Female 258 60 (23.3) 0.9 (0.4, 1.9) 0.83 0.8 (0.3, 1.9) 0.65 Caregiver level of education No educationa 10 0 (-) Complete primary school 425 95 (22.3) 1.0 1.0 Complete secondary or higher 80 26 (32.5) 2.4 (0.8, 6.8) 0.10 2.2 (0.6, 7.9) 0.20 Caregiver age <20 yearsa 43 7 (16.3) 1.0 1.0 20–30 years 307 80 (26.1) 2.9 (0.6, 13.8) 0.18 2.2 (0.3, 13.6) 0.40 31–40 years 111 21 (18.9) 1.4 (0.3, 7.5) 0.67 0.7 (0.1, 5.5) 0.79 41–50 years 41 9 (21.9) 1.9 (0.3, 13.7) 0.53 1.5 (0.1, 16.1) 0.72 >50 years 13 4 (31.0) 4.7 (0.3, 71.8) 0.26 4.3 (0.1, 115.6) 0.39 Number of children under ﬁve years in household One childa 210 53 (25.2) 1.0 1.0 two children 305 68 (22.3) 0.8 (0.4, 1.8) 0.63 1.0 (0.4, 2.6) 0.98 Socio-economic category of household Quintile 1 (most poor)a 117 23 (20.0) 1.0 1.0 Quintile 2 (very poor) 105 13 (12.4) 0.4 (0.1, 1.7) 0.24 0.3 (0.1, 1.5) 0.15 Quintile 3 (poor) 117 29 (25.0) 2.0 (0.6, 6.7) 0.25 1.5 (0.4, 5.9) 0.56 Quintile 4 (less poor) 98 32 (32.6) 3.5 (1.0, 12.3) 0.05 2.7 (0.6, 11.9) 0.19 Quintile 5 (least poor) 78 24 (31.0) 3.2 (0.8, 12.3) 0.08 1.8 (0.4, 8.6) 0.43 Distance to nearest public health facility <1 kma 37 11 (29.7) 1.0 1.0 1-<2km 217 48 (22.1) 0.5 (0.1, 2.2) 0.36 0.3 (0.1, 1.9) 0.20 2-<3km 123 31 (25.2) 0.7 (0.1, 3.2) 0.61 0.4 (0.1, 2.9) 0.40 3-<4km 68 16 (23.5) 0.4 (0.1, 2.4) 0.31 0.2 (0.02, 1.9) 0.16 (Continued) Behaviour Change Communication and Fever Treatment Seeking Behaviour Table 5. Univariable and multivariable logistic regression analysis of factors predicting prompt (within 48 hours) treatment of fever with AL among children < 5 years of age in 2010. Variable Low use of government health facilities is not universal, however, and a study undertaken in Zambia reported almost half (49.8%) of febrile children accessed a public health facility for fever treatment [22], while public health facility utilization of 50% for fever treatment has been reported in Tanzania, Namibia, Mozambique, Djibouti, Sudan and Liberia [23]. A study undertaken in Tanzania following the roll out an intervention similar to haraka upesi reported that more half (58%) of febrile children accessed a health facility as the first source for fever treatment, however, general health facility attendance among children was already high (76%) at baseline [24]. In general, in Kenya the reported use of the PLOS ONE \| DOI:10.1371/journal.pone.0130305 July 2, 2015 12 / 19 ariable logistic regression analysis of factors predicting prompt (within 48 hours) treatment of fever with AL in 2010. PLOS ONE \| DOI:10.1371/journal.pone.0130305 July 2, 2015 13 / 19 Variable government sector as the first source of treatment for febrile children ranges from 29.3% to 33.6% [23,25], lower than in Tanzania. In the current study undertaken in Bondo district Kenya, at baseline in 2009, 39.5% of the caregivers reported that their first treatment action for a febrile child was to take them to a government health facility and this dropped to 22.3% in 2010. A key factor that has been found to influence treatment seeking behaviour for a febrile ill- ness is the availability of drugs [24, 25–29]. Understandably, most caregivers will seek treat- ment from sources where they are sure of receiving treatment with a drug. Nationwide stock outs of AL at public health facilities were reported at the onset of this study between 2008 and 2009 and AL availability was variable throughout the entire study period. The political situa- tion in Kenya at the onset of the study and delays in AL procurement affected AL supply in Bondo district. Supply side problems in the public sector have been widely document as a key factor affecting its use [27, 30–32]. It therefore seems likely that fluctuations in AL supply might have led to a lack of patient confidence in our study population in the public sector’s ability to provide treatment for fever which contributed to the drop off in use of government health facilities as the first source of treatment that was observed in the post-intervention period. A more surprising observation was the increase between 2009 and 2010 in the number of reports of first treatments of a febrile child being undertaken with drugs sourced from within the household. It is possible that, in response to the fluctuations in AL supply mentioned previ- ously, caregivers had become inclined to hoard AL packs in the household leading to the observed increase in the proportion of home treatments (and home treatment with AL) in the post-intervention survey. Of concern is the fact that only one third of the AL treatments sourced from within the home were reported to have started with complete blisters packs while two thirds of AL drugs obtained within the households were incomplete packs. The presence of incomplete AL packs in the household and their use in subsequent febrile episodes has Table 6. of febrile children N (%) febrile children with outcome OR (95% CI) p- value OR (95% CI) p- value 4-<5km 54 13 (24.1) 0.6 (0.1, 3.6) 0.59 0.3 (0.03, 2.6) 0.27 5km 16 2 (12.5) 0.2 (0.1, 3.2) 0.23 0.1 (0.002, 1.7) 0.10 a Reference group doi:10.1371/journal.pone.0130305.t005 Table 6. Final multivariable regression of factors predicting prompt (within 48 hours) treatment of fever with AL among children < 5 years of age in 2010- (factors with p-value <0.05). Variable OR (95% CI) p-value Child age <1 yeara 1.0 1 year 1.4 (0.3, 6.0) 0.63 2 years 4.6 (1.2, 18.2) 0.03 a Reference group Table 5. (Continued) Univariable Multivariable Variable No. of febrile children N (%) febrile children with outcome OR (95% CI) p- value OR (95% CI) p- value 4-<5km 54 13 (24.1) 0.6 (0.1, 3.6) 0.59 0.3 (0.03, 2.6) 0.27 5km 16 2 (12.5) 0.2 (0.1, 3.2) 0.23 0.1 (0.002, 1.7) 0.10 a Reference group Table 5. (Continued) Table 5. (Continued) Behaviour Change Communication and Fever Treatment Seeking Behaviour government sector as the first source of treatment for febrile children ranges from 29.3% to 33.6% [23,25], lower than in Tanzania. In the current study undertaken in Bondo district Kenya, at baseline in 2009, 39.5% of the caregivers reported that their first treatment action for a febrile child was to take them to a government health facility and this dropped to 22.3% in 2010. A key factor that has been found to influence treatment seeking behaviour for a febrile ill- ness is the availability of drugs [24, 25–29]. Understandably, most caregivers will seek treat- ment from sources where they are sure of receiving treatment with a drug. Nationwide stock outs of AL at public health facilities were reported at the onset of this study between 2008 and 2009 and AL availability was variable throughout the entire study period. The political situa- tion in Kenya at the onset of the study and delays in AL procurement affected AL supply in Bondo district. Supply side problems in the public sector have been widely document as a key factor affecting its use [27, 30–32]. It therefore seems likely that fluctuations in AL supply might have led to a lack of patient confidence in our study population in the public sector’s ability to provide treatment for fever which contributed to the drop off in use of government health facilities as the first source of treatment that was observed in the post-intervention period. A more surprising observation was the increase between 2009 and 2010 in the number of reports of first treatments of a febrile child being undertaken with drugs sourced from within the household. It is possible that, in response to the fluctuations in AL supply mentioned previ- ously, caregivers had become inclined to hoard AL packs in the household leading to the observed increase in the proportion of home treatments (and home treatment with AL) in the post-intervention survey. Of concern is the fact that only one third of the AL treatments sourced from within the home were reported to have started with complete blisters packs while two thirds of AL drugs obtained within the households were incomplete packs. The presence of incomplete AL packs in the household and their use in subsequent febrile episodes has Table 5. (Continued) Univariable Multivariable Variable No. Behaviour Change Communication and Fever Treatment Seeking Behaviour Table 7. AL availability (all AL pack sizes) assessed by telephone interviews with health facilities (TI) or directs observation through physical audit (PA) at 33 government facilities in 2009 and 41 government and mission facilities in 2010 in Bondo district. Note: Emergency supplies sent on 22nd July 2010 to facilitate survey. AL 18 tablets (25-<35kgs) blister packs were not available for this emergency supply. In June and July 2010, six and twelve blis- ter packs were Coartem Dispersible; Table 7. AL availability (all AL pack sizes) assessed by telephone interviews with health facilities (TI) or directs observation through physical audit (PA) at 33 government facilities in 2009 and 41 government and mission facilities in 2010 in Bondo district. Note: Emergency supplies sent on 22nd July 2010 to facilitate survey. AL 18 tablets (25-<35kgs) blister packs were not available for this emergency supply. In June and July 2010, six and twelve blis- t k C t Di ibl Table 7. AL availability (all AL pack sizes) assessed by telephone interviews with health facilities (TI) or directs observation through physical audit (PA) at 33 government facilities in 2009 and 41 government and mission facilities in 2010 in Bondo district. Note: Emergency supplies sent on 22nd July 2010 to facilitate survey. AL 18 tablets (25-<35kgs) blister packs were not available for this emergency supply. In June and July 2010, six and twelve blis- ter packs were Coartem Dispersible; Number of health facilities Any AL blister packs in stock AL 6 tablets blister pack in stock AL 12 tablets blister pack in stock AL 18 tablets blister pack in stock AL 24 tablets blister pack in stock All AL blister packs in stock N n (%) n (%) n (%) n (%) n (%) n (%) January 2009 (TI) 33 25 (75.8) 16 (48.5) 22 (66.7) 15 (45.4) 19 (57.6) 10 (30.3) May 2009 (TI) 33 33 (100) 14 (42.4) 33 (100) 15 (45.4) 33 (100) 14 (42.4) June 2009 (PA) 33 33 (100) 33 (100) 33 (100) 31 (93.9) 33 (100) 31 (93.9) October 2009 (TI) 33 31 (93.9) 31 (93.9) 31 (93.9) 31 (93.9) 30 (90.9) 30 (90.9) January 2010 (TI) 41 40 (97.6) 27 (65.8) 37 (90.2) 37 (90.2) 37 (90.2) 24 (58.5) April 2010 (TI)* 41 38 (93.0) 19 (46.3) 25 (61.0) 29 (70.7) 37 (90.2) 17 (41.5) May 2010 (TI)* 41 32(78.0) 13(31.7) 18(43.9) 17(41.5) 33(80.5) 8(19.5) June 2010 (TI)* 41 37 (95.0) 35 (89.7) 35 (89.7) 34 (87.2) 36 (92.3) 33 (84.6) July 2010 (PA)* 41 41 (100) 41 (100) 41 (100) 18 (43.9) 41 (100) 18 (43.9) August 2010 (PA)* 41 39 (95.1) 39 (95.1) 38 (93.0) 10 (24.4) 37 (90.2) 10 (24.4) Includes three new government health facilities established and commissioned in January 2010 through the Constituency Development Fund (CDF) initiative and ﬁve mission health facilities doi:10 1371/journal pone 0130305 t007 Includes three new government health facilities established and commissioned in January 2010 through the Constituency Development Fund (CDF) initiative and ﬁve mission health facilities doi:10.1371/journal.pone.0130305.t007 implications for correct dosing as it suggests that febrile children are receiving inadequate doses. AL is taken over a three-day period and has a complex dosage scheduled. Several studies have reported varying adherence rates for ACTs of between 38.7% and 93% [33–45] and poor adherence to AL has been suggested as a major constraint to its effectiveness [37, 44, 46]. In the current study reported adherence to AL increased from around two thirds (67%) to over 80% among caregivers who sourced their treatment from the public health facility but this increase was not reflected in overall adherence rates, perhaps due to the decline in the use of the govern- ment sector. Variable Final multivariable regression of factors predicting prompt (within 48 hours) treatment of fever with AL among children < 5 years of age in 2010- (factors with p-value <0.05). Variable OR (95% CI) p-value Child age <1 yeara 1.0 1 year 1.4 (0.3, 6.0) 0.63 2 years 4.6 (1.2, 18.2) 0.03 a Reference group doi:10.1371/journal.pone.0130305.t006 Table 6. Final multivariable regression of factors predicting prompt (within 48 hours) treatment of fever with AL among children < 5 years of age in 2010- (factors with p-value <0.05). PLOS ONE \| DOI:10.1371/journal.pone.0130305 July 2, 2015 14 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0130305 July 2, 2015 Acknowledgments We thank Sumaya Sanya for survey supervision and data entry. We also wish to thank Terry Muchoki and Evans Odhiambo of PSI and Lillian Achieng, Norah Ochiel, Victoria Adhiambo and Beatrice Miringu of Experiential Marketing Agency (EXP), Kenya for providing details of the intervention, Victor Alegana for help producing maps for use in the field and Viola Kirui for analysis of travel distances and production of maps used in this publication. We acknowl- edge Professors Abdisalan Mohamed Noor and Dejan Zurovac for their comments on earlier versions of the manuscript. Special thanks are conveyed to Dr. Elizabeth Juma for advice and comments during the designing of this study and to Dr. Charles Mwandawiro for logistics and administrative support. This paper is published with the permission of the director KEMRI. Conclusion Overall the data suggest that, during the pre- and post-intervention period the biggest barrier to effective treatment for febrile children under five in Bondo district was the infrequent use of the government health facilities as the first step in treatment seeking. There was evidence of a significant shift in treatment seeking behaviour away from the government sector (the inter- vention target delivery point) and the likely reasons for the shift in source of treatment relates to the implementation context mentioned above (drug stock-outs) which lay outside of the control of the intervention. These data demonstrate the importance of understanding the implementation context when analysing and interpreting intervention outcomes. The context may be the driver of how and whether the intervention works. They also demonstrate that in contexts where treatment for febrile illnesses is available from more than one sector, interven- tions that target only one sector will struggle to produce population level improvements in prompt and effective treatment. To make a real impact upon effective case management of febrile children at the national level, cross-sectoral interventions are needed. These are inter- ventions across the potential sources of fever treatment and include public and mission, private and retail sectors. The evaluation design included a post-intervention comparison to measure changes in the fever treatment seeking indicators. We made adjustments for multiple comparisons using the Bonferroni procedure [0.001 (0.05/34)]. However, the analyses presented in this study, do not allow the attribution of any of the observed changes in the primary and secondary indicators to reported exposure to the intervention. That is, the analysis provides an adequacy inference with no attempt to attribute the observed changes to the haraka upesi intervention. The Intervention reported in this study was a complex intervention implemented under routine operational conditions. Such interventions present particular problems for evaluators, as they contain several interacting components, they often involve a number of different behav- iours and they tend targets different levels within a system (for example household and health facility). In addition to the complexity of the intervention, a key strategy in the BCC campaign PLOS ONE \| DOI:10.1371/journal.pone.0130305 July 2, 2015 15 / 19 Behaviour Change Communication and Fever Treatment Seeking Behaviour was the use of mass media (radio). Where mass media are employed it is difficult to identify an appropriate control group that is not exposed to the intervention. Under these conditions it was not possible to employ a probability evaluation. In this evaluation we undertook a process evaluation to explore the way in which the intervention had been implemented and outcome data in this study was collected using pre- and post-cross-sectional community surveys to assess changes in caregiver treatment seeking behaviour. Overall, the evaluation cannot completely isolate the effect of the haraka upesi intervention In this evaluation we undertook a process evaluation to explore the way in which the intervention had been implemented and outcome data in this study was collected using pre- and post-cross-sectional from those of other concurrent processes or activities. Furthermore, the use of pre- and post-intervention evaluation design does not account for the changes that occurred between the two time points that were not related to the intervention. The time interval between intervention implementa- tion and evaluation has been has also been highlighted as a methodological challenge in the evaluation of BCC (www.cpc.unc.edu/measure/prh/rhindictators/crosscutting/bcc accessed 25/ 07/2013). References 1. Roll Back Malaria (2008) Global Malaria Action Plan. Geneva. http://www.rbm.who.int/gmap/gmap.pdf. 2. Snow RW, Marsh K (2010) Malaria in Africa: progress and prospects in the decade since the Abuja Declaration. Lancet 376: 137–139. doi: 10.1016/S0140-6736(10)60577-6 PMID: 20417552 3. United Nations Children Fund (2013) Invest in the future: Defeat malaria; World Malaria Day 2013. Focus on Africa. New York, United Nations Children Fund. 4. Amin AA, Zurovac D, Kangwana BB, Greenfield J, Otieno DN, Akhwale WS, et al. (2007) The chal- lenges of changing national malaria drug policy to artemisinin-based combinations in Kenya. Malar Journal 6: e72. 5. Pharmacy and Poisons Board (PPB) and Division of Malaria Control (2007) Antimalarial Medicines in Kenya—availability, quality and registration status: A baseline study undertaken prior to widespread distribution of Artemether-Lumefantine (AL) in Kenya. Ministry of Health, Republic of Kenya, November 2007. 6. Zurovac D, Ngigi J, Akhwale WS, Hamer DH, Snow RW (2008) Translation of artemether-lumefathrine treatment policy into paediatric clinical practice: an early experience from Kenya. Tropical Medicine and International Health 13: 99–107. doi: 10.1111/j.1365-3156.2007.01980.x PMID: 18291008 7. Gitonga CW, Amin AA, Ajanga AA, Kangwana BB, Noor AM, Snow RW(2008) The use of artemetherlu- mefantrine by febrile children following national implementation of a revised drug policy in Kenya. 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W4293078635.txt	https://www.researchsquare.com/article/rs-1671781/latest.pdf	en		Experimental investigation of kinetic parameters of bamboo and bamboo biochar using thermogravimetric analysis under non-isothermal conditions	Research Square (Research Square)	2,022	cc-by	7,858	Experimental investigation of kinetic parameters of bamboo and bamboo biochar using thermogravimetric analysis under non-isothermal conditions Priti Jagnade (  priti.jagnade@gmail.com ) Maharana Pratap University of Agriculture and Technology College of Technology and Engineering https://orcid.org/0000-0002-8189-5971 N. L. Panwar Maharana Pratap University of Agriculture and Technology Chitranjan Agarwal Maharana Pratap University of Agriculture and Technology Research Article Keywords: Bamboo, pyrolysis, biochar, kinetics, TG/DTG evaluation, activation energy Posted Date: June 8th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1671781/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Experimental investigation of kinetic parameters of bamboo and bamboo biochar using thermogravimetric analysis under non-isothermal conditions Priti Jagnade1 and N. L. Panwar1, Chitranjan Agarwal2 1 Department of Renewable Energy Engineering, College of Technology and Engineering, Maharana Pratap University of Agriculture and Technology, Udaipur 313001 Rajasthan, India 2 Department of Mechanical Engineering, College of Technology and Engineering, Maharana Pratap University of Agriculture and Technology, Udaipur 313001 Rajasthan, India Abstract The subject of this research is the thermogravimetric analysis of bamboo and bamboo biochar in an inert environment at 10, 20, and 30 °C/min. The physicochemical characterizations of bamboo and bamboo biochar were carried out as per the standard methods. Vacuum pyrolysis was used for the bamboo biochar. The FWO (Flynn-Wall-Ozawa) and KAS (Kissinger–Akahira– Sunose), methods determined thermodynamic and kinetic parameters within the active pyrolysis zone. Thermal degradation bamboo biomass undergoes several steps of loss of mass, including moisture loss, passive and active pyrolysis. Between 180 °C and 395 °C, the active pyrolysis zone accounted for 50 to 55 percent of the mass loss. For FWO and KAS models, bamboo biochar had lower activation energy values (99.23 and 96.07 kJ/mol) than bamboo biomass (262.5303 and 266.62 kJ/mol). The findings of the study for bamboo and its biochar revealed a significant opportunity in the agro industry for designing and building pyrolysis reactors for long-term biofuel generation. Keywords: Bamboo, pyrolysis, biochar, kinetics, TG/DTG evaluation, activation energy  Corrosponding author Email: priti.jagnade@gmail.com (Priti Jagnade) 1 1. Introduction Fossil fuel energy is valuable for economic and long-term growth [1]. On the other hand, increased population and industrialization have played a significant role in inflating global energy demand, resulting in uncontrolled energy use. Moreover, the release of hazardous gases (CO2, SOx, NOx) due to the indiscriminate burning of fossil fuel reserves has a negative impact on the environment [2]. Therefore, the use of renewable energy increases as the global energy demand rises [3]. Among the various sustainable energy sources like solar, biomass, hydropower, and wind, biomass is an environmentally friendly and carbon-neutral energy sources [4]. It's also been proven that biomass releases fewer gaseous pollutants during combustion [5]. Many countries have large forests and agricultural areas where biomass resources provide about 40–50% of the required energy in many developing countries [6]. Biomass production in India ranges from 450 to 500 million tonnes. Biomass accounts for 32 percent of the country's overall primary energy consumption [7]. Biomass has the potential to supplement coal to the tune of 260 million tonnes. Every year, this could result in a savings of around Rs. 250 billion [8]. There are mainly two processes for biomass conversion: biological and thermochemical conversion processes [9]. In thermochemical conversion, pyrolysis is the most feasible process that extracts energy from biomass into various products like biochar, bio-oil, and syngas [10]. Biomass carbonization is influenced by reaction conditions such as heating rate, biomass composition, biomass particle size, pressure, and residence time. The application of these biomass technologies for energy generation necessitates a thorough knowledge of biomass thermal decomposition properties (such as cellulose, hemicelluloses, and lignin) and reaction kinetics, which are important for better understanding and improving pyrolytic conversion. Thermogravimetric analysis is one of the most common methods for examining pyrolysis reactions' kinetic and thermodynamic parameters [11]. Thermogravimetric investigations, which are required for estimating the kinetic factors of solid-state processes, record variations in mass loss as a function of temperature and time. The two most common methods for quantitatively describing biomass pyrolytic kinetics are model-free (isoconversional) and model-fitting [12]. Model-free techniques are the most widely used in kinetics analysis because the kinetic parameters are obtained from data gathered at various heating rates, 2 making them more reliable, ideal, and accurate for kinetic analysis than single heating rate or model-fitting methods [13]. As a result, iso-conversional methods, FWO and KAS are frequently used to estimate the thermodynamic and kinetic parameters of the pyrolysis process of various biomass like activation energy, enthalpy frequency factor, Gibbs free energy, entropy and reaction order etc. [14] [15]. Compared to woody biomass, bamboo has a high yield and a short growth cycle, making it a promising renewable biomass [16]. Bamboo is used for various applications like making bamboo houses, mats, knives, activated carbon, papermaking, and many others [17]. In recent times, a new approach of employing bamboo as an alternative energy source to replace fossil fuels that have gone out of stock has been introduced to the list [18]. Bamboo can potentially be used to generate energy in the future [16]. This new approach must be thoroughly investigated to maximize the use of bamboo biomass while avoiding or minimising any potential risks to mankind and the environment. However, the range of research on bamboo (woody biomass) is relatively restricted. As a result, studying bamboo's pyrolysis properties is extremely beneficial to comprehension the process conversion and utilizing bamboo as a biofuel. The main components of bamboo biomass are cellulose, hemicellulose, and lignin, with 35–45 percent, 15– 20 percent, and 15–25 percent, respectively. Chen et al. studied the effects of rate of heating at 5, 10, 20, and 300C/min on the product properties of moso bamboo. They observed that the pyrolysis technique can be broken down into several steps and is similar to that of other biomasses. Hemicelluloses, cellulose, and lignin decompose at temperatures ranging from 200 to 380 degrees Celsius, 250 to 380 degrees Celsius, and 180-900 degrees Celsius, respectively [19]. There are some researchers examined kinetics analyses of bamboo wasete. Mallick et al. examined the kinetic analysis of bamboo waste where the Eα (average activation energies) values for the FWO and KAS methods were found to be 181.973 kJ/mol and 183.113 kJ/mol for the degree of conversion 0.1–0.9 [20]. In addition to this, Poletto et al. investigated the average activation energy of pine and eucalyptus wood. They found that the values for FWO for the rate of conversion 0.1–0.8 were 191 and 208 kJ/mol, respectively [21]. The present study investigated the thermal degradation of bamboo and bamboo biochar produced from vacuum pyrolysis by using thermogravimetric. The bamboo and bamboo biochar were thermally degraded at 10, 20 and 30 °C/min. The KAS (Kissinger-Akahira-Sunose) and FWO (Flynn-Wall-Ozawa method) are two iso-conversional methods used to calculate Ea 3 (activation energy) at 0.1 to 0.9 conversion rate. In addition, Thermodynamic variables such as enthalpy, entropy, and Gibbs free energy were also calculated using iso-conversional models. 2. Methodology 2.1 Feedstock preparation The bamboo (B) was acquired from the experimental learning field of MPUAT, Udaipur, India. Bamboo wood can be cut into 2-5 cm pieces to produce biochar. The bamboo pieces were dried in a solar dryer for 24 hours to remove excess moisture. For physicochemical properties and thermogravimetric analysis, the materials are crushed and sieved to acquire particle sizes of less than 100 microns. 2.2 Biochar production Two kilograms of biomass feedstock were pyrolyzed at 600 °C for 60 minutes under reduced pressure of 18–25 kPa in a vacuum pyrolysis reactor. It was found that the bamboo biomass (B) was carbonised and turned into bamboo biochar (BB). Both samples were ground in a grinder to obtain the smallest particle size possible. The powdered B and BB samples were kept in an airtight container to prevent further moisture attained. 2.3 Physicochemical properties The moisture, ash, and volatile matter of bamboo and bamboo biochar samples were determined using ASTM D3174, ASTM D3173, and ASTM D3175 standard procedures, respectively [22]. The HHV (higher heating value) of B and BB was obtained using a bomb calorimeter. The cellulose, hemicellulose, and lignin percentages in B feedstock were obtained from TGA using the DTG curve. Using an elemental analyser, the available carbon (C), hydrogen (H), nitrogen (N), and oxygen (O) content in B and BB were examined. To achieve the average value, all of the abovementioned tests were performed three times using the standard error experiment. 2.4 Thermo-gravimetric analysis The produced B and BB material were analysed in a thermogravimetric analyzer, (Hitachi STA7300) obtain a thermal decomposition pattern of feedstock. In TG analysis, temperature from 30 to 900 °C, the finely ground material is heated at 10, 20, and 30 °C/min. To avoid undesired oxidation reactions inside the pyrolysis zone, nitrogen (N2) gas was provided at 80 ml/min flow rate in all experimental runs. With the help of TG data, the differential thermo gravimetric values (DTG) were updated using Origin Pro software. 4 2.5 Kinetic analysis The kinetic analysis of feedstock gives crucial data for developing and optimizing biofuel generation systems. Using non-isothermal iso-conversional methods, the kinetic analysis of bamboo and its biochar obtained from vacuum pyrolysis was carried out. Biomass pyrolysis is a thermochemical conversion process that can be described by equation 1: k (T) Feedstock(Bamboo) → Where, k- constant rate Soild biochar + Volatiles (Gas + Tar) (1) Under non-isothermal conditions, the conversion rate from solid (biochar) state to volatile (Gas+ liquid) product following reaction can be used to described [23]. dα dt = k (T) f (α) (2) Where, α (degree of conversion) can determined by the mass-loss given as follows, [24]: α= m0 − mT (3) m0 − mf Constant rate depend on temperature ‘T’ which is given by Arrhenius equation Ea (4) Ea (5) k(T) = Ae−(RT) Combining Equations (2) and (4) gives ) k(T) = Ae−(RT f(α) Where, m0, mf - Initial and final mass mT - Instantaneous mass T - Temperature, K A - Pre-exponential factor (s-1); Ea - the apparent activation energy, kJ.mol-1; R - Gas constant, J.mol -1.K-1. According to Apaydin-Varol et al., the f (α) (expression of the function) is used to describe (n) order of reactions and is directly proportional to the concentration of non-degraded material [25]. As a result, expression of the function is mathematically expressed as follows: f(α) = (1 − α)n (6) Substituting equation (6) for equation (5) yields the following equation. 5 Ea k(T) = Ae−(RT) f(1 − α)n (7) T = T0 + βt (8) During the non-isothermal method, the β (heating rate) remains linear to increase the temperature. dT = βdt (9) Where, T0, are initial temperature. dα = dT dα 1 (10) dt β Where; dt/dT = (1/β) and dα/dt = isothermal reaction rate, and dα/dT = non-isothermal rate of reaction. Equation (7) can be substituted into Equation (10) to obtain formula of the rate law for non-isothermal conditions: dα dT = A −( Ea ) e RT β f(1 − α)n (11) This Equation (11) refers to the biomass decomposition. The kinetic parameters of the are calculated using this expression and was integrated a g(α) = ∫0 g(α) = (1− α)n AEa βR dα = A T ∫ e β T0 ∞ E − a ∫X u−2 e−u du = RT dT AEa βR P(X) (12) (13) Where, P(x) and Ea/RT are the exponential integral. 2.6 Iso-conversional methods For non-isothermal thermogravimetric analysis, fitting models or free models can be used to evaluate kinetic parameters. As a function of temperature or conversion, the kinetic parameters are determined. In the iso-conversional method, reaction rate is solely determined by thermogravimetric data and temperature, and it required minimum three different heating rates [26]. 6 Complex processes involving a series of chemical reactions are described using isoconversional approaches. Their exact reaction mechanism, however, is unknown. Therefore, differential or integral approaches can be used to describe iso-conversional methods. The current research examined kinetic parameters from two iso-conversional methods: FWO (Flynn–wall– Ozawa) and KAS (Kissinger–Akahira–Sunose). 2.6.1 Flynn–Wall–Ozawa (FWO) method The Flynn–Wall–Ozawa is a model-free iso-conversional method. For obtains the activation energy (Eα) the ploting ln βi versus 1/Tαi for a given value of conversion (α) at different heating rates (β), In (βi ) = In ( AEα RG(α) ) − 5.331 − 1.052 Eα RTαi (14) Where βi and αi the rate of heating for a given value of i and g (α) is constant. The slope 1.052 𝐸𝛼 𝑅𝑇𝛼𝑖 is used to estimate Eα (activation energy). 2.6.2 Kissinger–Akahira–Sunose (KAS) method KAS is also model free iso-conversional method for calculating the Eα (activation energy) as given as follows. In verses βi T2 αi =( AR )− Eα G(α) Eα (15) RTαi The activation energy calculated using the slope of the equation derived from In 1 Tαi βi T2 αi . The slope of the straight line (Eα/R) using for calculated activation energy. In comparison to FWO, it provides more precise or exact activation energy estimates [27]. 2.6.3 Thermodynamic parameters FWO and KAS models have been used to estimate Eα (activation energy), and also used to calculate thermodynamic parameters such as A (pre-exponential factor), ΔH (enthalpy), ΔG (Gibbs energy), and ΔS (entropy) etc. listed by below equation 16,17,18 and 19, respectively [15]. A = βEα exp Eα ) RTm RTm 2 ( 7 (16) ∆H = Eα − RTα ∆G = Eα + RTm ln ( ∆S = ( (17) Tm kB ∆H− ∆G Tm ) hA ) (18) (19) Where, Tm, kB and h are the peak temperature, Boltzmann constant and plank constant, in DTG curve respectively. 3. Results and discussions 3.1 Characteristics of bamboo and bamboo biochar Biomass characterization is crucial in determining its physico-chemical properties, which are significant in deciding its ability as a source of fuel. Table 1 summarises the physicochemical and calorific values of B and BB determined in the current study. Bamboo has a moisture content of 5.26 percent, which is less than 10% and makes it acceptable for thermal processing [28]. According to Table 1, bamboo biomass has lower ash content and a higher volatile matter content than other agricultural residues, 2.18% and 77.12%, respectively. Biomass fuels with low ash and high volatile matter are more suitable for thermochemical conversion [29]. When feedstock has a higher ash percentage, it usually has an inverse proportional with biochar heating value of the biochar produced [30]. It also slows down the burning process and causes aggregation and fouling [20]. The heating value of bamboo biomass is 18.50 MJ/kg. Bamboo has a C (carbon) 45.05%, H (hydrogen) 5.57 %, N (nitrogen) 0.56 %, and an O (oxygen) 39.42 %. The low percentage of nitrogen in bamboo means it produces fewer nitrogen oxide emissions during combustion. [20]. Compared to raw bamboo biomass, bamboo biochar has contained less percentage of ash and volatile matter content. Therefore, thermochemical conversion might be able to remove some of the oxygen-based chemical groups in organic waste [31]. The produced biochar had a heating value of 28.25 MJ/kg, greater than the other biochar sample produced from agro-waste [32]. As shown in Table 2, the combined hemicellulose and cellulose content of bamboo biomass is around 67.96 percent, with hemicelluloses and cellulose accounting for 45.34 percent and 22.62 percent, respectively, and a lower lignin content of 21.83 percent. Endothermic reactions are involved in the decomposition of biomass with high lignin content [33]. 8 Table 1: Physico-chemicals properties of bamboo and its biochar Analysis Proximate analysis Moisture content Volatile matter Ash content Fixed carbon HHV (MJ/kg) Ultimate analysis C H O (by difference) N S Bamboo (present study) Moso bamboo [34] Bamboo Biochar (Present study) Moso bamboo Biochar [19] 5.26 77.12 2.18 15.44 18.50 8.67 74.81 2.56 13.96 - 1.3 6.29 5.36 87.05 27.8 5.27 4.89 89.84 28.25 45.05 5.57 39.42 0.56 0.03 44.87 5.73 38.32 0.71 0.01 88.34 2.01 8.33 1.2 0.12 89.71 1.19 7.91 1.05 0.14 Table 2: Lignocellulosic percentages of bamboo Cellulose Hemicellulose Lignin Extractives References 45.34 46.45 ± 3.0 22.62 19.23 ± 3.0 21.83 18.17 ± 2.0 10.21 - Present study [20] 3.2 Thermogravimetric analysis of bamboo and bamboo biochar The thermal behaviour of bamboo and its biochar was evaluated in a thermogravimetry furnace at rate of heating of 10, 20, and 30 0C/min was shown in Fig 1 and Fig 2 respectively. In the TG analyzer's thermal performance, bamboo and bamboo biochar go through three different phases: moisture removal, devolatilization, and biochar formation. Biomass mainly consists of, cellulose, hemicellulose, and lignin. During pyrolysis, they respond differently at specific temperatures and heating rates. Hemicelluloses, cellulose, and lignin decompose at temperatures ranging from 200 to 380 degrees Celsius, 250 to 380 degrees Celsius, and 180-900 degrees Celsius, respectively [19]. Thermal decomposition of bamboo mainly occurs in three different temperature stages, viz., drying, devolatilization, and char formation. At temperatures below 200°C, the physically and externally bound moisture from bamboo and its biochar is removed in the initial phase. The second stage entails of pyrolysis, hemicellulose, and cellulose thermally decompose, with the decomposition rate reaching its maximum. The second phase is divided into 9 two parts: left and right sides. Left side indicates the decomposition of hemicelluloses. At moderate temperatures, biochar degrades more slowly; this could be due to the char's low volatile component content [35]., The bamboo biochar indicates the highest temperature of degradation in the second region As shown in Fig. 2, which was possible due to the biochar's low volatile content and high availability of fixed carbon, which causes decomposition to occur at a higher temperature [36]. This research looked into how the temperature range in the bamboo pyrolysis process and the mass loss rate were influenced by the heating rate according to the DTG and TG curves represented in Fig 1 and Fig. 2, respectively. Furthermore, increasing the heating rate may cause the DTG and TG curves very as the temperature rises, but the total mass loss remains constant. Due to the low thermal conductivity of biomass, the effects of heat and mass transfer increase as the pyrolysis rate increases [37]. As a result, the TG curves shifted to a higher temperature. Overlapping curves indicate a complex composition of hemicellulose, cellulose, and lignin in bamboo biomass [38]. Table 3 summarises the intensity of decomposition and the corresponding temperatures for the various heating rates of bamboo. The first degradation peak in the DTG curve is depicted in Fig. 1, which is separated into three stages: Stage I (30 to 180 0C) is dehydration stage in this stage some moisture and extractives are released, Stage II (180 0C to 395 0C) about 50 to 55 % of mass loss occurred this is the active pyrolysis zone where hemicellulose and cellulose decompose, with hemicellulose decomposition causing the first peak of left side, and Stage III (3950C to 579 0C), lignin degradation occurred in this zone, as well as some endothermic and exothermic reactions takes placed between organic compounds. Lignin degradation begins at 420 0C and reaches a broad peak before slowing down to a closing carbonization temperature of 580 0C. Lignin has better heat stability requires a higher temperature (100–900 0C) to degrade completely of the slower rate of reaction [39] [40] [41]. The rate of devolatilization was almost stable after 600 0C, indicating that the pyrolysis had completed this stage, indicating the biochar's formation. This means that the devolatilization process has completely stopped after 600 0C. The most mass degradation occurred in second stage of the curve. As a result, stage II is considered active pyrolysis for determining the kinetics, and stages I and III are considered passive pyrolysis zones [42]. Thermal performance of bamboo biochar was accomplished at 10, 20, and 30 °C/min with the help of a thermogravimetric analyser. Fig. 2 shows that the first peak shows at the 10 temperature of 40 to 60 0C at which biochar is released with moisture. At temperatures of 170 to 390 0C, the peak totally disappeared when it was compared with raw bamboo. The least mass loss (1.5-2%) was observed at temperatures ranging from 390 to 480 0C, indicating the highest thermal stability of obtained biochar [43]. When the temperature was raised from 480 to 740 °C, the maximum weight loss was observed, which is about 75–78%, whereas there was no loss of mass in the bamboo biochar when the temperature was raised above 740 °C. These studies indicate that the pyrolysis process had no effect on the characteristics of biochar after a period of time [44]. A similar graph pattern was found by [45] in their kinetic and thermal study. Table 3: Pyrolysis parameters relevant to heating rates for bamboo biomass Bamboo Parameters Stage I Stage II Stage II β, ◦C/min 10 20 30 10 20 30 10 20 30 Ti,0C ( Initial temperature) 30 30 30 171 175 180 380 385 395 Tf, 0C (final temperature) 171 175 180 380 385 395 565 570 579 11 Tm, ◦C (Temperature at max decomposition) 52 63 67 335 340 344 482 483 493 DTG max, %/min (max decomposition rate) 1.33 3.09 4.69 6.79 20.79 38.49 2.19 6.60 13.14 Temperature (°C) 0 100 200 300 400 500 600 700 800 900 120 TG (%) 90 60 30 (Passive pyrolysis ) Stage I Stage II 10°C/min 20°C/min 30°C/min Stage III 0 (Active pyrolysis ) -30 DTG (%/min) -60 Cellulose peak Hemi-cellulose 40 30 Lignin peak 20 Dehydration peak Char formation 10 0 0 100 200 300 400 500 600 Temperature (°C) 700 800 900 Fig. 1 Thermal decomposition behaviour of raw bamboo at heating rate of 10 0C/ min, 20 0C/min and 30 0C/min showing different peak of dehydration, devolatilization, and biochar formation. 12 Temperature (°C) 0 100 200 300 400 500 600 700 800 TG (%) 100 900 1000 10 °C/min 20 °C/min 30 °C/min 80 60 40 20 Stage I Stage III Stage II DTG (%/min) 0 6 4 2 0 0 100 200 300 400 500 600 700 800 900 1000 Temperature (°C) Fig. 2 Thermal decomposition behaviour of bamboo biochar at heating rate of 10 0C/ min, 20 0C/min and 30 0C/min for analyzing the mass loss at different stages. 3.2 Kinetic analysis Iso-conversional methods were used to determine the kinetic parameters of bamboo and bamboo biochar such as A (pre-exponential factor) and Eα (activation energy). The apparent activation energy of bamboo and bamboo biochar determined with FWO and KAS were shown in the Fig. 3(a & b) and Fig 4 (a & b), respectively. Fig 3 (a & b) and Fig 4 (a & b) shows all lines are nearly parallel, indicating that the apparent activation energy can be closed and possibly indicating single reaction mechanisms [46]. As per, the FWO model, Eα can be evaluated from the slope at increasing degrees of conversion estimated from the linear plots of ln(β/T2) versus 1/T as appear in Fig. 3 (a & b), using equation(14). In the KAS method Eα was calculated using the and eq (15), Fig 4(a & b) depict the linear plots of ln(T) versus 1/T. The activation energies for bamboo were determined to be within the range of 183 –327 kJ/mol using the FWO and KAS models of 0.1–0.9 in the conversion range, respectively. The average activation energies for bamboo using the Flynn–wall–Ozawa and Kissinger–Akahira–Sunose models were observed to be 262.5303 and 266.62 (KJ/mol) respectively. The difference in all average values of activation 13 energy noted is less than 5%, which justifies its reliability and accuracy [47]. The activation energy values obtained in this study were found to be very close to timber, which was determined by FWO and Starink models to be 300-222 KJ/mol respectively [48] and higher side to those of bamboo subfamily (200-225 KJ/mol), and bamboo waste (181.97- 183 KJ/mol) Flynn–wall–Ozawa and Kissinger–Akahira–Sunose models, respectively [34] [20]. Several factors influenced the activation energy values, including the different kinetics models, heating rate, type of biomass, and particle size [49]. The model-free methods' Eα (activation energy) differ due to estimates and calculations used to solve the temperature integral. These findings suggest that bamboo could be used as a feedstock for biomass thermal conversion to bioenergy. Table 4 illustrate that as the activation energy increased as the degree of conversion rate (α) increased from 0.1 to 0.9, the, indicating that the complex reaction may have occurred at a higher temperature [50], thermal cracking and devolatization of wood components occur at this stage, implying a series of reaction [51]. The Eα values increased from 189 to 326 KJ/mol and from 189 to 335 KJ/mol for FWO and KAS, shown in Fig 5 (a & b). The linear correlation coefficients for determining the activation energy ranged from 0.88 to 0.99, which was nearly suitable for liner plots. The FWO and KAS methods were used to compute the activation energy values for bamboo biochar generated at 600 °C in order to estimate its thermal potential for energy generation via combustion. As shown in Table 5, the average values Eα bamboo biochar of for the FWO and KAS models were 99.23 and 96.07 kJ/mol, respectively. Bamboo biochar had a lower Eα value than raw bamboo, indicating that the biochar produced from the biomass can easily react with the low energy requirement shown in Fig 5b. As biochar has high carbon contain, higher heating value, higher lignin composition, and higher aromaticity, it easily reacts with a low-energy supply [47] . Fig 5 (a & b) shows the activation energy change for bamboo and bamboo biochar verses degree of conversion using the FWO and KAS methods. The pre-exponential factor (A) is crucial for biomass pyrolysis optimization. For the model-free kinetic approach, Eα and A factor have a mathematical relationship such that changing (Eα) according to the pyrolysis extent changes (A) as well [52]. In the current research, the pre-exponential factor was calculated by using Coats-Redfern method as given in equation (12). The values of A (pre-exponential factor) from FWO and KAS ranges for 2.37 × 1013 to 4.35× 1024 S-1, 2.79 × 1013 to 2.31× 1025 S-1 respectively. The wide variation in pre-exponential 14 factor with respect to the degree of conversion rate denotes the feedstock natural structure composition complexity and the complex pyrolysis reaction [53]. The pre-exponential factor is the intersection of three different parameters, such as activate energy, gas constant, and heating rate. The value of pre-exponential is greater than 109 S-1 indicates the reactive system simple complex reaction and the value 109 S-1 indicates less reactive with a closed complex reaction [54]. The pre-exponential factor values for all KAS and FWO models were summarised in Table 4 using Eq (12). (b) FWO 3.6 3.4 3.2 3 2.8 2.6 2.4 2.2 2 0.1 0.1 0.2 3.6 0.2 0.3 0.3 3.2 log(β) log(β) (a) FWO 0.4 0.5 0.6 0.4 2.8 0.5 2.4 0.6 0.7 0.7 1.5 1.7 (1/T) x 1000 1.9 2 0.75 0.8 1 1.25 (1/T) x 1000 0.9 0.8 1.5 0.9 Fig. 3 (a & b) Linear plots of ln (β/T2) versus 1/T for raw bamboo biomass and bamboo biochar respectively using FWO method for calculating the activation energy. (a) KAS 1.7 -8.8 (b) KAS 0.8 0.1 0.2 1 1.2 0.4 -9.8 0.5 0.6 -10.3 -10.4 0.4 0.5 -10.8 0.6 -11.2 0.7 0.8 (1/T) X 1000 0.1 0.3 0.7 -10.8 1.4 0.2 -10 0.3 -9.3 ln(β/T²) 1.9 ln(β/T²) 1.5 0.8 -11.6 0.9 (1/T) X 1000 15 0.9 Fig 4 (a & b) Linear plots of ln(T) versus 1/T for raw bamboo biomass and bamboo biochar respectively using KAS method for calculating the activation energy. Table 4 ‘Eα’ (activation energy) and ‘A’ (pre-exponential factor) for the bamboo biomass significant to degree of conversion (α) at 10 0C/min. Conversion FWO KAS factor α Eα Eα A(S-1) R2 (KJ/mol) 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 189.13 206.82 225.70 247.48 264.75 279.45 303.75 318.72 326.99 Average 262.53 A(S-1) R2 2.79 × 1013 8.87 × 1014 3.61× 1016 2.61× 1018 7.72× 1019 1.36× 1021 1.59 × 1023 2.96× 1024 2.31× 1025 0.9928 0.9883 0.9854 0.9845 0.9800 0.9726 0.9717 0.9660 0.8687 - (KJ/mol) 2.37 × 1013 6.74 × 1014 2.38 × 1016 1.44 × 1018 3.71 × 1019 5.87 × 1020 5.59× 1022 9.26× 1023 4.35× 1024 0.9934 0.9893 0.9866 0.9857 0.9814 0.9744 0.9734 0.9680 0.8757 - 189.98 208.27 227.91 250.64 268.65 283.93 309.33 324.94 335.92 266.62 Table 5 ‘Eα’ (activation energy) for the bamboo biochar significant to degree of conversion (α) at 10 0C/min. Conversion factor FWO KAS α Eα (KJ/mol) R2 Eα (KJ/mol) R2 0.10 173.87 0.7554 174.59 0.7277 0.20 134.35 0.9163 133.02 0.9003 0.30 118.55 0.9427 116.40 0.9289 0.40 102.74 0.9456 99.77 0.9302 0.50 94.84 0.9427 91.45 0.9233 0.60 79.03 0.9370 74.83 0.9108 0.70 71.13 0.9251 66.51 0.8869 0.80 63.22 0.9210 58.20 0.8720 0.90 55.32 0.9123 49.88 0.8483 Average 99.23 - 96.07 - 16 FWO KAS (b) 160 ΔH(KJ/mol) Eα (KJ/mol) 200 (a) 400 350 300 250 200 150 100 50 0 FWO KAS 120 80 40 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Degree of conversion (α) 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Rate of conversion (α) Fig 5 (a & b) Activation energy value changes with respect to degree of conversion (α) for FWO and KAS models for bamboo biomass and bamboo biochar respectively. 3.3 Thermodynamic parameters analysis Figure 6, 7 and 8 shows changes in enthalpy (ΔH), Gibbs free energy (GΔ), and entropy (ΔS) for both bamboo biomass as a function of conversion rate ‘α’ using the FWO and KAS methods. The interactions between constituents become more intense during the pyrolysis process as the heating rate increases [55]. A low heating rate, such as 10 °C/min, was proposed to mitigate the effects of such an interaction. The change in enthalpy during the pyrolysis process indicates a difference in energy between biomass and its end products [15]. The ΔH values of bamboo increased with conversion degrees, which fell between 183 to 330 kJ/mol for both FWO and KAS methods, as shown in Table 6. The change in enthalpy value for bamboo biomass rises as the rate of conversion rises, as shown in Fig 6, and for bamboo biochar it first increased for the rate of conversion of 0.1–0.3 and then decreased to 0.4–0.9, respectively which shown Table 6. The average enthalpy values for bamboo biomass FWO and KAS are 257.10 and 261.19, respectively. In Tables 4 and 6, a minimal difference in energy (5 kJ/mol) was detected for activation energy and enthalpy value, which was linked to the production of an activated complex, implying that the lowest amount of energy necessary for effective bamboo pyrolysis [34]. Pawar et al. discovered a minimal energy difference between change in enthalpy (224 kJ/mol) and activation energy (229 kJ/mol) for pyrolysis of coconut shell biomass [47]. The ΔH and E values were in satisfactory correlation, indicating formation of simple product. 17 The Gibbs free energy (ΔG) states that increases energy in the system as the state of the activated product and the nature of the reactant changes [15]. The ΔG change ranged from 182 to 186 kJ/mol, less than the activation energy. The maximum ΔG value for rate of conversion 0.1, indicating that the system received excessive thermal energy at the start of the pyrolysis process for raw bamboo shown in Fig 7 contrary for bamboo biochar the maximum ΔG value for rate of conversion 0.9 shown in Table 7. The average value of ΔG for bamboo biomass was around 184.41 and 184.33 kJ/mol, respectively, for the FWO and KAS methods. A minimal difference in Gibbs free energy values calculated using FWO and KAS techniques implies the creation of an activated complex, which could be effective in the treatment of flow-related problems. The value of ΔG slightly decreased for the conversion rate of 0.1–0.9, respectively, for both the methods. The change in ΔG (Gibbs energy) value proportional to α (rate of conversion) is shown in Fig 7. The positive ΔG value for bamboo biomass indicates that the reaction was nonspontaneous and would need more energy to complete. The change in entropy indicates the degree of disorder in a chemical process. For FWO and KAS methods, a minimal entropy value was determined at a conversion rate of 0.1, which rises to 0.9, as illustrated in Table 6. For raw bamboo the value of ΔS for the FWO and KAS method was around -3.68 and - 2.34 kJ/mol for ‘α’ of 0.1 and then increased to 211.94 and 225.84 kJ/mol for a conversion rate of 0.9 respectively shown in Fig 8. The average value of entropy for FWO and KAS was 111.29 and 117.67 respectively. For bamboo biochar, all the values of ΔS found negatives show that the rate of conversion is lower, as shown Table 7. When compared to the product of thermal degradation, a positive value of entropy (ΔS) indicates that the degree of conversion of product is higher, whereas a negative value of ΔS shows that the conversion rate of the product was lower [56]. The presence of both positive and negative values of entropy suggests the presence of a complex thermal conversion process of biomass into a variety of products [42] [29]. Table 7 summarises the enthalpy ΔH, Gibbs energy (ΔG), and entropy (ΔS) of bamboo biochar derived from bamboo biomass using the FWO and KAS, at 10 0C/min rate of degree conversion (α) respectively. At 6000C, biochar made from bamboo biomass showed a negative entropy value, indicating biomass feedstock has a lower degree of disorder. Almost comparable results obtained from other biomass, for ΔH, ΔG, and ΔS were determined for wheat straw biochar [45], prosopis juliflora biochar [47], bamboo [34]. 18 Table 6 Thermodynamic parameters of bamboo biomass Conversion factor α 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 Average FWO KAS ΔH(KJ/mol) ΔG (KJ/mol) 183.70 201.38 220.26 242.05 259.32 274.02 298.32 313.29 321.56 257.10 186.10 185.62 185.14 184.64 184.28 183.98 183.53 183.27 183.13 184.41 ΔS (KJ/mol) -3.68 24.14 53.77 87.89 114.90 137.85 175.74 199.07 211.94 111.29 ΔH(KJ/mol) ΔG (KJ/mol) 184.55 202.84 222.48 245.21 263.22 278.50 303.90 319.51 330.49 261.19 186.08 185.58 185.09 184.57 184.20 183.90 183.43 183.16 182.98 184.33 ΔS (KJ/mol) -2.34 26.42 57.25 92.84 120.99 144.84 184.44 208.74 225.84 117.67 Table 7 Thermodynamic parameters of bamboo biochar Conversion factor 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 Average KAS ΔH(KJ/mol) ΔG (KJ/mol) 165.46 125.95 110.14 94.33 86.43 70.62 62.72 54.82 46.91 90.82 299.84 302.01 303.06 304.26 304.93 306.47 307.35 308.34 309.46 305.08 ΔS (KJ/mol) -132.90 -174.12 -190.79 -207.61 -216.09 -233.24 -241.93 -250.73 -259.66 -211.90 ΔH(KJ/mol) ΔG (KJ/mol) 166.19 124.62 107.99 91.36 83.05 66.42 58.11 49.79 41.48 87.67 350 299.80 302.09 303.21 304.51 305.24 306.93 307.92 309.04 310.33 305.45 (a) 300 ΔH(KJ/mol) α FWO 250 200 150 FWO 100 KAS 50 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Rate of conversion (α) 19 ΔS (KJ/mol) -132.14 -175.51 -193.07 -210.80 -219.74 -237.85 -247.06 -256.39 -265.89 -215.38 Fig. 6 Enthalpy (ΔH) value changes with respect to rate of conversion (α) for FWO and KAS models for bamboo biomass 187 (b) ΔG(KJ/mol) 186 FWO 185 KAS 184 183 182 181 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Rate of conversion(α) Fig. 7 Gibbs free energy (ΔG) value changes with respect to rate of conversion (α) for FWO and KAS models for bamboo biomass 250 (c) ΔS(KJ/mol) 200 150 FWO 100 KAS 50 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 -50 Rate of conversion (α) Fig. 8 Entropy (ΔS) value Changes with respect to rate of conversion (α) for FWO and KAS models for bamboo biomass Table 8 shows how bamboo biomass's thermodynamic and kinetic parameters compare to those obtained for other wood and agricultural residues. Bamboo has lower activation energy than other woody biomass and is very close to, having a value that is very similar to that of other bamboo species. Lower activation energy for bamboo indicates that it can react effectively with a small amount of energy, implying that bamboo biomass can be efficiently utilised for bio-energy production. Furthermore, knowledge of thermodynamic variables would aid in the proper design of thermochemical conversions. 20 Table 8 Comparison of the thermodynamic and kinetic parameters obtained for bamboo to other wood and agricultural residues Feedstock Models Heating rate 0 C/min Bamboo FWO, KAS 10,20,30 Mustard stalk FWO, KAS, Starink Prosopis Juliflora Cedar Activation energy kJ/mol Thermodynamic parameters References Enthalpy kJ/mol Gibbs energy kJ/mol 262.53,266.62 257.10 261.19 184.41 184.33 10, 20, 30, 40 173.83, 173.18, 172.94 168.68, 168.03, 167.78 176.37, 176.39, 176.40 [57] FWO, KAS, Starink Friedman 10,20,30 150.52, 150.47, 149.51, 147 147.88 146.47 146.43 143.89 172.79 172.74 172.71 172.40 [47] FWO, Criado 5, 10, 20, 40 188-205 - - [50] FWO, Starink’s 5, 15, 25, 35 300, 222 - - [48] KAS FWO, 10, 15, 20 30 183.113 181.973 - - [20] Bamboo subfamily DAEM (distributed activation energy model) 10, 40, 70 201.59, 220.49, 224.47 170-250 200 - 250 [34] Cotton stalk FWO KAS 15, 25, 35, 45 55 142.93 145.39 - - [40] Tectona grandis (teak) Bamboo waste Present study 4. Conclusion Kinetic analysis of bamboo and its biochar using thermogravimetric under nonisothermal conditions was performed at defined rates of heating 10, 20, and 30 0C/min of temperature 30 to 900 0C. Bamboo and bamboo biochar are found to have higher heating values of 18.50 and 28.25 MJ/kg, respectively. Thermal decomposition of bamboo mainly occurs in three different temperature stages, viz., drying, devolatilization, and char formation. The major thermal decomposition occurred at a temperature of 180 0C to 395 0C, called the active pyrolysis zone. Flynn–wall–Ozawa and Kissinger–Akahira–Sunose methods were used to evaluate the kinetic and thermodynamic parameters. The activation energy was determined using isoconversational methods such as the FWO and KAS models, which gave the average values for bamboo of 262.53 and 266.62 (KJ/mol) and for bamboo biochar of 99.23 and 96.07 kJ/mol, respectively. The A (pre-exponential factor) was measured within the range of 1013 to 1025 S -1. 21 The positive values for both ΔH and ΔG for the total degree of conversion suggest that thermal decomposition of bamboo biomass occurs in a non-spontaneous way. The small difference in enthalpy and activation energy indicates that biomass pyrolysis is feasible Authors’ contributions Priti Jagnade conducted an experimental study, prepared a draft manuscript, and analysed the constructive discussion data. Narayan Lal Panwar and Chitranjan Agarwal contributed to writing the manuscript and interpreting the data. All authors read and approved the final paper. Acknowledgments Priti Jagnade sincerely acknowledged Chhatrapati Shahu Maharaj Research Training and Human Development Institute (SARTHI), Pune, for providing Research Fellowship. 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https://openalex.org/W3047409708	https://handbook.pubpub.org/pub/assignment-playing-agency/download/pdf	English	null	Playing with Historical Purpose and Agency	null	2,020	cc-by	2,066	Visualizing Objects, Places, and Spaces: A Digital Project Handbook Playing with Historical Purpose and Agency Playing with Historical Purpose and Agency Visualizing Objects, Places, and Spaces: A Digital Project Handbook Roles: Sean Smith is co-creator of this curriculum and has taught this project in History 172 Early US History Survey in a large lecture section and in a small honors program section and has taught it in History 306 Playing the Past an upper-division history course at CSULB. Jeffrey Lawler is co-creator of this curriculum and has taught this project in History 173 US History Survey Since 1865 in a large lecture section with student facilitators, in History 473 an upper-division California History course and has taught it in our games specific upper-division course History 306 Playing the Past at CSULB. Authors: Sean Smith is a full time lecturer at CSU, Long Beach, and Co-Founder/Director of the CSULB Center for the History of Video Games and Critical Play. Jeffrey Lawler is also a full time lecturer at CSU, Long Beach, and Co-Founder/Director of the CSULB Center for the History of Video Games and Critical Play. Technology-Dependent Learning Outcomes Was there anything this assignment taught students that you felt they wouldn't have been able to learn through other types of class assignments? The creation of a choice-based game subverts students’ understanding of the historical process by engaging them directly in manufacturing possible historical choices that are based on real and reliable sources and historical events. This creative process provides students with a sense of agency and helps them create a narrative contingent on their own interpretations, an interpretation rooted in a solid historical methodology and traditional historical thinking skills, yet presented in a novel fashion. Moving the historical creation and narrative process over to students has the potential to reify students’ connection to the past and its meaning, thus allowing students to engage with the past on their own terms while maintaining strong methodological underpinnings rooted in research, analysis, and contextual contingency. Adding games discourse and integrating game engines such as Twine and other digital forms into class projects has provided a number of ways to help students think about and relate to the past. Specifically, we see Twine or game creation in general as a tool that provides students applicable historical methods of analysis, offers them agency in narrative creation, helps them develop an understanding of contingency and context, and reminds them that agency and choice are important elements when creating a representation of the past. Creating historically rooted games also helps students distinguish the difference between what happened in the past and how we tell accurate and meaningful stories that represent a fuller picture of the history of the United States. In particular, having students engage in game creation teases out the difficulty and necessity of forming meaningful narratives about the past. As students research and formulate ideas about their games, the process of what evidence to use and connect becomes readily apparent. This process is markedly different than writing an essay and allows our students to struggle with the past and the varied ways historians tell stories. The affordances of digital projects used in our classrooms allow students to engage in a more diverse history and find their own meaning in multiple interpretations of the past. Most students also find a way to tell a story within their game that speaks to ideas and moments that are important to them. What did you want students to be able to do by completing this assignment What did you want students to be able to do by completing this assignme What did you want students to be able to do by completing this assignment? 1. Formulate a historical question, prepare research, and select appropriate primary and secondary sources. 1. Formulate a historical question, prepare research, and select appropriate primary and secondary sources. 2. Evaluate and analyze a variety of sources and craft a historical interpretation/argument. 3. Construct a historically plausible game in Twine or other game engine using traditional history research that conveys a clear and appropriate narrative. 2 Visualizing Objects, Places, and Spaces: A Digital Project Handbook Playing with Historical Purpose and Agency Technology-Dependent Learning Outcomes In fact, recent games include historical narratives about a variety of marginalized groups: The Black Panther Party, The Lavender Scare, Early 20th century immigration, or racial issues 3 Visualizing Objects, Places, and Spaces: A Digital Project Handbook Playing with Historical Purpose and Agency with Jazz during the 1920s in America. (To see examples of student work go to http://twinegames.criticalplay.net) with Jazz during the 1920s in America. (To see examples of student work go to http://twinegames.criticalplay.net) What is the course title and level? What is the course title and level? What is the course title and level? This assignment has been successfully implemented in our freshman-level United States History Survey courses. It is also the core research project for our upper- division history course History 306 Playing the Past: Games as Historical Narrative, Public Memory, and Cultural Representations. The iterations for each course differ in expectation and explanation depending on a variety of skill levels such as lower or upper-division, non-major or major, and honors or non-honor courses. Some of the lower division courses have integrated games as a pedagogical tool throughout the semester with the explicit idea that skills, research, content, and game creation will be integrated. What kinds of prior knowledge is necessary to complete this assignment? How do students gain this knowledge? For many of our students, their interest in history emerged from the games they played in their primary grade classrooms and the games that they currently play on their own game consoles and PCs. These games have had a deep impact on their historical understanding and have shaped their beliefs about history and historical study. In fact, an ongoing survey of the authors’ undergraduate students (an informal classroom poll at the beginning of each semester of nearly 800 students from the fall semester of 2017 to the spring semester of 2019 in both upper-division and lower- division history courses) revealed nearly seventy percent of them became interested in history or the history degree program at our university because of a video game they had played or are currently playing. While it is encouraging that video games bring students to history classes and help to foster an interest in studying the past, their influence remains problematic. This assignment hopes to adjust this prior knowledge and bring it more in line with academic understandings of the past. Students are already conditioned to read games uncritically by their past classroom experiences and the nostalgia they have for these games. Additionally, their consumption of contemporary history games with interpretations that serve the developers’ agenda or narrative interests creates a prior historical knowledge that they bring to the classroom rooted in simplistic often heritage-based interpretations of the past. How much time did you allot to this project? This is a time intensive semester-long project and how to incorporate technology, historical concepts, and research skills depend on whether it was assigned in an upper- division or lower-division class. After spending several semesters teaching this assignment in both semester-long and in a shorter time frame, we found digital game creation works best when 8 - 12 weeks are dedicated to the project. To succeed in the project students need time to do proper research and analysis, initial storyboards or passage cards, timelines, learning the technical aspects of their chosen game engine, writing the game, play testing, and final draft. What is the course title and level? The games are written with notions of 4 Visualizing Objects, Places, and Spaces: A Digital Project Handbook Playing with Historical Purpose and Agency popular history and whether intended or not are loaded with historical myth or allusions that players internalize. Our students are clearly not equipped with the skills necessary to read video games critically and gauge their historical value. History is often conflated by students as a story well written and told. But the study of history is more than just the creation of a narrative of past events, it is a body of skills and knowledge that lead to historical thinking. Assignment Description Students build historically plausible games in Twine, RenPy, or other available digital tools to help them think analytically and historically about the past. To accomplish this task they must complete several steps of historical research including collection, analysis, and synthesis of primary and secondary sources. Students organize these sources and their research into both storyboards and often we use timelines to set the historical context. The narrative structure of the games allows students to more clearly discern historical choice, agency, and the non-teleological nature of the past. The digital game creation process provides students with a non-traditional approach to the history class that challenges students to re-think cause and effect and the difficulty in creating meaning from the past. These games are collected and archived for play and comment on a class website. Support & Training What kinds of support or training did you provide to help students learn to use new techniques or specialized tools? 5 Playing with Historical Purpose and Agency Visualizing Objects, Places, and Spaces: A Digital Project Handbook Class time is spent teaching research methods including, the use of the internet and library for finding and evaluating primary and secondary sources, the application of Zotero for collection of their data, the formulation of historical questions, modeling narrative creation, instruction in some light coding in Twine and HTML/CSS, all while making certain that the game is historically plausible. Importantly, we must be vigilant in the reinforcing of the idea that these are discipline-specific historical accounts in a playable form. They get both digital skills and traditional historical thinking skills. Resources Did you need any specialized equipment, tools, or human resources to make this assignment feasible? If so, please describe. The game project is best suited in one of our University’s Active Learning Classrooms. These rooms facilitate group collaboration and provide access to a variety of technology that students and faculty can use to enhance and more actively engage and collaborate with during class time. We encourage students to bring their own devices and have seen students succeed using smartphones, tablets, and laptop computers. The rooms also allow for faculty to screen share in technological creation and programming processes in a group setting. It also allows for collaborative critiques and group gameplay, furthering their games and literacy and enhancing their more traditional historical research. The library staff, and in particular the history librarian, provide a strong resource for our students regarding research and a variety of digital tools. Active Learning Classroom, CSU, Long Beach. Photo: Authors Active Learning Classroom, CSU, Long Beach. Photo: Authors Assessment How did you assess or grade this project? Many components of the projects are assessed and graded for this assignment, including their historical research and bibliography (use of Zotero or other note- 6 Visualizing Objects, Places, and Spaces: A Digital Project Handbook Playing with Historical Purpose and Agency taking/research collection skills), historical questions and thesis, storyboards, game drafts, reflections and explanation of their use of integration of historical materials, and their final project. Students are provided with clear and appropriate guidelines for completion of each portion; they are also afforded the opportunity of providing feedback during game testing of all the games. Rubrics and peer reviews are used for the development and completion of the game. Challenges & Opportunities If you assigned this project again, would you change anything? If so, what? If you assigned this project again, would you change anything? If so, what? This assignment is constantly changing and being adapted to improve student comprehension and game creation. Each semester is different from the next with specific changes and adaptations being made based on students’ games literacy and historical skills. We also struggle, especially in the lower division survey courses with questions of breadth and depth. This assignment offers students a deep dive into a specific subject often at the expense of breadth. Describe any trouble spots or complications someone else might want to be aware of before trying a similar assignment in a course of their own. The use of Twine and other game engines in the classroom has not proven to be a panacea, but when used in very particular circumstances and with sufficient engagement and feedback students do benefit.
https://openalex.org/W4200294240	https://cubicjournal.org/index.php/cubic/article/download/40/38	English	null	Blended Learning Strategies for Advertising Design Studies	Cubic Journal/Cubic journal	2,021	cc-by	5,519	Introduction cess. The teachers are the domain experts who guide and advise students on their projects. Stu- dent–instructor interaction is central to stu- dio-based educational practices. In this setting, on-the-spot communication between students and teachers is spontaneous and contagious, but also viewed as “off the lip” (Meyer 2003, 61). Stu- dents must remember what has been said and be mentally and verbally quick to respond and clarify their responses. The astronomical development of technology has brought profound challenges to design edu- cation. With the market of personal handheld devices becoming more mature and unlimited authentic resources becoming available online, learning can occur anywhere, anytime. Thus, there has been a rapid increase in accredited online courses offered by universities around the world. Technology communication allows stu- dents to control the time, place, and pace of their learning. Students are increasingly demanding a quality learning experience with convenience and flexibility. The role of the teacher is to foster a learning environment that is learner-centered and focused on the process of delivering a qual- ity learning experience (Beetham and Sharpe 2013, 31-48). Blended learning is the integration of synchro- nous (face-to-face) and asynchronous (text-based Internet) learning experiences (Garrison and Kanuka 2004, 96). What distinguishes blended learning from traditional classroom-based and online courses is the combination of in-class teaching and out-class learning through com- puter-based technologies. It is characterised by the use of multiple instruction and delivery channels that can retain the best of face-to-face and online learning experiences. Asynchronous Internet communication has the ability to facil- itate an important reflective element because it emphasises written communication. Writing encourages reflection and thinking both crea- tively and critically. Although some competency is required to write skillfully, all students are pro- vided with an opportunity to learn how to clearly express themselves in written form. The learning experience is one of the core com- ponents of student satisfaction and academic success. Thomas Fischer (2004) argued that design studio disciplines need to move to the next stage of their existence in terms of what they can deliver. New models are now emerg- ing in response to changing needs. There may be a virtual studio where design students learn by doing things remotely. Technologies have changed not only how students learn, but also ways that students expect to learn and behave. How should design educators adapt to the needs and challenges of this new technological era? Introduction Communication can also provide a permanent record and thus expand learning time. Students can revisit instructors’ comments as needed. The communication is accurate and no information is lost. Written comments are often less intuitive and better thought through because instructors can think, research, and provide feedback. The most well-known model of blended learning is Anthony Picciano’s Blending with Purpose Mul- timodal Framework (Picciano et al. 2013, 2). Pic- ciano’s framework comprises six objectives for educators to take into account for planning their teaching design and delivery, which include con- Blended Learning Strategies for Advertising Design Studies Gladys Lam Wai Ling Technological developments have brought profound challenges to design education. To understand how design educators adapt to new technological directions, this article examines student feedback from advertising design courses that apply blended learning approaches. This study identified three blended learning strategies conducive to meaningful learning: timely and meaningful feedback; engagement with real world tasks; and support from expert tutors. This article also discusses potential resistance and challenges in implementing instruction in blended technological environments. #blended learning approach #design studio pedagogy #student learning experience #student perception #meaningful feedback \| 45 Gladys Lam Wai Ling . Blended Learning Strategies for Advertising Design Studies Gladys Lam Wai Ling . Blended Learning Strategies for Advertising Design Studies Background Design education has a long tradition of using studio pedagogy, in which teachers provide feedback and suggestions to students on their designs throughout the creative process. This is done face-to-face. Such communication gener- ates energy and enthusiasm that helps students remain motivated throughout the design pro- 46 \| 46 \| tent, social/emotional, dialectic/questioning, col- laboration, synthesis/evaluation, and reflection. The essence of this model is the ability to meet the needs of a wide range of students with dif- ferent backgrounds, learning styles and person- ality types. There is evidence that blended learn- ing has the potential to be more effective and efficient at constructing meaningful learning experiences than traditional learning methods (Kintu et al. 2017, 11). Therefore, blended learning has become an essential approach to the future of education. However, success depends on a well-designed strategy to effectively integrate Internet technology with the most desirable and valued aspects of face-to-face learning. ing environments in which technology is pres- ent. In addition, David Boud and Michael Prosser (2002) argued that high quality learning activi- ties must demonstrate four principles: engage- ment of learners; acknowledgement of context; challenge for learners; and the involvement of practice. Blended learning offers opportunities to deliver on several of these principles. Design studio pedagogy has a long tradition of offering project-based learning and mentor- ing support to students. With the introduction of technology-facilitated classroom manage- ment platforms that allow chat rooms, forum discussions, and blogging for community learn- ing, learning support has never been lacking. However, research on applying blended learn- ing strategies to project-based studies has found that faculty members and students do not bene- fit from using eLearning systems (Ma 2016). In a study of engaging creative media students’ moti- vation, the author suggested that faculty should give students more power over their learning process with their projects because autonomy is a primary motivator (Oh et al. 2018). Learners live in a digital world where they can retrieve infor- mation easily and communicate with almost anyone. Flexibility and convenience are increas- ingly important in the technological age, and it is inevitable that educators will adapt to this new direction. Thus, more research is required to gain a deeper understanding of students’ percep- tions on effective blended learning approaches to design education. An effective blended learning setting requires the design of learning tasks, learning support, and learning resources (Herrington et al. 2005). Background Learning support refers to the capacity to inter- act with systems, peers, and tutors in the learn- ing process. Students often turn to their peers for company and seek support and advice from their tutors to guide their projects. Providing this support in a blended learning setting establishes a sense of community and promotes higher-or- der thinking and conceptual development that is often not achievable in an individual learning setting (Brook and Oliver 2004). What is needed in a blended learning setting is not only the use of technology but a blended learning strategy. Such a strategy is a deliber- ate set of learning activities and an environment that engages learners in a process that results in the required learning outcomes. Jane Herrington and Tom Reeves identified ten design principles that characterise authentic learning tasks: real- world relevance; ill-defined tasks; complex tasks; opportunities to examine, collaborate and reflect; going beyond domain specifics; integration with assessment; creating valuable products; and allowing for competing solutions. These ten design principles also apply to effective learn- CUB I C JO U R N AL .No.4. Pe d ago gy · Crit iq ue · Trans fo rmat io n Research Questions This study examines blended learning strategies for project-based advertising design courses. It aims to find out the determining factors in stu- dent satisfaction and understand the essence of the relationship between students’ learning experience and the blended technological world. \| 47 ladys Lam Wai Ling . Blended Learning Strategies for Advertising Design Studies Describe some good points about the course; 2) Describe some areas of the course that could be improved; 3) other comments. The quantitative data indicated students’ levels of satisfaction regarding the overall teaching effectiveness of the courses, but the data did not provide much information on the core factors behind this satisfaction rating. There- fore, the main data was derived from the students’ individual written narratives. Unlike reflective jour- nal assignments, formative feedback by participants at the end of a course is a way to evaluate the effec- tiveness of the teaching and improve the course in all dimensions, such as preparation, pedagogy, deliv- ery, and learning environment. All of the responses were anonymous. Because of the nature and timing of the survey, the students had no reason to please the instructor to obtain a good grade. Thus, the nar- ratives were true to the respondents’ experience and based on their personal judgment. The research questions that guide the study are as follows: 1. How do undergraduates perceive and experience their advertising design courses? 2. What blended learning approaches do students find effective? Gladys Lam Wai Ling . Blended Learning Strategies for Advertising Design Studies Results and Discussion Three themes were identified from the analysis of the data: meaningful and timely feedback, real world activities and supportive expert teachers. Meaningful in this context refers to giving feedback that students appreciate, feedback that provides value to them, and helps them fulfill their pur- pose for the course. In examining the technology that facilitated the learning environment, students appreciated “meaningful comments” and “prompt replies.” One student cited an incident in which she “was desperate” and wrote an email to her teacher late at night. She was pleased to receive a quick reply. With technology, students can approach their instructors with their own design problems any- time and anywhere through technology-mediated communication. Teachers can provide help when students are most in need. Giving students what they need when they need it does not mean that they are being encouraged to ask frivolous ques- tions. It is meant to show consideration of their needs when they call for support. Technology facil- itated communication enables teachers to differen- tiate the individual needs of students. It facilitates personalised learning and student-centered edu- cation. It also avoids an overabundance of opin- ions because it is the learner who invites the feed- back. Some students do not like tutors to intrude on their creative endeavors. Teachers must learn when to give comments and when to stop giving them. Technology-mediated communication helps teachers identify such needs. “Nice and responsible tutor. Always have a quick email reply, very appreciate =]” The students also enjoyed seeking the teacher's “professional advice” outside class and viewed this as “valuable guidance.” The students said that the teacher “judges right and criticises right” and her comments were “constructive,” “clear,” “useful,” and “inspiring.” “This class gives us many chances in practicing execution, it is a great chance for us to make an improvement in doing advertising. The lecturer gives a big freedom for us to develop our creativ- ity and also gives us many opinions in our works. That's great!!!” “She is creative. And she really knows how to art direct. She judges right and criticises right.” Clear and precise feedback is paramount in design education, whether it is in a traditional face-to face or technologically mediated settings. Writ- ten feedback requires special attention and skills. To achieve clear feedback, it is better to write in short paragraphs or in point form. Different stages of the creative process require different formative feedback. Research Context and Methodology The sample was collected from advertising design courses at the School of Communication, Hong Kong Baptist University. Formal written feed- back was solicited from students at the end of their courses over eight academic years. These courses were offered by the Communication Studies Department to undergraduate students enrolled in the bachelor’s of social science (Hons) program in communications, majoring in either public relations and advertising or digital graphic communication. The design courses under inves- tigation included Advertising Design and visual- isation, Advanced Advertising Design and Vis- ualisation, Advertising Copywriting and Guerrilla Advertising. The courses ran for 13-14 weeks, three hours a week, with an average enrollment of 24 students. These students were Year 2 and Year 3 communication students majoring in either advertising (PRA) or digital graphic communica- tion (DGC). The average ratio of female to male in the classes was approximately 7:3. Participa- tion was voluntary with the response rate ranging from 28 percent to 88 percent. The method used was qualitative inquiry with the phenomenological approach. The study of phenomenology pertains to the analytical and descriptive experience of individuals, emphasis- ing their first-hand descriptions of phenomena (Creswell 2013). During the analysis, excerpts and quotes were grouped based on the latent mean- ings expressed by each participant. Through clus- tering the invariant constituents, or themes, found in the narrative descriptions are uncovered during the reduction process. Only themes that are representative by each class of participants are checked against the overarching topic, which in this case is blended technology. By outlining the reoccurring and prominent themes across all participants, common themes were identified such that only dominant phenomena with high consistency were considered. Finally, the most essential elements that informed the experiences were conceptualised. In this case, the individual textural-structural descriptions of each partici- pant were not applicable. A composite description of the “meanings and essences of the experience, representing the group as a whole” was presented The feedback was collected during the last week of the lessons and released to the course instruc- tor within two months. The feedback was collected via online questionnaires, with eight questions to assess aspects such as course preparation, deliv- ery, and learning environment on a five-point Lik- ert-scale. Research Context and Methodology The questionnaire also included three descriptive questions to invite respondents to describe their experience in their own words: 1) 48 \| 48 \| “I learnt a lot from Gladys' class, no matter adver- tising knowledge from her real field experience or from her fruitful teaching. I think Gladys is really a good, responsible teacher and she treats us very well. For example, one time I was desperate in cre- ating new ideas for our print ad, I wrote to her through student mail [and] unexpectedly got the answer from her very soon as it is almost very late at night. However, Gladys being a strong passionate and dedicated teacher, she gives me a prompt reply plus offering very meaningful comments on my print ad. I am so glad to have such a great teacher and I hope to continue learning from her :))” instead (Moustakas 1994, 121). The narratives have remained in their original language and a selected few have been presented in the findings. CUB I C JO U R N AL .No.4. Pe d ago gy · Crit iq ue · Trans fo rmat io n Results and Discussion They said that “having a real campaign” was “really great.” It helped them “learn by experiencing the real situation” and cre- ated opportunities for them “to think deeply about the practi ject to be ing our ow ing.” Laun learn “mo course mo “meaning to join a r esting,” an Students rilla Adve “Watch an exc challen “It's ni feedba edge of “Lots o date h visit th examp thorou paign ing the The stude in relevan talk” and tening to they were their “futu “Miss and in me lea lovely always in the pare m 1. Choosing the best potential idea and backing it up with reasons. 2. If nothing appears to be appropriate, pro- vide direction. 3. Offer suggestions, if it appears to be help- ful. 4. Encourage idea development. Students of PRA and DGC from the course of Gue- rilla Advertising gave the following comments: During the idea execution phase, students ask for advice on design layouts and production. During the idea execution phase, students ask for advice on design layouts and production. Comments could be made on refining the art direction and copywriting. The core task is to make sure that the idea can be effectively con- veyed through appropriate executions. For clar- ity and elaboration, both parties could attach layouts, examples and reference links to their communications. “Watching our own project be shown to society is an exciting time for me. The final project is really a challenge to me.” “It's nice to have our work launched, and receiving feedback from the public. Got more practical knowl- edge of launching a campaign.” Technologies have caused a revolution in cre- ative production through the Internet and this has materialised in real world activities. Anyone can post his or her creative work on YouTube, social media and many other online platforms. The Internet has become a dynamic medium for interconnecting people and co-creating. These changes in social systems have transformed the ways designs develop, based on knowledge, col- laborative processes, and cross-disciplinary prac- tices (Sanders and Stappers 2008, 8-9). Design educators can use social media to design all sorts of simulated tasks based on real world activities. Meetings with “real” people or launching “live” projects enables students to better understand the societal context and their own potential as prospective professionals. “Lots of examples to help us understand this up-to- date hot topic. Gladys Lam Wai Ling . Blended Learning Strategies for Advertising Design Studies Results and Discussion During the idea-generating phase, stu- dents ask for advice on the potential of their ideas from a pool of rough concepts. Students of PRA and DGC from the Advertising Design and Visualization course wrote the follow- ing comments: \| 49 ladys Lam Wai Ling . Blended Learning Strategies for Advertising Design Studies Under normal circumstances, feedback includes: Under normal circumstances, feedback includes: the practical problems.” They also found the pro- ject to be “really challenging” and said that “watch- ing our own project be shown to society was excit- ing.” Launching a “real” campaign helped students learn “more practical knowledge” and “made the course more interesting.” They remarked that it was “meaningful” and “valuable” to have an opportunity to join a real world competition that was “so inter- esting,” and said it “really inspired us to learn.” Under normal circumstances, feedback includes: 1. Choosing the best potential idea and backing it up with reasons. 2. If nothing appears to be appropriate, pro- vide direction. 3. Offer suggestions, if it appears to be help- ful. 4. Encourage idea development. During the idea execution phase, students ask for advice on design layouts and production. Comments could be made on refining the art direction and copywriting. The core task is to make sure that the idea can be effectively con- veyed through appropriate executions. For clar- ity and elaboration, both parties could attach layouts, examples and reference links to their communications. Technologies have caused a revolution in cre- ative production through the Internet and this has materialised in real world activities. Anyone can post his or her creative work on YouTube, social media and many other online platforms. The Internet has become a dynamic medium for interconnecting people and co-creating. These changes in social systems have transformed the ways designs develop, based on knowledge, col- laborative processes, and cross-disciplinary prac- tices (Sanders and Stappers 2008, 8-9). Design educators can use social media to design all sorts of simulated tasks based on real world activities. Meetings with “real” people or launching “live” projects enables students to better understand the societal context and their own potential as prospective professionals. In this study, the students considered their learning effective by launching their projects on the Inter- net and joining competitions. Results and Discussion By inviting client briefings or professional judgments, students can learn from a diversity of people and benefit from different opinions of those from different backgrounds. Involving industry professionals to provide a few important creative reviews before the final critique would be ideal. Virtual judging can also save commuting time for busy practitioners. “She is really kind and always ready to help with her great competency of advertising. I love her!” “She is really kind and always ready to help with her great competency of advertising. I love her!” Design educators are usually domain experts. Expertise in the field helps cultivate critical and creative thinking skills in the students. However, not all experts are good teachers. Merely being an expert is not enough. The students looked for a dedicated, passionate, and supportive expert. Joe Ruhl (Ruhl 2015) argued that teachers should pos- sess two loves; love for the subject and love for the kids. It is “genuine, decisional and puts the other person first” (ibid., 47) kind of love that motivates and inspires students in a powerful way. What is keeping educators from integrating blended learning? Formative feedback is labour intensive, both for the learners and the tutors. Online feed- back for teachers is more labour intensive than face-to-face communication due to the amount of time required to respond to questions. To provide feedback, teachers must regularly read and com- ment on the students’ postings. Educators have often said that written communication may not be as effective as speaking face-to-face. However, writ- ten communication could become clearer if the core subjects were presented in points supported by references. Even without that, written commu- nication allows students to fill in knowledge gaps through their own inquiries and gives them access to unlimited online information. The final theme being supportive expert tutors refer to those who have extensive knowledge, experience and ability in a particular design profession. When the students were asked to describe some good points about their course, they repeatedly mentioned their teacher. The students perceived the teacher as “very warm- hearted” and “passionate,” “dedicated” and “always ready to help.” She “used her extra time” “to give support” to the students. Results and Discussion It is really great to have a chance to visit the advertising firm and get the really updated example to understand this trend of advertising thoroughly. The practical part of having a real cam- paign can make everyone learn through experienc- ing the real situation.” The students also showed interest in participating in relevant creative industries events such as “guest talk” and “agency visit.” They said that they like lis- tening to “real design field experience” because they were keen on preparing their “portfolio” for their “future design career.” “Miss Gladys' lesson is always eventful, innovative and interesting. Her homework and project made me learn a lot and it was rewarding. The most lovely part of Miss Gladys’ lessons were that she always shared a lot of her real design experiences in the field, which prepared me massively to pre- pare my future design career path.” In this study, the students considered their learning effective by launching their projects on the Inter- net and joining competitions. They said that “having a real campaign” was “really great.” It helped them “learn by experiencing the real situation” and cre- ated opportunities for them “to think deeply about 50 \| Participating in a real competition was very chal- lenging and practical at the same time to the stu- dents as they could put their experience into their portfolio. “As Gladys is a professional creative advertiser, I would love to seek her continued experience sharing in her real-advertising field. Listening to her experi- ence is really fruitful to me :))” “I think it is so great that the lecturer is an expert in this field. I really admire her and I hope to continue to attend the classes that she teaches.” In the traditional classroom, the teacher often collaborates with industries and invites practi- tioners to brief the students or provide face-to- face critiques. In the technological age, these activities can be done in a virtual environment. Creative reviews are critical in design educa- tion because they simulate professional practice. Instructors need to systematically evaluate the effectiveness of advertising campaigns to pro- mote students’ critical thinking. Students need to nurture critical thinking skills to inform the right decisions when choosing the best poten- tial idea. Although the mentor is usually a pro- fessional expert, creative work is subjective in nature. CUB I C JO U R N AL .No.4. Pe d ago gy · Crit iq ue · Trans fo rmat io n Results and Discussion The students said the teacher was “really kind” and “treated us very well.” They admired her because she was “a veteran” with “lots of industry experience” that “gives us a lot of inspiration.” The students expressed their admiration and hopes to con- tinue to be taught by her. Interactions with students after class take up teachers’ personal time. It is the teacher’s decision whether to embrace the students’ participation \| 51 ladys Lam Wai Ling . Blended Learning Strategies for Advertising Design Studies not have a history of documented conversations. outside the classroom or to end the interaction after class. Students learn this on the first teach- ing day when the teachers establish the classroom rules. Imagine if teachers spoke about the rules on the first day of the class and either required the students to send emails at least one day prior to an appointment or welcomed students to drop by their office for advice. Given that the core learn- ing in design education is to complete a project, for a teacher, making himself or herself accessible is important. Students can find someone to turn to for support and guidance and respond to their questions during the creative process. However, this is not always possible for teachers and profes- sor-track educators who have a heavy workload and a demanding publishing schedule. To address the concerns above, universities could support teachers by providing release time and recognizing that technological interactions are time-consuming. They could also provide instruc- tor training when technological tools are intro- duced. In a teaching culture in which adaptabil- ity has become the golden currency, it is important to support teachers who are guiding learning in a new environment and are learners themselves. If resources are provided in a personalised way to both teachers and learners, blended learning can evolve dynamically, in a managed way, toward a more interactive and successful pedagogy. What matters is not only how technology can be inte- grated, but how learning can occur in an enhanced and engaged way. Technological glitches have also hindered the willingness of teachers to adopt the blended approach. Although there have been classroom management platforms such as Moodle and Blackboard, students and teachers might not feel comfortable using them as channels for commu- nication. These e-platforms require online logins and take time to load. Even then, notifications are not always available. Results and Discussion Blogs for students’ reflec- tions, for instance, required a long time to load and could not be downloaded as a file. It was not user friendly and some interfaces could only be displayed properly on a computer, not a hand- held device. People are creatures of behaviour and once they become used to a certain platform, they stick to it. For example, most of the students pre- ferred to communicate via email, Facebook Mes- senger or WhatsApp. These communication tech- nologies are very convenient and reliable in terms of their pop- up notifications. Educators should allow for flexibility and not limit communica- tions to a specific platform. If the platform is too rigid or abrupt, a change may produce resistance and restrict interactivity. Thus, as long as teaching and learning activities take place, we should not limit them to classroom management platforms, although this would mean that universities would Gladys Lam Wai Ling . Blended Learning Strategies for Advertising Design Studies Conclusion A qualitative enquiry into advertising design courses revealed three effective blended learning strategies: meaningful timely feedback, real world tasks, and supportive expert tutors. Design edu- cation fosters learning by devising projects and helping students learn through feedback provided throughout the creative process. The most impor- tant skills teachers should have include know- ing how to facilitate learning, design meaningful activities, and create an appropriate environment beneficial to students’ learning experience. When using technology, the quality of the experience is more important than the use of the technol- ogy itself. Technologies overcome barriers of dis- tance and time to bring everyone together, help- ing students learn. We need instructors who have instruction skills in both traditional classrooms and virtual environments so that they can han- dle students’ changing expectations, behaviours, and needs. Many educators still insist on face-to- face communication in their teaching and learning activities. They should learn the positive impact of 52 \| 52 \| relinquishing control to the learner. An instructor’s decision to implement a blended learning envi- ronment and use technology in his or her course depends on the faculty’s preparedness to effec- tively facilitate and manage both online and face- to-face discussion and interaction. Beyond that, it requires teachers to be flexible, committed, and have a positive mindset (Markoff 2014). It also calls for school support and leadership to facilitate change and overcome resistance. CUB I C JO U R N AL .No.4. Pe d ago gy · Crit iq ue · Trans fo rmat io n Limitations and Potential Further Investigations The study was limited to an examination of stu- dents’ perception of their experience in advertis- ing design courses, primarily with a focus on the development of technology. While individual per- ception is useful to understand factors behind the phenomena examined, self-reported data includes a degree of subjectivity. To further under- stand design teaching with technology, future research may consider studying a broader context for the learning experience in design education, for instance: mistakes and learning processes (Wenzel 2002), design making and thinking (Mitcham 2001), critique and learning experience (Hokanson 2012). \| 53 Gladys Lam Wai Ling . Blended Learning Strategies for Advertising Design Studies Gladys Lam Wai Ling . Blended Learning Strategies for Advertising Design Studies Bibliography Mitcham, Carl. “Dasein versus design: The problematics of turn- ing making into thinking.” International Journal of Technology and Design Education 11, no.1 (2001): 27-36. Beetham, Helen, and Rhona Sharpe. Rethinking Pedagogy for a Digital Age: Designing for 21st Century Learning. London: Routledge, 2013. Moustakas, Clark. Phenomenological Research Methods. London: Sage Publications, 1994. Boud, David, and Michael Prosser. “Key Principles for High Quality Student Learning in Higher Education: A framework for Evaluation.” Educational Media International 39, no. 3 (2002): 237-245. Oh, Jae E., Jeffrey C. F. Ho, Chris Shaw, and Justin Chan. “Engag- ing Creative Media Students’ Motivation: The Influence of Auton- omy, Peer Relationships, and Opportunities in the Industry” World Journal of Education 8, no. 6 (2018). https://doi.org/10.5430/wje. v8n6p1. Brook, Christopher, and Ron Oliver. 2004. “Online Learning Com- munities: Exploring the Impact of Group Size on Community Development.” Paper presented at Ed-Media 2004, World Confer- ence on Educational Multimedia, Hypermedia & Telecommunica- tions, Lugano, Switzerland, June 21-26, 2004. Picciano, Antony G., Charles D. Dziuban, and Charles R. Graham. Blended Learning: Research Perspectives. Volume 2. New York: Rout- ledge, 2013. Creswell, John W. Qualitative Inquiry and Research Design: Choosing among 5 Traditions. San Francisco, CA: Sage Publications, 2013. Ruhl, Joe. “Teaching Methods for Inspiring the Students of the Future: What’s love got to do with it?” Youtube TEDx Talks. Published on May 27, 2015. https://www.youtube.com/ watch?v=UCFg9bcW7Bk. Fischer, Thomas. “The Past and Future of Studio Culture.” Appeared in the October 15 2004 issue of ArchVoices. Fischer, Thomas. “The Past and Future of Studio Culture.” Appeared in the October 15 2004 issue of ArchVoices. Garrison, Randy D., and Heather Kanuka. “Blended Learning: Uncovering its Transformative Potential in Higher Education.” Internet and Higher Education 7, no. 2 (2004): 95–105. Sanders, Elizabeth B. N., and Pieter J. Stappers. “Co-creation and the New Landscapes of Design.” CoDesign 4, no.1 (2008): 5-18. Wenzel, Thomas J. “AC Educator: Using mistakes as learning opportunities.” Analytical Chemistry 74, no.15 (2002): 439-4 Herrington, Jane, Tom C. Reeves, and Ron Oliver. “Creating Authentic Learning Environments through Blended Learning Approaches.” In Handbook of Blended Learning: Global Perspectives, Local Designs, ed. Curtis Bonk and Charles Graham. (New York: Jos- sey Bass, 2005), 502-517. Bio Gladys Lam worked in advertising for 16 years and served as a creative director of international 4As agencies, winning numer- ous awards worldwide before joining the Department of Com- munication Studies, Hong Kong Baptist University in 2006. She taught a variety of advertising design and creativity subjects and supervised honours projects. In addition, Gladys introduced new courses for the school and interdisciplinary general educa- tion, presenting the paper in the Global Conference on General Education and University Curriculum Reform at City University of Hong Kong in 2012. She has also published research find- ings in American Academy of Advertising Global Conference Proceedings. Her current research interests include advertis- ing visual and copy strategies, advertising design and creativity education. Hokanson, Brad. “The design critique as a model for distrib- uted learning.” In The Next Generation of Distance Education, Boston: Springer, 2012. Kintu, Justice M., Chang Zhu and Edmond Kagambe. “Blended Learning Effectiveness: The Relationship between Student Char- acteristics, Design Features and Outcomes.” International Jour- nal of Educational Technology in Higher Education 14, no.7 (2017). DOI 10.1186/s41239-017-0043-4 Ma, Henry. “Study of Blended Learning Approach for Project-Based Learning.” Paper presented at the Asia-Pacific Social Sciences Conference, Kyoto, Japan, November 22-24, 2015. Markoff, Monique. “Blended Learning and the Future of Educa- tion.” Youtube TEDx Talks. Published on May 6, 2014. https:// www.youtube.com/watch?v=Mb2d8E1dZjY. DOI: 10.31182/cubic.2021.4.037 DOI: 10.31182/cubic.2021.4.037 Meyer, Katrina A. “Face-to-face versus Threaded Discussions: The Role of Time and Higher-order Thinking.” Asynchronous Learning Networks 7, no. 3 (2003): 55–65.
https://openalex.org/W3194839164	https://pure.eur.nl/ws/files/41409226/1_s2.0_S0147176721001164_main.pdf	English	null	Length of stay, acculturation and transnational medical travel among Polish migrants in the Netherlands	International journal of intercultural relations	2,021	cc-by	9,747	A R T I C L E I N F O Keywords: Transnationalism Patient mobility Medical tourism Health care seeking behavior Cross-border healthcare An important aspect of the transnational lives of Polish migrants in the Netherlands is their frequent use of healthcare services in Poland. Transnational care use may be detrimental for the continuity and the quality of the care migrants receive. The current study aims to shed light on the antecedents of migrants’ doctor visits in Poland. Drawing on a representative population- based sample of Polish migrants in the Netherlands (n = 1,082), logistic regression is used to assess whether length of stay in the Netherlands is negatively associated with the likelihood of doctor visits in Poland. The KHB decomposition method is used to determine the extent to which this potential association is mediated by three specific acculturation factors: ethnic identification, trust in the Dutch healthcare system and Dutch language proficiency. The models show that migrants who stayed in the Netherlands longer were less likely to visit doctors in Poland. Mediation analyses indicated that this effect was largely attributable to their greater Dutch lan guage proficiency compared to their counterparts who arrived in the Netherlands more recently. Strong ethnic self-identification as Polish and lower trust in the Dutch healthcare system were also associated with a higher likelihood of visiting doctors in Poland. However, no significant mediation of the effect of length of stay via ethnic self-identification or Dutch language profi ciency was found. The findings suggest that voluntary language programs may foster inclusion of Polish migrants in the Dutch healthcare system and reduce the need migrants perceive to seek care in their country of origin. Thijs van den Broek Thijs van den Broek Erasmus School of Health Policy and Management, Erasmus University Rotterdam, Postbus 1738, 3000 DR Rotterdam, the Netherlands Erasmus School of Health Policy and Management, Erasmus University Rotterdam, Postbus 1738, 3000 DR Rotterdam, the Netherlands E-mail address: vandenbroek@eshpm.eur.nl. Contents lists available at ScienceDirect Contents lists available at S Available online 12 August 2021 0147-1767/© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). E-mail address: vandenbroek@eshpm.eur.nl. https://doi.org/10.1016/j.ijintrel.2021.08.002 Received 23 September 2020; Received in revised form 21 July 2021; Accepted 2 August 2021 International Journal of Intercultural Relations 84 (2021) 210–219 International Journal of Intercultural Relations 84 (2021) 210–219 Theoretical background and hypotheses Approximately one in four migrants of Polish origin in the Netherlands frequently use health care services in Poland (Gijsberts et al., 2018). The use of health care services in the country of origin is also high among migrants of Polish origin residing in other Western-European countries, such as the United Kingdom (Horsfall, 2019; Osipoviˇc, 2013) and Norway (Czapka, 2010). Osipoviˇc (2013) emphasized the marked differences in healthcare utilization patterns within the group of Polish migrants in the United Kingdom, with some subgroups having a strong tendency to seek health care in the country of origin and others not. In particular, she pointed to differences between migrants who recently arrived in the destination country and migrants who had been in the destination country for several years, and argued that “it is crucial to include the aspect of change over time in studies of migrant health care seeking behavior” (p. 111). Czapka and Sagbakken (2016) also noted that migrants of Polish origin in Norway tended to adapt their health care seeking practices over time. Navigating the health care system of the destination country can be especially challenging for recently arrived migrants (Leduc & Proulx, 2004), which may make the use of health care services in the country of origin particularly attractive for them. Therefore, it may be expected that length of stay in the Netherlands is negatively associated with the likelihood of visiting doctors in Poland (Hypothesis 1). The association between length of stay in the Netherlands and Polish migrants’ likelihood of visiting doctors in the country of origin may, in part, be expected to run via acculturation factors. Acculturation refers to the extent to which migrants over time may (or may not) (a) learn behaviors from the new culture and (b) shed features of their original culture (Berry, 1992). Berry’s (1992, 2005) seminal acculturation model distinguishes four acculturation strategies that vary on these two dimensions. The integration strategy is char acterized by high maintenance of the heritage culture in combination with strong participation in the larger society of the destination country. In the marginalization strategy, maintenance of the heritage culture and participation in the destination society are instead both weak. Migrants adopting an assimilation strategy aim to establish relations in the larger society of the destination country, while doing little to maintain their heritage culture and identity. The opposite is the case for migrants adopting a separation strategy. Theoretical background and hypotheses Acculturation is not just multidimensional in terms of the independence of the orientations towards the heritage culture and to wards the culture of the destination country, but also with regard to its various subdomains, whereby identification, values and practices can be distinguished (Schwartz, Unger, Zamboanga, & Szapocznik, 2010). Ethnic identity refers to the extent to which persons explored what their ethnic group means to them, and to the extent they feel attached to their ethnic group (Phinney, 1990). It may be expected that migrants in the Netherlands who identify strongly as Polish have a relatively strong tendency to visit doctors in Poland. This is illustrated by a migrant of Polish origin in the UK quoted in Goodwin, Polek, and Goodwin’s (2013) study who preferred a hospital stay in Poland over one in the UK, and expressed that the reason for this was that “when someone is ill, he wants to be [..] at home’’ (p. 164). Given that Polish migrants may identify more strongly with destination country with increased length of stay (Diehl, Fischer-Neumann, & Mühlau, 2016), it may therefore be hypothesized that the negative association between length of stay and the likelihood of visiting doctors in Poland is (partly) attributable to differences in ethnic identification (Hypothesis 2). i With increased length of stay in the destination country, migrants’ cultural values and beliefs may also change (Schwartz et al., 2010). Given the focus of the current study on health care services use, values regarding health care provision may be particularly relevant here. When migrants arrive in the destination country, they bring particular values and beliefs about how health care services should be provided and they tend to compare local healthcare practices with those in the healthcare practices in the country of origin (Goodwin et al., 2013; Leduc & Proulx, 2004; Sime, 2014). When aspects of the healthcare system in the destination country do not correspond with their values and beliefs and what they are used to, this may result in skepticism and distrust towards the destination country’s healthcare system. Sime (2014) has argued that “distrust towards the effectiveness of care […] may lead many migrants to view health care in transnational terms as a better and ‘safer’ approach, rather than to rely on local provision” (p. 92) (cf. Czapka & Sagbakken, 2016; Goodwin et al., 2013). Introduction (2003) defined continuity of care as “the degree to which a series of healthcare events is experienced as coherent and consistent with the patient’s medical needs and personal context” (p. 1221). When migrants use health care services in the country of origin in addition to the destination country, then this may hamper the exchange of information between the different care providers who interact with them. It may also be a barrier to the consistency and coherence of the management of the treatment of health conditions they may have, and to relational continuity between patient and provider (Haggerty et al., 2003). Together, this may result in suboptimal quality of the care these migrants receive. Scholars have pointed out the substantial within-group differences in migrants’ use of healthcare services (Leduc & Proulx, 2004; Osipoviˇc, 2013). Research on drivers of transnational medical travel among migrants has been mostly qualitative (Villa-Torres et al., 2017). Although findings from this qualitative work by design cannot be generalized, the thick descriptions provided by these studies have highlighted various factors that may shape migrants’ tendency to visit doctors in their country of origin. Most notably, the qualitative work suggest that the likelihood of doctor visits in the country of origin declines with increasing length of stay in the receiving country. The current study aims to extend this qualitative work by testing in population based sample of Polish migrants whether length of stay is indeed systematically negatively associated with the likelihood of visiting doctors in the country of origin, and by exploring the mediating role of acculturation factors in this association. Introduction The number of migrants of Polish origin in the Netherlands has been rising rapidly since Poland became a member of the European Union. The number of migrants of Polish origin in the Netherlands increased from less than 36,000 in 2004 to almost 200,000 in 2020 (Source: Statistics Netherlands). Luthra, Platt, and Salamo´nska (2016) noted that relatively many Polish migrants in the Netherlands can be labelled “committed expats”, as they tend to be committed to an international life from the very onset of their migration. While actively developing a new life in the Netherlands, they also maintain multiple linkages to their country of origin (cf. Schiller, Basch, & Blanc, 1995). For instance, they tend to maintain strong bonds and frequent contact with parents and family in Poland (Conkova, 2019; Karpinska & Dykstra, 2019) and a large majority frequently consumes Polish online and traditional media (Gijsberts, Andriessen, Nicolaas, & Huijnk, 2018). The current study focuses on a different aspect of the transnational lives that many Polish migrants in the Netherlands live, namely transnational medical travel. Polish migrants in the Netherlands are known to frequently visit doctors in their country of origin (Gij Polish migrants in the Netherlands are known to frequently visit doctors in their country of origin (Gijsberts et al., 2018). Such T. van den Broek International Journal of Intercultural Relations 84 (2021) 210–219 transnational medical travel may be detrimental for the continuity of the care they receive (cf. Kemppainen, Kemppainen, Skogberg, Kuusio, & Koponen, 2018). Haggerty et al. (2003) defined continuity of care as “the degree to which a series of healthcare events is experienced as coherent and consistent with the patient’s medical needs and personal context” (p. 1221). When migrants use health care services in the country of origin in addition to the destination country, then this may hamper the exchange of information between the different care providers who interact with them. It may also be a barrier to the consistency and coherence of the management of the treatment of health conditions they may have, and to relational continuity between patient and provider (Haggerty et al., 2003). Together, this may result in suboptimal quality of the care these migrants receive. transnational medical travel may be detrimental for the continuity of the care they receive (cf. Kemppainen, Kemppainen, Skogberg, Kuusio, & Koponen, 2018). Haggerty et al. Theoretical background and hypotheses Although qualitative studies suggest that some migrants of Polish origin appreciate the egalitarian patient-GP communication in Norway and the United Kingdom (Czapka & Sagbakken, 2016; Goodwin et al., 2013), a GP commu nication style that is less authoritarian than what Polish migrants are used to in their country of origin may also raise a sense of cultural unease and doubts about the GP’s competence (Osipoviˇc, 2013). Values and beliefs about how health care services should be provided, and therefore the discrepancy between these values and beliefs and the way the healthcare system of the destination country works, may change with increased length of stay in the destination country. Madden et al. (2017), for instance, noted that some of their respondents said they had become more open to the United Kingdom’s approach to self-care and medication use after staying in the country for several years, despite having strong reservations when they first arrived. Similarly, Czapka and Sagbakken (2016) observed that many migrants of Polish origin who had been in Norway for many years had changed their views about the use of antibiotics among children. Polish migrants may also increasingly appreciate physicians’ more friendly and egalitarian communication with patients (Czapka & Sagbakken, 2016; Goodwin et al., 2013; Sime, 2014). All this may make Polish migrants who stayed in the Netherlands longer reappreciate the Dutch healthcare system, resulting in waning distrust. Consequently, it may be expected that the negative association between length of stay and the likelihood of visiting doctors in Poland is (partly) attributable to differences in trust in the Dutch healthcare system (Hypothesis 3). Finally, language is a key aspect of the cultural practices subdomain of acculturation (Schwartz et al., 2010; Solis, Marks, Garcia, & Shelton, 1990). Migrants’ Dutch language proficiency tends to improve with increasing length of stay (Geurts & Lubbers, 2017). Dutch language proficiency improvements may, in turn, result in a declining tendency over time to seek health care in the country of origin, as qualitative studies suggest that language barriers are a driver for migrants’ transnational medical travel (Villa-Torres et al., 2017). Sime (2014) argued, for instance, that limited language proficiency harmed migrants’ ability to find information on health services (cf. Osipoviˇc, 2013) and the quality of their interactions with medical practitioners (cf. Goodwin et al., 2013), and that this made some of them feel more reassured when they used healthcare services in the country of origin. Theoretical background and hypotheses Aspects of the Dutch healthcare system that migrants of Polish origin may have reservations about – and that thus may lower Polish migrants’ trust in the Dutch healthcare system – are the role of the general practitioner (GP), antibiotics prescription practices, and the style of communication between physicians and patients. In the Dutch healthcare system, the GP serves as a gatekeeper. This means that a referral from the GP is required for inpatient hospital care (Boerma, Van der Zee, & Fleming, 1997; Kroneman, 2011). 211 T. van den Broek International Journal of Intercultural Relations 84 (2021) 210–219 Qualitative studies conducted in Norway (Czapka & Sagbakken, 2016) and the United Kingdom (Madden, Harris, Blickem, Harrison, & Timpson, 2017), where the GP also has a gatekeeping role (Kroneman, 2011), suggest that many Polish migrants do not appreciate this way of organizing the healthcare system. Many migrants of Polish origin reportedly dislike it when GPs treat a broad spectrum of diseases, because, in their view, a lack of specialization is likely to negatively affect the quality of the doctors’ work (Czapka & Sagbakken, 2016). Also, migrants sometimes perceive it as a cost-cutting measure and a sign of inefficiency when the GP acts as a gatekeeper restricting access to specialists (Sime, 2014). Madden et al. (2017) even noted resentment among several of their Eastern-European respondents in the United Kingdom about the power held by the GP, and the way this limited their access to the specialist care they felt they needed. In addition to the gatekeeping role of the GP, antibiotics prescription practices are much more restrictive in the Netherlands than in Poland (Goossens, Ferech, Vander Stichele, & Elseviers, 2005; Kroneman et al., 2016). A larger reluctance of GPs to prescribe anti biotics is sometimes perceived by migrants as unhelpful and dismissive (Madden et al., 2017). Czapka and Sagbakken (2016) noted that “some Polish migrants equate serious treatment by a physician with a prescription” (p. 9) and that a reluctance to prescribe can undermine Polish migrants’ trust and confidence in the health care system of the destination country. i Moreover, patient-GP communication in the Netherlands has become notably more egalitarian since the late 20th Century (Butalid, Verhaak, Boeije, & Bensing, 2012). Theoretical background and hypotheses In a similar vein, Czapka and Sagbakken (2016) described how Polish migrants in Norway preferred to visit doctors in their country of origin, rather than in the destination country, because they felt “more secure at the doctor’s in terms of linguistic competence” (p. 7). These considerations lead to the expectation that the negative association between length of stay and the likelihood of visiting doctors in Poland can (partly) be explained by language proficiency differences (Hypothesis 4). Data and methods Sample Control variables A range of potential confounders were controlled for to reduce the room for bias in the estimates of the effects of interest. These are variables that are plausibly predictive of healthcare visits to Poland as well as of one or more of the explanatory variables of interest (length of stay, trust in the Dutch healthcare system, and language proficiency). Failure to account for such variables may bias results, because estimates of effects of interest may be attributable to the confounders. Health status determines the need for care, which is, in turn, one of the most important antecedents of health care services use (Andersen & Newman, 1973). Although Polish migrants in the Netherlands tend to have relatively good mental and physical health in comparison to the general Dutch population without a migration background (Dagevos, 2011; Gijsberts et al., 2018), research has shown that health and psychosocial wellbeing declines with enduring length of stay (Lubbers & Gijsberts, 2019; Van den Broek & Grundy, 2017). A dichotomous variable distinguishing respondents who reported having “good” or “very good” health from their counterparts who reported that their health was “fair”, “bad” or “very bad” was therefore included. Experiences of discrimination is a known antecedent of healthcare use in the country of origin (Goodwin et al., 2013; Kemppainen et al., 2018). It also has a complex interrelation with length of residence in the destination country. Migrants in the Netherlands tend to report more experiences of discrimination with increased length of stay (McGinnity & Gijsberts, 2018). However, there is also evidence that experiences of discrimination are associated with return migration (Yilmaz Sener, 2019), which may imply out-selection of mi grants subjected to experiences of discrimination among those with lengthy residence spells in the destination country. Respondents were asked how often they had personally experienced discrimination by Dutch persons. Response categories were “never”, “almost never”, “now and then”, “often” and “very often”. Categories were collapsed, whereby respondents who reported that they “never” or “almost never” had experienced discrimination by Dutch persons were distinguished from their counterparts who reported having experienced discrimination at least now and then. In addition to these potential confounders, a range of basic socio-demographic background characteristics will also be adjusted for. Outcome variable The outcome variable of interest is whether or not respondents had visited a doctor in Poland in the last year. Respondents were asked whether they had visited Poland in the 12 months prior to the interview. Those who answered affirmatively were subsequently asked whether they had been to Poland in the last 12 months to visit a doctor. Based on these two questions, a dichotomous variable was derived. Respondents who reported having visited doctor in Poland in the last 12 months were coded as 1 and their counterparts who reported not having been to Poland in the last 12 months, or having been to Poland in the last 12 months, but not having visited a doctor while being there were coded as 0. Sample The data used in the current study are from the Survey Integration Minorities (SIM) (Dutch: Survey Intergratie Minderheden). SIM is a repeated cross-sectional survey aimed at providing insights on the structural and socio-cultural position of people with a migration background in the Netherlands (Van Thiel, Hooijmans, Schothorst, & Rozema, 2015). The study was commissioned by the Dutch Ministry of Social Affairs and Employment and conducted by the Netherlands Institute for Social Research, with data collection taking place at intervals of approximately five years. The third round of SIM (SIM2015) was collected between January and June 2015. In contrast to prior SIM rounds, the third round data was also collected among a subsample of migrants of Polish origin. Other groups interviewed were people with Turkish, Moroccan, Surinamese, Antillean, Somali and native Dutch backgrounds. Statistics Netherlands provided a sample of Polish-born adults aged 15 and older who registered in a Dutch municipality after January 2004 to facilitate data collection among the Polish subsample of the SIM2015 survey. It is important to note that, in the Netherlands, newcomers with the intention of staying longer than four months are required to register in the municipality where they reside. 1,129 migrants of Polish origin participated in the survey (response rate: 44.7 %). Two modes of data collection were used: a web survey (53.2 %) and computer-assisted face-to-face interviews (46.8 %). Regardless of the mode of data collection, respondents could choose to participate in a Polish language version (84.1 %) or a Dutch language version (15.9 %) of the survey. The Polish origin subsample contained 47 respondents (4.2 %) with missing values on at least one variable of interest: ethnic self- identification (n = 21), reported experiences of discrimination (n = 13), length of stay (n = 8), and educational attainment (n = 6). After listwise deletion a final analytical sample of 1,082 respondents remained. Supplied analytical weights were used to adjust for systematic non-response. The Polish origin subsample contained 47 respondents (4.2 %) with missing values on at least one variable of interest: ethnic self- identification (n = 21), reported experiences of discrimination (n = 13), length of stay (n = 8), and educational attainment (n = 6). After listwise deletion a final analytical sample of 1,082 respondents remained. Supplied analytical weights were used to adjust for systematic non-response. 212 International Journal of Intercultural Relations 84 (2021) 210–219 T. van den Broek Explanatory variables Length of stay in the Netherlands was included as a continuous variable. It was derived by deducting the year in which respondents reportedly moved to the Netherlands for the first time from the year in which the interview took place. A log transformation was performed to account for the strongly positively skewed distribution of this variable. i Ethnic self-identification was measured with a single question: “Do you more strongly feel Polish or Dutch?”. Respondents who answered that they felt “completely Polish" or "more Polish than Dutch" were coded as self-identifying strongly as Polish. Respondents who answered that they felt “as much Polish as Dutch”, “more Dutch than Polish” or “completely Dutch” were coded as not self- identifying strongly as Polish. Trust in the Dutch healthcare system was measured with a single question “How much trust do you currently have in the healthcare system in the Netherlands?” Respondents were asked to indicate their level of trust on a 10-point scale, with a score of one indicating “very little trust” and a score of ten indicating “very high trust”. i With regard to language skills, respondents were asked “How often do you have language difficulties when having a conversation in Dutch?” Response categories were “often”, “sometimes” and “never”. Respondents could also indicate that they did not speak Dutch at all. Respondents for whom this was the case – approximately 13 percent of the Polish origin sample – were coded as having difficulties with the Dutch language often. Control variables In addition to a dichotomous variables for sex (women vs men), employment status (currently in paid employment vs not currently in paid employment) and partner status (partnered vs unpartnered), and categorical variables for age, educational attainment, and level of urbanization were included in the models. Age categories were 24 and younger; 25−34 years old; 35−44 years old; and 45 years and older. Three categories of educational attainment were distinguished: none or low; intermediate; and high. These categories were pre- coded. The more detailed information on education followed in Poland as well as in the Netherlands on which the pre-coded values were based was not made available in the public release version of the SIM2015 dataset. Level of urbanization of the municipality of registration was originally measured with a five-category classification: very strongly urbanized; strongly urbanized; moderately urbanized; weakly urbanized; rural. Given the lower numbers of respondents in the latter three categories these were collapsed into one category for low level of urbanization. Measures Outcome variable Methods In addition to basic univariate and bivariate descriptive analyses, a series of logistic regression models were estimated. In the first model, the likelihood of healthcare visits to Poland was regressed on length of stay in the Netherlands and the aforementioned range of 213 International Journal of Intercultural Relations 84 (2021) 210–219 T. van den Broek control variables. In subsequent models the proposed mediators (ethnic self-identification, trust in the Dutch healthcare system and Dutch language proficiency) were added. Karlson, Holm and Breen’s KHB decomposition method (Kohler, Karlson, & Holm, 2011) was used to formally test the extent to which the effect of length of stay on the likelihood of healthcare visits to Poland was mediated by, respectively, ethnic self-identification, trust in the Dutch healthcare system and Dutch language proficiency. The KHB method is suitable for the analysis of mediation in logistic regression and other non-linear models, because it accounts for attenuation bias that may occur in such models. control variables. In subsequent models the proposed mediators (ethnic self-identification, trust in the Dutch healthcare system and Dutch language proficiency) were added. Karlson, Holm and Breen’s KHB decomposition method (Kohler, Karlson, & Holm, 2011) was used to formally test the extent to which the effect of length of stay on the likelihood of healthcare visits to Poland was mediated by, respectively, ethnic self-identification, trust in the Dutch healthcare system and Dutch language proficiency. The KHB method is suitable for the analysis of mediation in logistic regression and other non-linear models, because it accounts for attenuation bias that may occur in such models. Notes: Data are from Survey on the Integration of Minorities (SIM) 2015; n = 1,082; data are weighted. Descriptive results Descriptive results Sample characteristics are provided in Table 1. One in four respondents reported having visited a doctor in Poland in the year prior to the interview. The group that had visited a doctor in Poland differed from the group that had not done so in several ways. Compared to their counterparts who had not visited a doctor in Poland, migrants who had embarked on such doctor visits, on average, had stayed in the Netherlands for a shorter period and had lower trust in the Dutch health care system. Also, a relatively large share of this group reported identifying strongly as Polish and having severe difficulties with the Dutch language. They also relatively often reported having had personal experiences of discrimination. Furthermore, migrants who had visited a doctor in Poland were relatively often female, highly educated and in less than good health. Results of multivariate analyses Table 2 presents the results of the logistic regression analyses. In Model 1, the likelihood of healthcare visits to Poland was regressed on length of stay in the Netherlands and a range of controls. As hypothesized, results indicated that length of stay was associated with a lower likelihood of having visited a doctor in Poland in the last year. Adjusted predictions are presented in Fig. 1 to facilitate an easier interpretation of the magnitude of this effect. These were calculated by setting the value of length of stay at distinct values while using observed values for each case for all other covariates included in Model 1. Based on these fixed and observed values of variables, the predicted probability of healthcare visits to Poland was then derived for each case, and subsequently the predicted values of all cases a Before log transformation. Table 1 Table 1 Sample characteristics; Percentages and means. All respondents visited doctor in Poland did not visit doctor in Poland Group difference Visited doctor in Poland 24.1 % Mean length of staya 6.3 5.7 6.6 F(1, 1080) = 10.6, p < .01 (standard deviation) (3.7) (3.0) (3.9) Self-identifies strongly as Polish 78.0 % 88.5 % 74.6 % χ2(1, n = 1082) = 19.3, p < .001 Mean trust in Dutch healthcare system 6.8 6.0 7.0 F(1, 1080) = 36.2, p < .001 (standard deviation) (2.5) (2.7) (2.3) Dutch language proficiency χ2(2, n = 1082) = 15.5, p < .001 Severe language problems 48.3 % 58.3 % 44.5 % Moderate language problems 40.4 % 35.1 % 42.7 % No language problems 11.3 % 6.7 % 12.8 % Female 51.6 % 64.8 % 47.4 % χ2(1, n = 1082) = 18.8, p < .001 Age: χ2(3, n = 1082) = 20.0, p < .001 < = 24 13.2 % 13.2 % 13.2 % 25−34 48.2 % 60.0 % 44.4 % 35−44 24.2 % 17.2 % 26.4 % > = 45 14.4 % 9.6 % 15.9 % Has partner 71.3 % 70.7 % 71.5 % χ2(1, n = 1082) = 0.1, p = .82 In paid employment 76.3 % 80.4 % 75.0 % χ2(1, n = 1082) = 2.9, p = .09 Educational attainment: χ2(2, n = 1082) = 18.2, p < .001 Low 39.0 % 32.3 % 41.0 % Mid 37.9 % 34.0 % 39.1 % High 23.1 % 33.6 % 19.8 % Level of urbanization χ2(2, n = 1082) = 3.2, p = .20 Low 44.4 % 42.9 % 44.9 % Mid 24.4 % 21.5 % 25.4 % High 31.1 % 35.6 % 29.7 % Poor self-reported health 15.8 % 20.6 % 14.3 % χ2(1, n = 1082) = 4.1, p < .05 Experiences of discrimination 46.1 % 54.4 % 43.5 % χ2(1, n = 1082) = 8.0, p < .01 Number of cases 1,082 253 829 Notes: Data are from Survey on the Integration of Minorities (SIM) 2015; n = 1,082; data are weighted. a Before log transformation. Sample characteristics; Percentages and means. 214 International Journal of Intercultural Relations 84 (2021) 210–219 T. van den Broek Table 2 Coefficient estimates from logistic regression analyses of medical visits to Poland. Model 1 Model 2 Model 3 Model 4 Coeff. [95 % CI] Coeff. [95 % CI] Coeff. [95 % CI] Coeff. Table 1 All this was done using the margins postestimation command in Stata 15.1 (Williams, 2 were averaged. All this was done using the margins postestimation command in Stata 15.1 (Williams, 2012). Model 1 furthermore showed that doctor visits in Poland were more likely for women than for men. Polish migrants with high educational attainment and those in paid employment were furthermore more likely to have visited doctors in Poland than their lower educated and non-employed counterparts. Finally, poor self-rated health and personal experiences of discrimination were associated with a higher likelihood of doctor visits in Poland. i Ethnic self-identification was added as an explanatory variable in Model 2. The model showed, as expected, that doctor visits to Poland were more likely for migrants who identified strongly as Polish than for their counterparts with a weaker self-identification as Polish. After the addition of ethnic self-identification, the coefficient estimate of length of stay became somewhat smaller in magni tude, but the KHB procedure did not show evidence of significant mediation (Δb = −0.117; 95 % CI: −0351, 0.116; p = .324). No support was thus found for the second hypothesis that the negative association between length of stay and the likelihood of visiting doctors in Poland could (partly) be attributed to differences in ethnic self-identification. Estimates of the control variables included in the model did not change substantially either between Model 1 and Model 2. Model 3 also included trust in the Dutch healthcare system as an additional explanatory variable. Consistent with expectations, the model showed that Polish migrants with a higher level of trust in the Dutch health care system were less likely to have visited a doctor in Poland in the last year. However, the addition of trust in the Dutch healthcare system did not attenuate the estimated effect of length of stay and the KHB procedure did not show evidence of significant mediation (Δb = 0.033; 95 % CI: −0.145, 0.211; p = .717). The hypothesis that the negative association between length of stay and the likelihood of visiting doctors in Poland could (partly) be attributed to differences in trust in the Dutch healthcare system was thus not supported. No substantial changes in the estimates of control variables included in the model could be observed between Model 2 and Model 3. Table 1 ii The final acculturation factor of interest – Dutch language proficiency – was added as an explanatory variable in Model 4. The model showed that Polish migrants who had severe difficulties speaking Dutch were more likely to have visited a doctor in Poland in the last year than their counterparts who only had moderate difficulties or no difficulties at all with the Dutch language. The coefficient estimate of length of stay became smaller and was no longer statistically significant after the addition of Dutch language proficiency to the model. The KHB procedure indicated that the mediation of the effect of length of stay on the likelihood of doctor visits in Poland via language proficiency was statistically significant (Δb = −0.234; 95 % CI: −0.458, −0.010; p < .05). These results provide empirical support for the fourth hypothesis that the negative association between length of stay and the propensity of visiting doctors in Poland was partly attributable to language proficiency differences. Again, no substantial change between Model 3 and Model 4 could be noted in the estimates of the control variables included in the model. i To provide a more intuitive insight in the magnitude of the effects of ethnic self-identification, trust in the Dutch healthcare system and language proficiency, adjusted predictions were again calculated based on Model 4 in Table 2 according to the procedure described above. The adjusted predictions of the likelihood of doctor visits to Poland are presented in Fig. 2. A strong self-identification as Polish was, on average, associated with a higher predicted probability of doctor visits in Poland by 10.2 percentage points (95 % CI: .042, .161; p < .01). A one-point increase on the ten-point scale of trust in the Dutch health care system was, on average, associated with a decline of 1.9 percentage points in the predicted probability of a doctor visit in Poland (95 % CI: −.030, −.009; p < .01). Having severe language difficulties as opposed to not having such difficulties was, on average, associated with a 12.8 percentage points (95 % CI: .045, .211; p < .01) higher predicted probability of a doctor visit in Poland. Table 1 [95 % CI] Length of stay (log) −0.412** [−0.717, −0.107] −0.315* [−0.628, −0.003] −0.362* [−0.677, −0.047] −0.148 [−0.494,0.198] Female 0.707* [0.359,1.056] 0.735* [0.384,1.085] 0.683* [0.330,1.035] 0.700* [0.345,1.056] Age: < = 24 Ref. Ref. Ref. 25−34 0.225 [−0.309,0.758] 0.204 [−0.334,0.743] 0.223 [−0.333,0.780] 0.083 [−0.477,0.643] 35−44 −0.368 [−0.984,0.247] −0.374 [−0.998,0.250] −0.300 [−0.940,0.341] −0.448 [−1.094,0.199] > = 45 −0.361 [−1.013,0.291] −0.343 [−1.009,0.322] −0.240 [−0.923,0.443] −0.450 [−1.145,0.244] Has partner −0.157 [−0.533,0.219] −0.177 [−0.553,0.200] −0.213 [−0.592,0.165] −0.259 [−0.639,0.121] In paid employment 0.495* [0.087,0.903] 0.433* [0.023,0.844] 0.424* [0.006,0.842] 0.429* [0.013,0.846] Educational attainment: Low Ref. Ref. Ref. Mid 0.097 [−0.280,0.474] 0.111 [−0.269,0.490] 0.094 [−0.292,0.480] 0.118 [−0.267,0.504] High 0.643 [0.234,1.052] 0.646 [0.232,1.061] 0.607 [0.197,1.018] 0.673 [0.253,1.092] Level of urbanization Low Ref. Ref. Ref. Mid −0.140 [−0.561,0.281] −0.166 [−0.590,0.258] −0.173 [−0.597,0.251] −0.174 [−0.600,0.253] High 0.286 [−0.073,0.644] 0.273 [−0.088,0.633] 0.262 [−0.103,0.627] 0.257 [−0.111,0.625] Poor self-reported health 0.625 [0.205,1.044] 0.670 [0.247,1.093] 0.652 [0.215,1.088] 0.616 [0.177,1.054] Experiences of discrimination 0.433** [0.116,0.749] 0.397* [0.078,0.716] 0.267 [−0.059,0.592] 0.262 [−0.066,0.589] Strongly self-identifies as Polish 0.883* [0.450,1.317] 0.842* [0.401,1.283] 0.694 [0.245,1.144] Trust in Dutch healthcare system −0.118 * [−0.183, −0.053] −0.121 *** [−0.187, −0.055] Dutch language proficiency Severe lang. problems Ref. Moderate lang. problems −0.419* [−0.772, −0.066] No language problems −0.854 [−1.495, −0.212] Intercept −1.636 * [−2.412, −0.861] −2.471 * [−3.337, −1.606] −1.475 [−2.484, −0.466] −1.347** [−2.368, −0.327] Pseudo R2 (McFadden) .076 .092 .104 .112 BIC 81,683.1 80,351.4 79,305.6 78,550.6 Notes: Data are from Survey on the Integration of Minorities (SIM) 2015; n = 1082; data are weighted; coefficient estimates with 95 % confidence intervals in brackets. * p < 0.05. Table 2 Coefficient estimates from logistic regression analyses of medical visits to Poland. Table 2 Coefficient estimates from logistic regression analyses of medical visits to Poland. on the Integration of Minorities (SIM) 2015; n = 1082; data are weighted; coefficient estimates with 95 % confidenc Notes: Data are from Survey on the Integration of Minorities (SIM) 2015; n = 1082; data are weighted; coefficient estimates with 95 % confidence intervals in brackets. * * p < 0.05. p < 0.01. *** p < 0.001. Fig. 1. Predicted probability of health care visits to Poland by length of stay. Fig. 1. Predicted probability of health care visits to Poland by length of stay. Fig. 1. Predicted probability of health care visits to Poland by length of stay. 215 T. van den Broek International Journal of Intercultural Relations 84 (2021) 210–219 were averaged. Discussion Also, the measure of ethnic self-identification was unidirectional, which means that it implicitly assumed that strong identification with the county of origin and a strong identification with the destination country were mutually exclusive (Arends-T´oth & Van de Vijver, 2004; Rudmin, 2009). This is at odds with Berry’s (1992, 2005) seminal bidimensional acculturation model. Future research on the links between acculturation and migrants’ transnational medical travel endeavors would be strengthened if surveys like the Dutch SIM study would collect more comprehensive and bi-lineal measures of acculturation in future waves (cf. Rudmin, 2009). Trust in the Dutch healthcare system was moreover measured with a single item. Recently, multi-item trust measures that acknowledge that healthcare trust comprises multiple dimensions – for instance trust in physicians’ competence, trust in the absence of a hidden agenda, and trust in the absence of discrimination – have been proposed (e.g., Schwei, Kadunc, Nguyen, & Jacobs, 2014; Sheppard, Huei-Yu Wang, Hurtado-de-Mendoza, Sutton, & LaVeist, 2019). Future studies using such multidimensional trust measures could explore whether specific dimensions of trust in the healthcare systems particularly shape migrants’ likelihood of visiting doctors in the country of origin. i The theoretical framework underlined the potential relevance of the cultural values subdomain of acculturation – and specifically of values and beliefs about how healthcare ought to be provided – for healthcare services use. Information on such values was, however, unfortunately not available in the data, and therefore trust in the Dutch healthcare system - conceptualized as an indicator of the concordance of migrants’ values and beliefs regarding healthcare provision with the Dutch healthcare system – was considered instead. Caution is called for as to whether trust in the Dutch healthcare system can indeed be perceived as a derivative of migrants’ values about how healthcare should be provided. This is because newly arrived migrants compare the healthcare system in the destination country not just with their values and beliefs, but also with with the system in country of origin (Goodwin et al., 2013, Leduc & Proulx, 2004; Sime, 2014). When there are aspects about the system in the country of origin that they do not appreciate, they may already evaluate the healthcare system of the destination country relatively positively upon arrival (R¨oder & Mühlau, 2012). Based on Eurobarometer data, Goodwin et al. noted that Poles have quite negative views about the healthcare system in their home country (Goodwin et al., 2013). Discussion A key aspect of the transnational lives led by Polish migrants in the Netherlands is their frequent use of healthcare services in their country of origin. Qualitative research suggests that migrants of Polish origin who recently arrived in the destination country are less likely to visit doctors in their country of origin than their counterparts who had been in the destination country for a longer period of A key aspect of the transnational lives led by Polish migrants in the Netherlands is their frequent use of healthcare services in their country of origin. Qualitative research suggests that migrants of Polish origin who recently arrived in the destination country are less likely to visit doctors in their country of origin than their counterparts who had been in the destination country for a longer period of Fig. 2. Predicted probability of health care visits to Poland by various acculturation factors. Fig. 2. Predicted probability of health care visits to Poland by various acculturation factors. 216 T. van den Broek International Journal of Intercultural Relations 84 (2021) 210–219 time (Osipoviˇc, 2013). The aim of the current study was to assess whether length of stay in the Netherlands is indeed systematically associated with a lower likelihood of doctor visits in Poland, and the extent to which this potential negative association is mediated by three acculturation factors: ethnic self-identification, trust in the Dutch healthcare system and Dutch language proficiency. ii As hypothesized, results indicated that migrants who stayed in the Netherlands longer were less likely to visit doctors in Poland. The analyses presented here moreover showed that a strong ethnic self-identification as Polish, low trust in the Dutch healthcare system and low Dutch language proficiency were associated with a higher likelihood of doctor visits in Poland. Moreover, the analyses provided evidence that the negative association between length of stay and the likelihood of visiting doctors in Poland was to a substantial extent attributable to improvements in Dutch language proficiency. Earlier research has highlighted that acculturation matters for access to healthcare in the destination country (e.g., Fassaert, Hesselink, & Verhoeff, 2009; Solis et al., 1990; Van der Stuyft, De Muynck, Schillemans, & Timmerman, 1989). The current study adds that, at least for Polish migrants in the Netherlands, acculturation also shapes the disposition towards medical visits in the country of origin. Discussion ii Although ethnic self-identification and lack of trust in the Dutch healthcare system were identified as important antecedents of health care visits to Poland, their inclusion in the models did not significantly attenuate the estimated negative effect of length of stay on the likelihood of doctor visits in Poland. This implies that ethnic-self-identification did not weaken and that migrants’ trust in the Dutch healthcare system did not improve substantially with increasing length of stay in the Netherlands. The absence of a substantial change with increasing length of stay on the identity (ethnic self-identification) and value (trust in the Dutch healthcare system) domains of acculturation in conjunction with considerable improvements in Dutch language proficiency – an aspect of the cultural practices domain of acculturation – underscore the point emphasized by Schwartz et al. (2010) that the various domains of accul turation are independent. p Several limitations of the current study should be considered. First of all, causality cannot be inferred from the current observa tional study given its cross-sectional design. Moreover, it is unfortunate that detailed information about the type of the healthcare used in Poland was not available. Given that costs considerations may also lead migrants to use care services in their country of origin (Czapka & Sagbakken, 2016; Migge & Gilmartin, 2011; Villa-Torres et al., 2017), it would be interesting to explore whether Polish migrants in the Netherlands are particularly likely to seek care in Poland for services that are not covered in the Netherlands as part of the basic benefits package of the country’s mandatory and universal social health insurance, for instance dental care (Kroneman et al., 2016). In this light, it is also unfortunate that the role of income in shaping migrants’ disposition towards doctor visits in the country of origin could not properly be assessed, given that information on income was missing for a very large part of the sample. The current study’s finding of a positive association between educational attainment and the likelihood of having visited doctors in Poland may reflect that people with higher levels of education typically have a greater financial ability to travel back to Poland. li The way in which several key concepts were operationalized constitutes another limitation of the current study. Most notably, the concept of acculturation arguably contains more domains than the three domains considered here (Arends-T´oth & Van de Vijver, 2004; cf. Schwartz et al., 2010). Discussion This may be related to the perceptions of corruption in the Polish healthcare system and of suboptimal efficiency of the management of healthcare visits (Czapka & Sagbakken, 2016; Goodwin et al., 2013). This may translate in a relatively positive evaluation of the Dutch healthcare system, also by migrants of Polish origin who recently arrived in the Netherlands. Finally, there are plausible mechanisms underlying an association length of stay and the likelihood of visiting doctors in Poland that could not be tested due to data limitations. Moreover, qualitative research highlighted the importance of insufficient familiarity with the destination country’s healthcare system, e.g. knowledge about entitlements, as a driver of doctor visits in the country of origin, particularly among recently arrived migrants (Czapka & Sagbakken, 2016; Glinos, Baeten, Helble, & Maarse, 2010; Migge & Gilmartin, 2011; Osipoviˇc, 2013). Unfortunately, it was not feasible to investigate whether increasing familiarity of the Dutch healthcare system partly mediated the association between length of stay and the likelihood of doctor visits in Poland, because information on knowledge 217 T. van den Broek International Journal of Intercultural Relations 84 (2021) 210–219 of the Dutch healthcare system was not collected. It should be noted, however, that earlier research suggests that familiarity with the Dutch healthcare system tends to be quite good among Polish migrants in the Netherlands (Dagevos, 2011). Madden et al. (2017) also reported good familiarity with the British healthcare system among Eastern-European migrants in the UK. The current study showed that Polish migrants, and particularly those who identify strongly as Polish, those with limited trust in the Dutch healthcare system and those with a poor command of Dutch, often use healthcare services in their country of origin. This may reflect poor inclusion of certain subgroups of Polish migrants in the Netherlands in the Dutch healthcare system, and may result in the receipt of care of suboptimal quality among these migrants due to fragmentation. 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https://openalex.org/W2803313196	https://nv.nltu.edu.ua/index.php/journal/article/view/687/676	Ukrainian	null	МЕТОД АВТОМАТИЧНОГО УСУНЕННЯ УДАРІВ ТА ВІБРАЦІЙ ПІД ЧАС БУРІННЯ	Naukovij vìsnik NLTU Ukraïni	2,015	cc-by	1,479	ння на долот ину. сті для забезп і вібрацій. Пр зують вібрац же, у цьому ви буріння. Алго авантаження н лот безп . Пр рац му ви А 3 ння 3 ння вібрацій під час бурі іння є: навантаже ачі бурового розчи умови її достатнос генерацію ударів и буріння сигналі оптимальності. Отж тимізації режиму ) містить зміну на к науково-технічних праць система переходить му лежать знання ек- ють на сьогодні буро- ю динамічною систе- уючих впливів і сиг- м середовищем. Ос- 4. Інформаційні технології галузі Рис. 1. Алгоритм автоматичного усунення ударів і с бур ь пода у, за ає на умов я до о ю оп ис. 1) Науковий вісник НЛТУ України. – 2015. – Вип. 25.10 новними керуючими параметрами під час частота обертів бурової колони; швидкість Швидкість подачі бурового розчину чення потрібного виносу шламу, не вплив неоптимальність режиму та незадовільні усунення яких свідчитиме про наближення падку вібрації виступають в ролі критерію ритм автоматичного усунення вібрацій (р долото та частоти обертів бурової колони. 5 5 аїни ків; ка" - - х а у - д , ння ле- ути им, по- на йно ми ого сне оз- д її от- як ви- ня, ний університет Укра ір. Т.М. Матвійк вська політехнік ід час похило-керо- оритмах, які врахо- а рішення подібних тримати швидше та я динаміки процесу мереж Петрі та да- дарів і вібрацій під основі мереж Петрі, ребує використан шення цієї пробл ає змогу досягну 00 м. Разом з ти оцесі глибинно-п собливий вплив ювати надзвичай дестабілізаційни го свердловинно ння, їх несвоєчас ризвести до серйо галом, наприклад рів і вібрацій істо ання ЕСП, таких буріння. Як прав ісля рейсу бурінн наук ист у ле ь на дин ючи сер ехні Асп рни а о тна итм ую ною керу шнім рол ими онн бур ть п К) з уд ист ча ум 7 - м ) 32 метрів ості ст ся таки ( льному в ( истеми; P ну в інший трі іння Петрі [9], та в загал множина станів си стеми з одного стан на основі мережі Пет аметрів режиму бурі них па у досяж описує них праць о в разі я і нав- К збіль- зовано- ням мо- 4. Інформаційні технології галузі Рис. 4. Граф досяжності станів процесу врахування технологічн режиму буріння Для визначення досліджуваних станів і побудови графу нів використовують модель на базі теорії мереж Петрі [8], яка о чином: Ммп= (П, Пр, Рб, М), галузі ності станів процесу врахування технологіч режиму буріння ня досліджуваних станів і побудови графу модель на базі теорії мереж Петрі [8], яка о Ммп= (П, Пр, Рб, М), галузі жності станів процесу врахування технологі режиму буріння ння досліджуваних станів і побудови граф модель на базі теорії мереж Петрі [8], яка Ммп= (П, Пр, Рб, М), те – Вип. 25.10 ї графів [8] і мереж ном: G = (Cm, P), системи [10]; Cm – можливі переходи си едставлення моделі н технологічних пара делей, які ґрунтуються на теорії падку формулюється таким чино де: G – граф досяжності станів множина ребер, яка відображає м Рис. 3. Схемна форма пред для алгоритму врахування емна ф тму вр а від нології г наченн ують м нології г наченн ують м них праць о в разі я і нав- К збіль- зовано- ням мо- 4. Інформаційні техн Рис. 4. Граф д Для визн нів використов чином: Рис. 3 для ал ситет України експертної оже запро- ого підхо- нення. рник ні ст БК тот трі мето ся з Ос- ня наванта- еральних – ових вібра- и наванта- приводить вністю зу- рацій. По- трами. Як- у конкрет- же раніше них прац о в ра я і нав К збіль зовано ням мо ово-технічн ни, тобто ншується ертів БК автоматиз ористанн ма мо ован усун шенн лате ерто ивши не п и пов вібр амет цій у є уж . 2). ння мод рмацій ації в ситуац у інфор их сис язку, як у інфор их сис язку, як и. – 201 ении ны мо анные намик мом бу тод, у емое б Teslyu Dire decisio illing re foc databa edge wnhol nhole 68 дстав спосо ктер п ступін влено На ос а таки ом під истика ов для ру мір інках, ння ст пеки, ній інф ов дл ру мір інках, ння ст пеки, ній ін 0 браций, получить реше дования динамики проц ируются на теории сет автоматического устран рации, алгоритм, модел cision Support Sys lling stem (DSS) for shocks a System architecture, o DSS incorporates real-t the design process, we u on. The proposed syste drilling of oilfield well drilling, shocks and vibra . викл. І.Р. Опірськи НУ "Ль для визначення об'єкті ї моделей, характер пр оделювання об'єкта, пр ння характеристик об'є цію моделей захисту ін ікації моделей захисту за способом реалізації, делювання об'єкта, за п уваного об'єкта. це створення ум еми з точки зор еба в таких оці ою відпрацюван ормаційної без ідними у сучасн й – ц систе Потр мето ії. інфо прові – ц исте Потр мето . інфо рові ма мо ован усун шенн лате ерто ивши не и по вібр амет цій у є уж . 2). чний уні азу зна система енційо собів у збільш они, а л ну обе менши -20 % оберти хання ми пара вібрац стовує я (рис. техн в ба ірник ні ст БК тот трі мето ся з – " 9 - о є ь і - 3 єктивної оці азливості а ичай, виник гічних ріше рема на рів труктурі фу ние быстрее и эффек- цесса устранения уда- тей Петри и дают воз- нения ударов и вибра- ль на основании сетей tem for Shocks a and vibration mitigation peration algorithm and time databases, rule-ba- use Bayesian Networks m works in an advisor s with modern MWD-, ations, database, know- ий, канд. техн. наук вівська політехнік в під час дослідження оцесів і явищ, які від- ризначення й специфі- єктів дослідження, які нформації в інформа- у наведено структурну за характером проце- призначенням об'єктів я об'є ри ура зазви ратег зокр фраст рни he X cous ич ибр остр устр кот ї галузі альної захисту сучасн еж зв'я України устране построен азработа вать дин равляем лова: ме правляе T.M., T tion in ment of d ional dri design ar wledge d t knowle ed in dow ms. SS, down 5]:061.6 но і пред такі як: с мі, харак ження, с Представ ержави. ахисту за арактеро рактери 4. Інформаційні технолог Основне при ки загального стан рівня захищеності під час аналізу зага під час організації Створення корпоративних мер ник НЛТУ едующем При этом п браций. Ра ь исследов клонно-упр ючевые сл аклонно уп atviykiv T s Mitigat e developm hole directi c diagram d expert know for expert can be use SS-system ywords: DS e. 056+3.75 ропонован захисту, т ся у систем ів дослідж юються. П ережах де моделей за темі, за ха ння, за хар ковий вісн ри после ивнее. П ов и виб ожность ий в нак Клю Петри, на Ma rations The n downh chematic ed and e modeling mode. It c WD-, RS Key edge base К 004.[0 Запр оделей з уваютьс а об'єкті загальню ійних ме одель м ів у сист осліджен Наук пр ти ро м ци П Vib in sc se m m LW le УДК м бу ка уз ці м сі до ь ar D я країни а ви- мате- про- веде- каза- у ви- ацій, і рі- варі- льтат шень, апро- вання ніка", versity ійків, НАН буро- итету Львів : T.M. 013. – s / O. – Pp. аїни ви- те- ро- де- за- ви- цій, рі- арі- тат нь, ро- ння ка", sity ків, АН уро- ету вів : .M. 3. – / O. Pp. во-технічних праць ernational Semina ve Theory (DIPED о устранения наклонно-уп- х алгоритмах, ударов и виб- обеспечивают, entat ional nal d 15. – Вип. ударов и одели и е модели ку проце бурении. удары и бурение. uk V.M ectional on-suppo is descr cused on ases. Du impleme le direct e directio влено озн оби реал підходу нь узагал о класиф снові кл им поділ дходу до ами досл дни ана пад с. 3 с. 4 роц і в уда плю Ре ття сите дни ана пад с. 3 с. 4 роц і в уда плю Ре ття ічний універс жина вхід ель записа цьому вип ду, на рис 2, а на рис инаміку пр ня ударів сунення у я. Він охо ої колони. а прийнят система мо у процесом "Львівська п illing // 7-th e олони / Т.М МЕ ім. Г.Є. П та ризику по іонального у та виробницт drilling autom (JGRCS). – I utomated Op Vol 27 Num к науков Vth Inte tic Wav еского аций в оенных анения у орые о ехні мно ої к ППМ оти Нац ки т in nce r A – V
https://openalex.org/W4313035812	https://uajnas.adenuniv.com/index.php/uajnas/article/download/151/176	Arabic	null	Integrals formulas involving confluent hypergeometric Functions of three variables Ф$_2^{(3)}$ and Ѱ$_2^{(3)}$	Mağallaẗ ğāmi'aẗ 'adan li-l-'ulūm al-ṭabīyyaẗ wa-al-taṭbīqiyyaẗ	2,018	cc-by-sa	6,998	Abstract The aim of this paper is to establish two general integral formulas involving confluent hypergeometric functions of three variables Ф2 (3) and Ѱ2 (3) with the help of two extension formulas for Lauricella’s functions of three variables 𝐹𝐴 (3) and 𝐹𝐷 (3) due to Atash [1] and Atash and Bellehaj [2]. Some applications of our main results are also presented. eywords: Integral formulas, Hypergeometric functions, Dixon’s theorem, Kummer’s theorem Introduction 1. Introduction The Lauricella’s functions of three variables ) 3 ( A F and ) 3 ( D F are defined and represented as follows [7]: ( ) 3 2 1 3 2 1 3 2 1 ) 3 ( , , ; , , ; , , , x x x c c c b b b a FA ! ! ! ) ( ) ( ) ( ) ( ) ( ) ( ) ( 3 3 2 2 1 1 0 , , 3 2 1 3 2 1 3 2 1 3 2 1 3 2 1 3 2 1 3 2 1 m x m x m x c c c b b b a m m m m m m m m m m m m m m m   = + + = (1.1) 1 3 2 1  + + x x x d ) 3 2 1 3 2 1 3 2 1 , , ; , , ; , , , ! ! ! ) ( ) ( ) ( ) ( ) ( ) ( ) ( 3 3 2 2 1 1 0 , , 3 2 1 3 2 1 3 2 1 3 2 1 3 2 1 3 2 1 3 2 1 m x m x m x c c c b b b a m m m m m m m m m m m m m m m   = + + = (1.1) 1 3 2 1  + + x x x and (1.1) and ( ) 3 2 1 3 2 1 ) 3 ( , , ; ; , , , x x x d b b b a FD ! ! ! ) ( ) ( ) ( ) ( ) ( 3 3 2 2 1 1 0 , , 3 2 1 3 2 1 3 2 1 3 2 1 3 2 1 3 2 1 m x m x m x d b b b a m m m m m m m m m m m m m m m   = + + + + = (1.2) 1 } , , max{ 3 2 1  x x x , ! ! ! ntegrals formulas involving confluent hypergeometric …...Ahmed A.Atash,Hussein S. Belleh ntegrals formulas involving confluent hypergeometric …...Ahmed A.Atash,Hussein S. Belleh Integrals formulas involving confluent hypergeometric Functions of three variables Ф2 (3)and Ѱ2 (3) Ahmed Ali Atash1 and Hussein Saleh Bellehaj2 1,2Department of Mathematics,, Faculty of Education-Shabwah Aden University, Aden, Yemen 1ah-a-atash@hotmail.com ,2bellehaj123@hotmail.com DOI: https://doi.org/10.47372/uajnas.2018.n1.a11 Univ. Aden J. Nat. and Appl. Sc. Vol. 22 No.1 – April 2018 143 1. Introduction ) ( ) ( ) ( ) ( ) ( 3 3 2 2 1 1 0 , , 3 2 1 3 2 1 3 2 1 3 2 1 3 2 1 3 2 1 m x m x m x d b b b a m m m m m m m m m m m m m m m   = + + + + = (1.2) 1 } { (1.2) where n a) ( is the Pochhammer’s symbol defined by where n a) ( is the Pochhammer’s symbol defined by    = − + + + = = ,...... 3,2,1 , )1 ( ).... 2 )( 1 ( 0 , 1 ) ( n if n a a a a n if a n (1.3)    = − + + + = = ,...... 3,2,1 , )1 ( ).... 1. Introduction ! ) ( ) ( ) ( ) ( 3 3 2 2 1 1 0 , , 3 2 1 3 2 1 3 2 1 3 2 1 3 2 1 m x m x m x b b b a m m m m m m m m m m m m   = + + = . (1.7) The Exton’s double hypergeometric functions are defined by [4] The Exton’s double hypergeometric functions are defined by [4] The Exton’s double hypergeometric functions are defined by [4]   = + + =         0 , 2 2 ! ! )) ' (( )) (( )) (( )) ' (( )) (( )) (( , ; ; ) ( ) ( ; ; ) ( ) ( : : ) ( ) ( ; : ; : n m n m n m n m n m n m n m d d c y x b b a y x d b d b c a D D C B B A X , (1.8) A   = + + =         0 , 2 2 ! ! )) ' (( )) (( )) (( )) ' (( )) (( )) (( , ; ; ) ( ) ( ; ; ) ( ) ( : : ) ( ) ( ; : ; : n m n m n m n m n m n m n m d d c y x b b a y x d b d b c a D D C B B A X , (1.8) (1.8) where the symbol m a)) (( denotes the product m j A j a ) ( 1 = . where the symbol m a)) (( denotes the product m j A j a ) ( 1 = . where the symbol m a)) (( denotes the product m j A j a ) ( 1 = . 1. Introduction 2 )( 1 ( 0 , 1 ) ( n if n a a a a n if a n (1.3) The Laplace integral representations ofLauricella functions ) 3 ( A F and ) 3 ( D F are given by Exton (see [3]): The Laplace integral representations ofLauricella functions ) 3 ( A F and ) 3 ( D F are given by Exton (see [3]): ( ) 3 2 1 3 2 1 3 2 1 ) 3 ( , , ; , , ; , , , x x x c c c b b b a FA   − − − − − − = 0 0 0 1 3 1 2 1 1 3 2 1 3 2 1 3 2 1 ) ( Γ ) ( Γ ) ( Γ 1 b b b t t t t t t e b b b 3 2 1 3 3 2 2 1 1 3 2 1 ) 3 ( 2 ) , , ; , , ; ( Ψ dt dt dt t x t x t x c c c a  , (1.4) 0 ) Re( ), Re( ), Re( 3 2 1  b b b 3 2 1 3 3 2 2 1 1 3 2 1 ) 3 ( 2 ) , , ; , , ; ( Ψ dt dt dt t x t x t x c c c a  , (1.4) 0 ) Re( ), Re( ), Re( 3 2 1  b b b and Univ. Aden J. Nat. and Appl. Sc. Vol. 22 No.1 – April 2018 143 Univ. Aden J. Nat. and Appl. Sc. Vol. 22 No.1 – April 2018 143 Integrals formulas involving confluent hypergeometric …...Ahmed A.Atash,Hussein S. 2.MainIntegralFormulas ) ( ) ( ) 4 / ( ) ( )' ( )1 ( 2 n n m n m n m n m m n m c d y x i b a a ay ( ) ) ( 2 2 ) 2 ( ) ( ) ( 2 ( ) ( ) ( ) 2 ( 2 2 1 2 1 2 1 ) 2 ( 2 j i b c n j i c c n b j i j i c n i i b j i c n b i j i n c + + −  + + +  − −   + + + − − −  − −  + +  − −   + + + ]) [ 1( ]) [ ( 2 2 2 1 + + + + − +  + + +  j j i i c b n c n D (2 1) ( ) ) ( 2 2 ) 2 ( ) ( ) ( 2 ( ) ( ) ( ) 2 ( 2 2 1 2 1 2 1 ) 2 ( 2 j i b c n j i c c n b j i j i c n i i b j i c n b i j i n c + + −  + + +  − −   + + + − − −  − −  + +  − −   + + + ]) [ ( ) ( ]) [ 1( ]) [ ( 2 1 2 1 2 2 2 1 , + + + − −   + + + − +  + + +   i j j i j i b n i c b n c n D , (2.1) for 2 ,1,0,1 ,2 ,3 − − − = i and 3,2,1,0 = j (2.1) and Univ. Aden J. Nat. and Appl. Sc. Vol. 22 No.1 – April 2018 144 and ( ) y x x d c b i b a FD , , ; ; , , , ) 3 ( − − Univ. Aden J. Nat. and Appl. Sc. Vol. 22 No.1 – April 2018 144 ( ) y D , , ; ; , , , Univ. Aden J. Nat. and Appl. 2.MainIntegralFormulas g By employing the generalized Dixon’s theorem [5] and the generalized Kummer’s theorem [6],Atash [1] and Atash and Bellehaj [2] derived the following two extension formulas for Lauricella’s functions of three variables ) 3 ( A F and ) 3 ( D F : ( ) y y x j i c c d b i b a a FA − + + − , , ; , , ; , ,' , ) 3 (    = +  = − − = 0 2 2 2 2 0 ! ! ) ( ) ( ) 4 / ( ) ( )' ( ) ( n n m n m n m n m m n m c d y x i b a a ( ) ) ( 2 1 2 ) 2 1( ) ( ( 2 1( ) ( ) ( ) 2 1( 2 1 2 1 2 1 j i b c n j i c n c b j i j i c n i i b j i c n i b + + −  + + + −  − −   + + + − − −  − −  + +  − + −  ]) [ 1( ) ( ]) [ ( ]) [ ( 2 2 1 2 1 2 1 2 1 , i j j i j i n b i c b n c n C + − −   + + + −  + − +   + + +    = + + +  = − − + − 0 1 2 2 1 2 2 0 ! ! 1. Introduction Bellehaj ( ) 3 2 1 3 2 1 ) 3 ( , , ; ; , , , x x x d b b b a FD ds s x s x s x d b b b s e a a s ) , , ; ; , , ( Φ ) ( Γ 1 3 2 1 3 2 1 ) 3 ( 2 0 1   − − = , (1.5) 0 ) Re(  a , (1.5) where the functions ) 3 ( 2 Φ and ) 3 ( 2 Ψ are the confluent hypergeometric functions defined by Srivastava[7] . ( ) ) 3 ( where the functions ) 3 ( 2 Φ and ) 3 ( 2 Ψ are the confluent hypergeometric functions defined by Srivastava[7] . ( ) 3 2 1 3 2 1 ) 3 ( 2 , , ; ; , , Φ x x x c b b b ! ! ! ) ( ) ( ) ( ) ( 3 3 2 2 1 1 0 , , 3 2 1 3 2 1 3 2 1 3 2 1 3 2 1 m x m x m x c b b b m m m m m m m m m m m m   = + + = (1.6) and ( ) 3 2 1 3 2 1 ) 3 ( 2 , , ; , , ; Ψ x x x b b b a ! ! ! ) ( ) ( ) ( ) ( 3 3 2 2 1 1 0 , , 3 2 1 3 2 1 3 2 1 3 2 1 3 2 1 m x m x m x b b b a m m m m m m m m m m m m   = + + = . (1.7) ) 3 2 1 3 2 , , ; ; , x x x c b b ! ! ! ) ( ) ( ) ( ) ( 3 3 2 2 1 1 0 , , 3 2 1 3 2 1 3 2 1 3 2 1 3 2 1 m x m x m x c b b b m m m m m m m m m m m m   = + + = (1.6) and ! ! First Integral Univ. Aden J. Nat. and Appl. Sc. Vol. 22 No.1 – April 2018 145 First Integral   − − − − − − − − 0 0 0 1 3 1 2 1 ' 1 3 2 1 ) ( Γ ) ( Γ )' ( Γ 1 b i b a t t t t t t e b i b a 3 2 1 3 2 1 ) 3 ( 2 ) , , ; , , ; ( Ψ dt dt dt yt yt xt j i c c d a − + +  .    = +  = − − = 0 2 2 2 2 0 ! ! ) ( ) ( ) 4 / ( ) ( )' ( ) ( n n m n m n m n m m n m c d y x i b a a ( ) ) ( 2 1 2 ) 2 1( ) ( ( 2 1( ) ( ) ( ) 2 1( 2 2 1 2 1 2 1 ) 1 2 ( 2 j i b c n j i c n c b j i j i c n i i b j i c n i b j i c n + + −  + + + −  − −   + + + − − −  − −  + +  − + −   + + − + ]) [ 1( ) ( ]) [ ( ]) [ ( 2 2 1 2 1 2 1 2 1 , i j j i j i n b i c b n c n C + − −   + + + −  + − +   + + +    = + + +  = − − + − 0 1 2 2 1 2 2 0 ! ! 2.MainIntegralFormulas Sc. Vol. 22 No.1 – April 2018 Univ. Aden J. Nat. and Appl. Sc. Vol. 22 No.1 – April 2018 144 Integrals formulas involving confluent hypergeometric …...Ahmed A.Atash,Hussein S. Belleha    = + +  = − = 0 2 2 2 2 0 ! !) 2 ( ) ( ) ( ) ( ) ( n n m n m n m n m m n m d y x c i b a    + − − − + + − + + − − + − −  + ) 1( Γ ]) [ ( Γ )) ( 1( Γ ) 1( Γ ) 2 1( Γ ) ( Γ 2 2 1 2 1 2 1 2 1 2 1 2 1 2 i b m i m i i b b i b m A i m i    + − + − − + − + + − − + − −  + ) ( Γ ]) [ ( Γ )) ( 1( Γ ) 1( Γ ) 2 1( Γ ) ( Γ 2 2 1 2 1 2 2 1 2 1 2 1 2 i b m i m i i b b i b m B i m i    = + + + + + +  = + − + 0 1 2 1 2 1 2 1 2 0 ! 2.MainIntegralFormulas !)1 2 ( ) ( ) ( ) ( ) ( n n m n m n m n m m n m d y x c i b a    + − + − − + − + + − − + − −   + + ) ( Γ ]) [ ( Γ )) ( 1( Γ ) 1( Γ ) 2 ( Γ ) ( Γ 2 2 1 2 1 2 1 2 1 2 1 2 1 1 2 i b m i m i i b b i b m A i m i    + − − − + − − + + − − + − −  + + ) ( Γ ]) [ ( Γ )) ( 1( Γ ) 1( Γ ) 2 ( Γ ) ( Γ 2 2 1 2 2 1 2 1 2 1 2 1 1 2 i b m i m i i b b i b m B i m i (2.2) for , 5 ,4 ,3 ,2 ,1 ,0      = i Integrals formulas involving confluent hypergeometric …...Ahmed A.Atash,Hussein where ] [x denotes the greatest integer less than or equal to x and x denotes the usual absolute value of x . The coefficient j i C , can be obtained from the table of j iA , given in [5] by replacing a and c by n 2 − and n c 2 1 − − and the coefficient j i D , can be obtained from the table of j iB , given in [5] by replacing a and c by 1 2 − −n and n c 2 − − respectively . The coefficients i A' and i B' can be obtained from the tables of iA and iB given in [6] by taking m a 2 − = and the coefficients i A" and i B" can be obtained from the same tables of iA and iB by taking 1 2 − − = m a . 2.MainIntegralFormulas ) ( ) ( ) 4 / ( ) ( )' ( ) ( n n m n m n m n m m n m c d y x i b a a ( ) ) ( 2 1 2 ) 2 1( ) ( ( 2 1( ) ( ) ( ) 2 1( 2 2 1 2 1 2 1 ) 1 2 ( 2 j i b c n j i c n c b j i j i c n i i b j i c n i b j i c n + + −  + + + −  − −   + + + − − −  − −  + +  − + −   + + − + ]) [ 1( ) ( ]) [ ( ]) [ ( 2 2 1 2 1 2 1 2 1 , i j j i j i n b i c b n c n C + − −   + + + −  + − +   + + +    = + + +  = − − + − 0 1 2 2 1 2 2 0 ! ! ) ( ) ( ) 4 / ( ) ( )' ( )1 ( 2 n n m n m n m n m m n m c d y x i b a a ay First Integral 2.MainIntegralFormulas The coefficients i A' and i B' can be obtained from the tables of iA and iB given in [6] by taking m a 2 − = and the coefficients i A" and i B" can be obtained from the same tables of iA and iB by taking 1 2 − − = m a . In (1.4) replacing 2 1 3 2 1 3 2 1 , , , , , , , x x c c c b b b and 3x by y x j i c c d b i b a , , , , , , ,' + + − and y − respectively and using the result (2.1), we get the following general integral involving confluent hypergeometric function of three variables ) 3 ( 2 Ψ : Univ. Aden J. Nat. and Appl. Sc. Vol. 22 No.1 – April 2018 145 First Integral   − − − − − − − − 0 0 0 1 3 1 2 1 ' 1 3 2 1 ) ( Γ ) ( Γ )' ( Γ 1 b i b a t t t t t t e b i b a 3 2 1 3 2 1 ) 3 ( 2 ) , , ; , , ; ( Ψ dt dt dt yt yt xt j i c c d a − + +  .    = +  = − − = 0 2 2 2 2 0 ! ! First Integral ) ( ) ( ) 4 / ( ) ( )' ( )1 ( 2 n n m n m n m n m m n m c d y x i b a a ay   − − − − − − − − 0 0 0 1 3 1 2 1 ' 1 3 2 1 ) ( Γ ) ( Γ )' ( Γ 1 b i b a t t t t t t e b i b a 3 2 1 3 2 1 ) 3 ( 2 ) , , ; , , ; ( Ψ dt dt dt yt yt xt j i c c d a − + +  .    = +  = − − = 0 2 2 2 2 0 ! ! ) ( ) ( ) 4 / ( ) ( )' ( ) ( n n m n m n m n m m n m c d y x i b a a ( ) ) ( 2 1 2 ) 2 1( ) ( ( 2 1( ) ( ) ( ) 2 1( 2 1 2 1 2 1 j i b c n j i c n c b j i j i c n i i b j i c n i b + + −  + + + −  − −   + + + − − −  − −  + +  − + −  ]) [ 1( ) ( ]) [ ( ]) [ ( 2 2 1 2 1 2 1 2 1 , i j j i j i n b i c b n c n C + − −   + + + −  + − +   + + +    = + + +  = − − + − 0 1 2 2 1 2 2 0 ! ! ) ( ) ( ) 4 / ( ) ( )' ( )1 ( 2 n n m n m n m n m m n m c d y x i b a a ay Univ. Aden J. Nat. and Appl. Sc. Vol. 22 No.1 – April 2018 145 Integrals formulas involving confluent hypergeometric …...Ahmed A.Atash,Hussein S. First Integral Bellehaj ( ) ) ( 2 2 ) 2 ( ) ( ) ( 2 ( ) ( ) ( ) 2 ( 2 2 1 2 1 2 1 ) 2 ( 2 j i b c n j i c c n b j i j i c n i i b j i c n b i j i n c + + −  + + +  − −   + + + − − −  − −  + +  − −   + + + ]) [ ( ) ( ]) [ 1( ]) [ ( 2 1 2 1 2 2 2 1 , + + + − −   + + + − +  + + +   i j j i j i b n i c b n c n D , (2.3) for 2 ,1,0,1 ,2 ,3 − − − = i and 3,2,1,0 = j . (2.3) Next, in (1.5),replacing 2 1 3 2 1 , , , , x x b b b and 3x by x x c b i b − − , , , , and y respectively andusing the result (2.2), we get the following general integral involving confluent hypergeometric function of three variables ) 3 ( 2 Φ : Next, in (1.5),replacing 2 1 3 2 1 , , , , x x b b b and 3x by x x c b i b − − , , , , and y respectively andusing the result (2.2), we get the following general integral involving confluent hypergeometric function of three variables ) 3 ( 2 Φ : Second Integral Second Integral ds ys xs xs d c b i b s e a a s ) , , ; ; , , ( Φ ) ( Γ 1 ) 3 ( 2 0 1 − −   − −    = + +  = − = 0 2 2 2 2 0 ! First Integral 5 ,4 ,3 ,2 ,1 ,0      = i ds ys xs xs d c b i b s e a a s ) , , ; ; , , ( Φ ) ( Γ 1 ) 3 ( 2 0 1 − −   − −    = + +  = − = 0 2 2 2 2 0 ! !) 2 ( ) ( ) ( ) ( ) ( n n m n m n m n m m n m d y x c i b a First Integral !) 2 ( ) ( ) ( ) ( ) ( n n m n m n m n m m n m d y x c i b a    + − − − + + − + + − − + − −  + ) 1( Γ ]) [ ( Γ )) ( 1( Γ ) 1( Γ ) 2 1( Γ ) ( Γ 2 2 1 2 1 2 1 2 1 2 1 2 1 2 i b m i m i i b b i b m A i m i    + − + − − + − + + − − + − −  + ) ( Γ ]) [ ( Γ )) ( 1( Γ ) 1( Γ ) 2 1( Γ ) ( Γ 2 2 1 2 1 2 2 1 2 1 2 1 2 i b m i m i i b b i b m B i m i    = + + + + + +  = + − + 0 1 2 1 2 1 2 1 2 0 ! !)1 2 ( ) ( ) ( ) ( ) ( n n m n m n m n m m n m d y x c i b a    + − + − − + − + + − − + − −   + + ) ( Γ ]) [ ( Γ )) ( 1( Γ ) 1( Γ ) 2 ( Γ ) ( Γ 2 2 1 2 1 2 1 2 1 2 1 2 1 1 2 i b m i m i i b b i b m A i m i    + − − − + − − + + − − + − −  + + ) ( Γ ]) [ ( Γ )) ( 1( Γ ) 1( Γ ) 2 ( Γ ) ( Γ 2 2 1 2 2 1 2 1 2 1 2 1 1 2 i b m i m i i b b i b m B i m i (2.4) for . Univ. Aden J. Nat. and Appl. Sc. Vol. 22 No.1 – April 2018 146 3. Applications The other special cases of (2.3) and (2.4) can be obtained by the similar manner . 3. Applications (3.8) (iv) Setting 0 = i in (2.4), we get     − − = −   − − y x c b d a X ds ys xs xs d c b b s e a a s , ; ; ; ; : : 0 1 ; ; 0 1 : : 1 1 ) , , ; ; , , ( Φ ) ( Γ 1 2 ) 3 ( 2 0 1 . (3.9) (v) Setting 1 − = i in (2.4), we get 0 =     − − + + + +     − − + y x c b d a X d ax y x c b d a X , ; ; ; ; 1 : : 1 1 0 1 ; ; 0 1 : : 1 1 , ; ; ; ; 1 : : 0 1 ; ; 0 1 : : 1 1 2 2 .(3.10) (vi) Setting 1 = i in (2.4), we get =     − − + + + +     − − + y x c b d a X d ax y x c b d a X , ; ; ; ; 1 : : 1 1 0 1 ; ; 0 1 : : 1 1 , ; ; ; ; 1 : : 0 1 ; ; 0 1 : : 1 1 2 2 .(3.10) (vi) Setting 1 = i in (2.4), we get ds ys xs xs d c b b s e a a s ) , , ; ; , ,1 ( Φ ) ( Γ 1 ) 3 ( 2 0 1 − −   − − ) ( 0 =     − − + + −     − − y x c b d a X d x a y x c b d a X , ; ; ; ; : : 1 1 0 1 ; ; 0 1 : : 1 1 , ; ; ; ; : : 0 1 ; ; 0 1 : : 1 1 2 2 , (3.11) which for x x − = and 1 + = b b gives the result (3.10). 3. Applications Bellehaj g g yp g , 3 2 1 3 2 1 ) 3 ( 2 0 0 0 1 3 1 2 1 ' 1 ) , , ; , , ; ( Ψ ) ( Γ ) ( Γ )' ( Γ 1 3 2 1 dt dt dt yt yt xt c c d a t t t e b b a b b a t t t −   − − − − − −     + − − = x y d a c c c b c b a X , 4 ; ;' ; ; , , , : : 1 1 ; ; 3 2 : : 0 1 2 2 1 2 1 2 1 .(3.6) (ii) Setting 1 ,0 = = j i in (2.3) , we get 3 2 1 3 2 1 ) 3 ( 2 0 0 0 1 3 1 2 1 ' 1 ) , , ;1 , , ; ( Ψ ) ( Γ ) ( Γ )' ( Γ 1 3 2 1 dt dt dt yt yt xt c c d a t t t e b b a b b a t t t − +   − − − − − −   2 3 2 1 3 2 1 ) 3 ( 2 0 0 0 1 3 1 2 1 ' 1 ) , , ;1 , , ; ( Ψ ) ( Γ ) ( Γ )' ( Γ 1 3 2 1 dt dt dt yt yt xt c c d a t t t e b b a b b a t t t − +   − − − − − − 3 2 1 3 2 1 ) 3 ( 2 0 0 0 1 3 1 2 1 ' 1 ) , , ;1 , , ; ( Ψ ) ( Γ ) ( Γ )' ( Γ 1 3 2 1 dt dt dt yt yt xt c c d a t t t e b b a b b a t t t − +   − − − − − −   + + + −   − = x y d a c c c b c b a X , 4 ; ;' ; ; 1 , , 1 , : : 1 1 ; ; 3 2 : : 0 1 2 2 1 2 1 2 1 ( )   + + + + − +   − + + + x y d a c c c b c b a X c c aby , 4 ; ;' ; ; ,1 ,1 1 ,1 : : 1 1 1 ; ; 3 2 : : 0 1 1 2 2 3 2 1 2 1 .(3.7)  + +  d c c c 4 ; ; 1 , , : 1 ; 3 : 0 2 2 2 ( )   + + + + − +   − + + + x y d a c c c b c b a X c c aby , 4 ; ;' ; ; ,1 ,1 1 ,1 : : 1 1 1 ; ; 3 2 : : 0 1 1 2 2 3 2 1 2 1 .(3.7) .(3.7) (iii) Setting 1 = = j i in (2.3), we get 3 2 1 3 2 1 ) 3 ( 2 0 0 0 1 3 2 2 1 ' 1 ) , , ; 2 , , ; ( Ψ ) ( Γ )1 ( Γ )' ( Γ 1 3 2 1 dt dt dt yt yt xt c c d a t t t e b b a b b a t t t − + −   − − − − − −   + + + + −   − = x y d a c c c b c b a X , 4 ; ;' ; ; ,1 ,1 2 , : : 1 1 ; ; 3 2 : : 0 1 2 2 3 2 1 2 1 ( ) ( )   + + + + −   − + + + − + x y d a c c c b c b a X c c y b c a , 4 ; ;' ; ; 2 , ,1 2 , : : 1 1 1 ; ; 3 2 : : 0 1 2 2 2 2 2 1 2 3 2 1 . Univ. Aden J. Nat. and Appl. Sc. Vol. 22 No.1 – April 2018 3. Applications 3. Applications In this section, we willuse in each case the following results [7]: ) ( Γ 3. Applications In this section, we willuse in each case the following results [7]:  ,2 ,1 ,0 , ) ( Γ ) ( Γ ) ( − −  + = a a n a a n (3.1) ( ) ( )n n n n a a a 2 1 2 1 2 1 2 2 2 ) ( + = (3.2)  ,2 ,1 ,0 , ) 1( )1 ( ) ( Γ ) ( Γ    − − = − a a a n a n n (3.3) ( ) ! 2 ! 2 3 2 n n n = (3.5)(i) Setting 0 = = j i in (2.3), we get ( ) ! 2 )! 1 2 ( 2 3 2 n n n n = + (3.5)(i) Setting 0 = = j i in (2.3), we get ( ) ! 2 )! 1 2 ( 2 3 2 n n n n = + (3.5)(i) Setting 0 = = j i in (2.3), we get Univ. Aden J. Nat. and Appl. Sc. Vol. 22 No.1 – April 2018 146 Univ. Aden J. Nat. and Appl. Sc. Vol. 22 No.1 – April 2018 Univ. Aden J. Nat. and Appl. Sc. Vol. 22 No.1 – April 2018 146 Integrals formulas involving confluent hypergeometric …...Ahmed A.Atash,Hussein S. Bellehaj ntegrals formulas involving confluent hypergeometric …...Ahmed A.Atash,Hussein S. Belleh ormulas involving confluent hypergeometric …...Ahmed A.Atash,Hussein S. Integrals formulas involving confluent hypergeometric …...Ahmed A.Atash,Hussein S. Bellehaj Integrals formulas involving confluent hypergeometric …...Ahmed A.Atash,Hussein S. Bellehaj Univ. Aden J. Nat. and Appl. Sc. Vol. 22 No.1 – April 2018 Acknowledgment g The authors are grateful to worthy referee for a careful checking of the details and for his valuable suggestion that improved this paper. Univ. Aden J. Nat. and Appl. Sc. Vol. 22 No.1 – April 2018 147 Univ. Aden J. Nat. and Appl. Sc. Vol. 22 No.1 – April 2018 References e e e ces 1. Atash, A. A. (2015). Extension formulas ofLauricella’sfunctions by applications of Dixon’s summation theorem, Applications and Applied Mathematics.10(2):1007-1018. 1. Atash, A. A. (2015). Extension formulas ofLauricella’sfunctions by applications of Dixon’s summation theorem, Applications and Applied Mathematics.10(2):1007-1018. pp pp 2. [2] Atash, A.A.and BellehajH. S. (2016).Transformation formulas of Lauricella’sfunction of the forth Kind of several variables.International Journal of Mathematics and its Applications, 4(1-D): 195-201. 2. [2] Atash, A.A.and BellehajH. S. (2016).Transformation formulas of Lauricella’sfunction of the forth Kind of several variables.International Journal of Mathematics and its Applications, 4(1-D): 195-201. pp 3. Exton, H. (1976). Multiple Hypergeometric Functions and Applications. Halsted Press, New York. 3. Exton, H. (1976). Multiple Hypergeometric Functions and Applications. Halsted Press, New York. 4. Exton, H.(1982). Reducible double hypergeometric functions and associated Integrals. AnFac. Ci. Univ. Porto, 63(1-4):137-143. 4. Exton, H.(1982). Reducible double hypergeometric functions and associated Integrals. AnFac. Ci. Univ. Porto, 63(1-4):137-143. 5. Lavoie, J. L., Grondin, F., Rathie, A. K. and Arora, K.(1994). Generalizations of Dixon’s theorem on the sum of a 2 3F . Mathematics of Computation, 62: 267- 276. 5. Lavoie, J. L., Grondin, F., Rathie, A. K. and Arora, K.(1994). Generalizations of Dixon’s theorem on the sum of a 2 3F . Mathematics of Computation, 62: 267- 276. 6. J. L. Lavoie, F. Grondinand A. K. Rathie, (1996).Generalizations of Whipple’s theoremon the sum of a 2 3F .Journal of Computational and Applied Mathematics, 72 :293-300. 6. J. L. Lavoie, F. Grondinand A. K. Rathie, (1996).Generalizations of Whipple’s theoremon the sum of a 2 3F .Journal of Computational and Applied Mathematics, 72 :293-300. 7. Srivastava, H.M. and Manocha, H.L.(1984). A Treatise on Generating Functions, Halsted Press, New York. 7. Srivastava, H.M. and Manocha, H.L.(1984). A Treatise on Generating Functions, Halsted Press, New York. 148 grals formulas involving confluent hypergeometric …...Ahmed A.Atash,Hussein S. Bellehaj صيغ تكاملية تتضمن الدوال الفوق هندسية ثالثية املتغريات Ф2 (3) وѰ2 (3) أحمد علي عت ش و حسين صالح بلحاج قسم،الرياضيات كلية التربية - شبوة، جامعة عدن DOI: https://doi.org/10.47372/uajnas.2018.n1.a11 امللخص هدف بحثنا هذا هو إ ثبات صيغ تكاملية عامة متضمنة الدوال الفوق هندسية ثالثية المتغيراتФ2 (3) وѰ2 (3) وذلك بمساعدة صيغتين لدوال الرسيال𝐹𝐴 (3) و𝐹𝐷 (3) ذات الثالثة المتغيرات والمعطا ة في [1] و[2] أ يضا تم عرض .بعض التطبيقات لنتائج بحثنا الرئيسية الكلمات:المفتاحية صيغ ،تكاملية الدوال الفوق هندسية، نظرية ديكسون، نظرية كومر. 149
https://openalex.org/W3118798776	https://durham-repository.worktribe.com/preview/1255531/34201.pdf	English	null	Toward an Integrative Geological and Geophysical View of Cascadia Subduction Zone Earthquakes	Annual review of earth and planetary sciences	2,021	cc-by	21,508	Abstract The Cascadia subduction zone (CSZ) is an exceptional geologic environment for recording evidence of land level changes, tsunamis, and ground motion that reveals at least 19 great megathrust earthquakes over the past 10 kyr. Such earthquakes are among the most impactful natural hazards on Earth, transcend national boundaries, and can have global impact. Reducing the societal impacts of future events in the U.S. Pacific Northwest and coastal British Columbia, Canada, requires improved scientific understanding of megathrust earthquake rupture, recurrence, and corresponding hazards. Despite substantial knowledge gained from decades of research, large uncertainties remain about the characteristics and frequencies of past CSZ earthquakes. In this review, we summarize geological, geophysical, and instrumental evidence relevant to understanding megathrust earthquakes along the CSZ and associated uncertainties. We discuss how the evidence constrains various models of great megathrust earthquake recurrence in Cascadia and identify potential paths forward for the earthquake science community. Keywords Subduction zone, Pacific Northwest, earthquake, recurrence, megathrust This draft manuscript is distributed solely for purposes of scientific peer review. Its content is deliberative and predecisional, so it must not be disclosed or released by reviewers. Because the manuscript has not yet been approved for publication by the U.S. Geological Survey (USGS), it does not represent any official USGS finding or policy. This draft manuscript is distributed solely for purposes of scientific peer review. Its content is deliberative and predecisional, so it must not be disclosed or released by reviewers. Because the manuscript has not yet been approved for publication by the U.S. Geological Survey (USGS), it does not represent any official USGS finding or policy. Toward an integrative geological and geophysical view of Cascadia subduction zone earthquakes Authors: Maureen A.L. Walton1 & Lydia M. Staisch2, Tina Dura3, Jessie K. Pearl4, Brian Sherrod4, Joan Gomberg4, Simon Engelhart5, Anne Tréhu6, Janet Watt1, Jon Perkins2, Robert C. Witter7, Noel Bartlow8, Chris Goldfinger6, Harvey Kelsey9, Ann E. Morey6, Valerie J. Sahakian10, Harold Tobin11, Kelin Wang12, Ray Wells2, Erin Wirth4 1US Geological Survey, Pacific Coastal and Marine Science Center 2US Geological Survey, Geology Minerals Energy and Geophysics Science Center 3Virginia Tech, Department of Geosciences, Blacksburg, VA 4US Geological Survey, Earthquake Science Center 5University of Durham, Department of Geography 6Oregon State University, College of Earth Ocean and Atmospheric Sciences 7US Geological Survey, Alaska Science Center 8University of Kansas, Department of Geology 9Humboldt State University, Department of Geology 10University of Oregon, Department of Earth Sciences University of Durham, Department of Geography 6Oregon State University, College of Earth Ocean and Atmospheric Sciences University of Oregon, Department of Earth Sciences 11University of Washington, Department of Earth and Space Sciences University of Washington, Department of Earth and Sp 12Geological Survey of Canada, Pacific Geoscience Centre Summary Phrases y 1. Despite outstanding geologic records of past megathrust events, large uncertainty of the magnitude and frequency of CSZ earthquakes remains 1. Despite outstanding geologic records of past megathrust events, large uncertainty of the magnitude and frequency of CSZ earthquakes remains 2. Here we outline current knowledge and promising future directions to address outstanding questions on CSZ rupture characteristics and recurrence 3. Integration of diverse datasets with attention to the geologic processes that create different records has potential to lead to major progress 3. Integration of diverse datasets with attention to the geologic processes that create different records has potential to lead to major progress 1. Introduction Subduction zones, where tectonic plates converge along plate boundary megathrust faults, produce some of the most devastating natural disasters globally: great (M>8.0) megathrust earthquakes and their corresponding hazardous phenomena (Fig. 1). The 2004 M 9.2 Sumatra earthquake and tsunami killed 250,000 people in 15 countries, producing an international disaster. Even well-prepared countries can suffer catastrophic damage and loss of life, as in the 2011 M9.0 Tōhoku earthquake and tsunami in Japan (McGuire et al., 2017). These two catastrophes took the world by surprise and showed a need for better understanding of the seismic cycle and rupture variability in subduction zones. The Cascadia subduction zone (CSZ) of western North America (Fig 1) presents a unique opportunity to address major outstanding questions in subduction zone science (Gomberg et al., 2017). With better understanding of these powerful and complicated tectonic systems, we may improve future hazard preparation and maintain the safety and economic viability of affected populations. Classic elastic theory (Reid, 1910) describes the subduction zone seismic cycle as a two- stage model in which the crust and uppermost-mantle deform elastically in response to far-field tectonic forces: 1) an interseismic period when strain accumulates (Fig. 2A), and 2) a coseismic period when an earthquake suddenly relieves the accumulated strain (Figure 2B). For a shallow- dipping subduction megathrust, gradual subsidence near the fault and uplift farther away characterizes interseismic upper plate deformation (Fig. 2A) and is followed by abrupt coseismic reversal of the deformation pattern (Fig. 2B). Global observations, however, reveal that the process of strain accumulation and release on faults is complex and that the recurrence interval for earthquakes can vary along a fault and through space and time (Sieh et al., 2008; Goldfinger et al., 2012; Kulkarni et al., 2013; Nocquet et al., 2017; Bilek & Lay, 2018). This presents challenges when trying to calculate future earthquake probabilities in order to prepare for and mitigate impacts from inevitable future events. The Cascadia subduction zone (CSZ) extends for more than 1300 km from Cape Mendocino in northern California to Vancouver Island in southwestern British Columbia (McCrory et al., 2012) and has been accumulating strain for 320 years since the last great earthquake in 1700 CE (Atwater et al., 2005; McCaffrey et al., 2013). 2.1. Onshore stratigraphic evidence of the earthquake deformation cycle The stratigraphy beneath Cascadia’s tidal wetlands reflects the strain accumulation and release of the earthquake deformation cycle (Fig. 2D). Bank sections and sediment cores preserve repeated sequences of organic-rich tidal wetland soils formed in the interseismic period, sharply overlain by tidal mud deposited following decimeter-scale coseismic subsidence (Fig. 3A & 3B; Darienzo et al., 1994; Atwater & Hemphill-Haley, 1997; Clague et al., 2000; Kelsey et al., 2002; Witter et al., 2003; Nelson et al., 2008). At some tidal wetland sites, sand and silt layers signaling high-energy tsunami inundation of the coast are evident at the soil-mud contact (Figs. 2D & 3B). In coastal lakes, landward thinning sand beds signal marine incursions from past tsunamis (Kelsey et al., 2005). Radiocarbon ages from pre- and post-earthquake and/or tsunami sediment bracket the timing of coseismic subsidence and/or tsunami inundation. Typical age uncertainty is on the order of a few hundred years; however, dendrochronological analysis of trees killed by rapid coseismic subsidence and marine inundation, particularly for events in the past 2000 years where sufficient wood has been preserved, has the potential to yield more precise ages (Fig. 2D; Atwater & Yamaguchi, 1991; Jacoby et al., 1995; 1997; Yamaguchi et al., 1997). The completeness of onshore geologic archives of coseismic subsidence and/or tsunami inundation depends on the creation and preservation thresholds at a site, termed evidence thresholds (Nelson et al., 2006). In order to exceed the creation threshold at a site, the evidence of coseismic subsidence and/or tsunami inundation must be distinct from similar evidence produced by local non-seismic processes (Nelson et al., 2006). In order to exceed the preservation threshold at a site, the balance among erosional and depositional processes must favor the preservation of coseismic subsidence and/or tsunami inundation evidence. Holocene relative sea level (RSL) history and evidence thresholds at each site along the CSZ control the length and completeness of onshore geologic archives of coseismic subsidence and tsunami inundation (Engelhart et al., 2015; Dura et al., 2016a). The longest geologic archives of coseismic subsidence and tsunami inundation are in central and southern Cascadia, where gradual RSL rise since ~5-7 ka produces the accommodation space in tidal wetlands necessary for preservation (Atwater & Hemphill-Haley, 1997; Witter et al., 2003). In northern Cascadia (e.g., Vancouver Island), gradual RSL fall since ~6 ka limits the preservation of coseismic subsidence evidence to the last ~1-2 ka, and typically only the last ~500 years (Fig. 1. Introduction The next CSZ earthquake could be another ~M9 that ruptures the entire margin like the 1700 CE event, but also might be a series of smaller events occurring in quick succession (Fig. 2C). While recent earthquakes help to inform forecasts of potential earthquakes in other subduction zones (e.g., Alaska in 1946, 1957, 1964, 1965; Chile in 1960 & 2010; Sumatra in 2004 & 2007; Japan in 2011), geologic records underpin our understanding of earthquake rupture parameters and CSZ earthquake hazard assessments (Hemphill-Haley, 1995; Atwater et al., 2005; Kelsey et al., 2002; Witter et al., 2003; Nelson et al., 2008; Goldfinger et al., 2012; Frankel et al., 2015). Fortunately, Cascadia coastal and submarine environments preserve different aspects of past earthquake processes over millennial time scales and feature some of the best prehistoric earthquake catalogs in the world (Hutchinson, 1992; Long & Shennan, 1998; Goldfinger et al., 2012; Engelhart et al., 2015; Dura et al., 2016a). ) The spatial and temporal robustness of geologic records in Cascadia provide a strong foundation to address outstanding questions on subduction zone science and earthquake recurrence-governing principles that remain elusive globally. However, questions about the timing and extent of past ruptures remain in Cascadia due to age-dating uncertainties resulting in non-unique interpretations of geologic records, unknown relative contributions of coseismic and postseismic motions, and unresolved structural and rheological controls on rupture extent. Furthermore, different rupture characteristics impact tsunami inundation, the extent of seismically triggered landslides, and the effects of geologic architecture on seismic wave amplification (Fig. 1; Geist, 2002; 2005; Frankel, 2013; Frankel et al., 2018; Wirth et al., 2018; Roten et al., 2019; Wirth & Frankel, 2019). In this review, we summarize the substantial knowledge gained over decades of subduction zone research in Cascadia, discuss subduction zone processes that create geologic archives of past earthquakes, and identify associated uncertainties and natural variability. We highlight remaining knowledge gaps in CSZ earthquake studies through a synthesis of available data and models and suggest pathways towards accurate interpretation of the earthquake deformation cycle model that incorporates both geological and geophysical datasets. 2. Cascadia subduction zone earthquake evidence over the millennia: Geologic observations 2. The CSZ preserves the most spatially and temporally complete geologic records of past great megathrust earthquakes in the world (Atwater & Hemphill-Haley, 1997; Kelsey et al., 2002; 2005; Witter et al., 2003; Nelson et al., 2006; Goldfinger et al., 2012; 2017). Widespread low-energy, ecologically sensitive tidal wetlands and estuaries and isolated coastal lakes are excellent recorders of decimeter-scale interseismic and coseismic deformation and tsunami inundation (Figs. 1 and 2D; Witter et al., 2003; Engelhart et al., 2015; Dura et al., 2016a). Additionally, nearshore marine environments receive ample sediment supply for the generation and preservation of seismically triggered turbidites (Fig. 1; Goldfinger et al., 2012). In this section, we summarize existing geologic evidence that constrains the timing and rupture characteristics of past Cascadia megathrust events. 2.1. Onshore stratigraphic evidence of the earthquake deformation cycle 4; Dura et al., 2016a). Evidence of tsunami inundation in northern Cascadia extends to ~3.5 ka (Goff et al., 2020). ) In order to distinguish stratigraphic contacts created by megathrust ruptures from other non-seismic processes (e.g., climate driven sea-level change, changes in estuary hydrography), researchers consider several criteria: (1) the suddenness of the change in environment across the contacts; (2) the lateral extent of sharp stratigraphic contacts; (3) significant environmental change evident in microfossil assemblages across sharp contacts; (4) the continuity of stratigraphic evidence within a site and across multiple sites; and (5) the coincidence of tsunami deposits with sudden stratigraphic change (Darienzo et al., 1994; Nelson et al., 1996; Shennan et al., 1996, 2016). Satisfying criteria 1-4 implies that an earthquake produces the decimeters of subsidence necessary to exceed the evidence threshold (Nelson et al., 2006). The additional presence of an overlying tsunami deposit (criteria 5) strongly supports an offshore rupture, rather than localized wetland depositional processes. The best-preserved and most widely documented megathrust earthquake in the onshore geologic record at the CSZ occurred in 1700 CE (Supplemental text; Nelson et al., 1995; Satake et al., 2003; Atwater et al., 2005; Goldfinger et al., 2012). Coastal wetlands spanning >1000 km of the CSZ preserve distinct soil-mud contacts, and anomalous accompanying silt or sand beds at the contacts signal sudden coseismic submergence and tsunami inundation of coastal environments (Figs. 1 & 2D; Atwater et al., 2005 & references therein). The 1700 CE tsunami propagated across the Pacific, causing inundation and damage along the coast of Japan (Satake et al., 2003; Atwater et al., 2005). Modeling of the arrival-time of tsunami waves documented in Japan, and dendrochronological dating of coastal trees simultaneously killed by coseismic subsidence in Washington, Oregon, and California, precisely constrain the age of the earthquake to January 26, 1700 CE (Atwater et al., 2005 & references therein). Tsunami modeling, along with the uniquely precise date and concurrence of evidence for this event, supports the inference that it was a full-margin, M8.7 - 9.2 rupture (Yamaguchi et al., 1997; Satake et al., 2003; Atwater et al., 2005; Nelson et al., 2020). Stratigraphic- and microfossil-based estimates of coseismic subsidence in 1700 CE aid in assessing the rupture characteristics of the event, such as slip distribution. 2.1. Onshore stratigraphic evidence of the earthquake deformation cycle Early stratigraphic- and microfossil-based estimates of coseismic subsidence in 1700 CE often have uncertainties in excess of a meter (Hemphill-Haley, 1995; Dura et al., 2016b), and therefore highly simplified uniform-slip rupture models were permissible by earlier datasets (Wang et al., 2003; Leonard et al., 2004, 2010). More recent statistically based transfer function analyses use empirical relationships derived from modern foraminifera samples to estimate past marsh elevations from fossil foraminifera assemblages and have reduced subsidence uncertainty to 0.3-0.5 m at some sites (Hawkes et al., 2011; Kemp et al., 2018), though uncertainties due to contamination from possible short-term postseismic deformation remain (Horton et al., 2017). The more precise microfossil-based subsidence estimates resolve slip variability along the CSZ in 1700 CE and result in more realistic heterogeneous rupture models (Wang et al., 2013; Wirth & Frankel, 2019). Gaining a deeper understanding of recurrence and slip behavior of past events along the CSZ requires geologic records that span multiple earthquake cycles (Leonard et al., 2004, 2010; Wirth & Frankel, 2019). Geologic studies in southern Washington and northernmost Oregon tidal wetlands (Shennan et al., 1996; Atwater & Hemphill-Haley, 1997; Nelson et al., 2006) document up to ten widely correlative buried soils representing coseismic subsidence over the last ~5000 years, with recurrence intervals between earthquakes ranging from a few decades to one millennium (average recurrence 500-540 years). In central and southern Oregon and northern California (Kelsey et al., 2002; Witter et al., 2003; Milker et al., 2016; Padgett et al., in review), tidal wetlands and coastal lakes preserve up to 12 earthquakes and/or tsunamis over the same ~5000 year time period (average recurrence ~390 years; Kelsey et al., 2002, 2005; Witter et al., 2003, 2012a). Geologic records reveal rupture patterns that suggest northern Cascadia commonly breaks in long ruptures, while southernmost Cascadia experiences more frequent ruptures of variable length (Nelson et al., 2006). Geologic records also show variable amounts of subsidence during successive earthquakes at some sites (Milker et al., 2016), and persistent low (Nelson et al., in review) or high (Kelsey et al., 2002) amounts of deformation at other sites. Along-strike structural barriers at the CSZ (see section 3.2) potentially control the along-strike variability in rupture length and coseismic deformation over multiple earthquake cycles documented in onshore geologic datasets. 2.2. Marine turbidite records Marine sediment cores in Cascadia record disturbance layers and evidence for turbidity currents, termed turbidites, generated from offshore coseismic ground shaking (Fig. 1B; Adams, 1990; Goldfinger et al., 2012). Turbidites can be found in abyssal channels, proximal canyons, fan systems, aprons, and slope basins, and typically consist of a sharp basal contact, a fine sandy- silty basal layer, and an upward-fining sequence of silt, mud, and clay (Fig. 3C). In southern Cascadia, subdued mud turbidites lack a sandy component in some locales (Goldfinger et al., 2012, 2013a). Turbidites result from the shaking produced by megathrust and crustal earthquakes, as well as non-earthquake related processes such as storms (Goldfinger et al., 2012; Gavey et al., 2017, Howarth et al., 2018; Mountjoy et al., 2018); thus, distinguishing between multiple sources of event beds requires sedimentological arguments or physical criteria, often site-specific. One physiographic test is to look for consistent Holocene stratigraphy among site types that lack connections to each other or to terrestrial sources. The confluence test is another physiographic criterion used along the Cascadia margin where multiple channel systems and turbidity current pathways lead away from the filled trench. The confluence argument suggests that if the turbidity currents travel synchronously down the tributary channels and coalesce into a single channel to travel as one large turbidity current, then a margin-wide event, such as a great earthquake, likely triggered the density flows (Adams, 1990; Goldfinger et al., 2012). If multiple events trigger turbidity currents, then the tributary channels and the main channel should contain different numbers of turbidite deposits. Most of the canyon systems of Cascadia are Pleistocene features, making Cascadia an ideal site for Holocene paleoseismology. There remains some debate about the Pleistocene to modern sediment routing in offshore channels and the infallibility of the confluence test (Atwater & Griggs, 2012; Atwater et al., 2014; Hill et al., 2020). While Holocene sediment supply is variable along the CSZ margin and can take a series of complex pathways that could obfuscate estimates of recurrence from the turbidite record (Atwater et al., 2014), Goldfinger et al. (2017) argue that consistent event-bed records among many site types and locales show that the earthquake signal commonly overprints local variability (see also Rong et al., 2014). Multiple tributaries to the Cascadia Channel contain 19 Holocene sandy turbidites, 13 of which post-date the ~7630 yr old Mazama ash (Fig. 4; Adams, 1990; Goldfinger et al., 2012). 2.1. Onshore stratigraphic evidence of the earthquake deformation cycle Tsunami deposits can provide clues about the time, location, and extent of the megathrust rupture source that complements other onshore paleoseismic evidence (Peters et al., 2007; Peterson et al., 2011). Earthquake-induced tsunamis occur when coseismic slip causes significant seafloor deformation and are sensitive to the depth and extent of rupture (Fig. 1D; Priest et al., 2014; Melgar et al., 2016). CSZ tsunami deposits generally consist of anomalous sandy to silty sediments extending kilometers inland from the shoreline, may contain marine microfossils, and often accompany coastal subsidence records (Fig. 4; Kelsey et al., 2002; 2005; Witter et al., 2003). Other tsunamigenic sources, such as crustal earthquakes and large submarine landslides, tend to produce localized tsunamis, whereas megathrust-generated tsunamis affect a broad region (Goldfinger et al., 2000; Garrison-Laney et al., 2017). At the CSZ, researchers use the inland extent, thickness, and grain size of tsunami deposits preserved along the CSZ to ground truth tsunami inundation simulations (Witter et al., 2013), estimate offshore slip during past tsunamigenic earthquakes (Witter et al., 2012a), and resolve the hydrodynamics of tsunami inundation (Witter et al., 2012b). 2.2. Marine turbidite records Downstream, the count remains 13 post-Mazma events in most cores, suggesting synchronous deposition. Heavy mineral suites and hydrodynamic modeling support the independence of the tributaries (Goldfinger et al., 2017) and the Adams (1990) confluence test. Juan de Fuca Channel, Hydrate Ridge slope basin, Rogue Apron, and Astoria Fan each contain 19 sandy turbidites (Fig. 4). These sandy turbidites share a common chronology estimated from 14C ages and depositional age models, and log correlation methods assist in correlating them along-strike (Enkin et al., 2013; Hamilton et al., 2015; Goldfinger et al., 2012, 2017). The 1700 CE earthquake is the youngest turbidite in nearly all marine cores (Fig. 4). Compilation of turbidite events and onshore subsidence and tsunami records suggests a recurrence interval of 500 - 530 years for margin-wide (~M9) megathrust earthquakes (Goldfinger et al., 2012). In southern Cascadia at Hydrate Ridge, Rogue Apron, and sites extending to Eel Canyon, a series of 12 - 22 fine-grained turbidites intercalated between hemipelagic sediments and sandy turbidites have been interpreted as more frequent and limited southern CSZ rupture (Goldfinger et al., 2012). p q p g Turbidite age estimates broadly overlap age ranges for onshore CSZ earthquake evidence, especially for the sandy turbidites representing the largest most widespread events (Witter et al., 2012a); however, some turbidites interpreted as earthquake-triggered events (e.g., T2) do not have corresponding onshore subsidence or tsunami evidence (Fig. 4). Differences in evidence thresholds can account for at least some discrepancies between onshore and offshore records (Nelson el al, 2006; Goldfinger et al., 2016). Onshore, subsidence thresholds may be as large as MW 8.4 (Nelson et al., 2006), while the turbidite record includes events at least as low as MW 7.1 (Goldfinger et al., 2019). For example, mud turbidites above the 1700 CE turbidite layer near Cape Mendocino likely correlate with the 1906 San Andreas and 1992 Petrolia earthquakes, suggesting that crustal M>7 earthquakes triggered these turbidity flows (Goldfinger et al., 2019). Thus, the turbidite record in southernmost Cascadia appears to include shorter CSZ ruptures as well as crustal earthquakes. The discrepancies in the datasets may alternatively suggest that not all margin-wide turbidites are seismically triggered, or that certain rupture characteristics optimize turbidite generation but do not generate onshore deformation and tsunamis. 2.3. Lacustrine turbidites and disturbance deposits Lakes from a variety of settings are uniquely sensitive to shaking from different types of seismic sources and often provide long, continuous sediment records ideally suited for paleoseismic investigation (Vandekerkhove et al., 2020; van Daele et al., 2019; Praet et al., 2017; Moernaut et al., 2007); recent work indicates increasing utilization of lacustrine records in Cascadia earthquake science (Morey et al., 2013; Goldfinger et al., 2017; Leithold et al., 2018). Turbidites in Oregon and northern California lakes are of a similar timing and frequency (Morey et al., 2013) as the record of offshore seismogenic turbidites (Goldfinger et al., 2012). Several studies suggest that lake sediments record locally generated ground shaking magnitude and source. Sedimentary records from Lake Washington, near Seattle, contain two event layers that coincide with known earthquakes, including the 1700 CE megathrust earthquake and an ~1100 yr old Seattle fault zone rupture; the other six events found in these records are from older earthquakes in the region and have recurrence intervals between 400 and 500 years, which may therefore indicate they were generated by megathrust rupture (Karlin et al., 2004). On the Olympic Peninsula, Lake Quinault sedimentary records contain three event layers in the last three thousand years (Leithold et al., 2018), suggesting either that only some CSZ earthquakes cause local ground shaking sufficient to create lacustrine disturbance events or that not all lakes are equally good earthquake recorders. Also on the Olympic Peninsula, Lake Crescent contains a sedimentary record with four major disturbance events that correlate to rupture along a nearby crustal fault, whereas thinner lake turbidite layers may be from megathrust, upper plate, and intraplate earthquakes that caused lesser local ground shaking (Leithold et al., 2019). On Vancouver Island, Effingham and Saanich inlets are deep anoxic inlets that effectively mimic lacustrine environments. Of the two records, the Saanich Inlet, well inland, shows evidence for nearly twice as many events (Blais-Stevens et al., 2011), whereas the Effingham inlet seems to record mainly plate boundary events. In addition, the Saanich Inlet record may suggest that some CSZ megathrust earthquakes rupture only the northern portion of the megathrust (Blais-Stevens et al., 2011). The difference in these records highlights the sensitivity of local response to seismic source type and shaking characteristics. 2.4. Other onland proxies of strong ground shaking Liquefaction from seismic shaking manifests as sedimentary intrusions (sills and dikes), vented sand deposits (Fig. 2D), soft sediment deformation, and lateral spreading. Previous surveys identify rare surficial liquefaction features in Cascadia (Obermeier, 1995; Takada & Atwater, 2004). Most evidence for seismically induced liquefaction in Cascadia comes from sedimentary outcrops along rivers and estuaries, such as swampy islands along the lower Columbia River and cut banks of the Chehalis River in southwestern Washington (Obermeier et al., 1993; Atwater, 1994; Obermeier, 1995; Obermeier & Dickenson, 2000; Takada & Atwater, 2004). Atwater (1994) describes outcrops on the banks on these islands with hundreds of centimeter-scale sand bodies intruding, and in some cases, venting onto the surface of a buried soil dated to the 1700 CE megathrust earthquake. Slices of subsurface deposits from the lower Columbia River show evidence of liquefaction from at least four great earthquakes in the past 2000 years (Takada and Atwater, 2004). Subduction zone earthquakes sometimes radiate strong shaking and trigger landslides over broad areas (Figs. 1F & 3D), as seen in the 1960 Chilean, 1964 Alaska, and the 2011 Tohoku earthquakes (Hansen, 1965; Veblen & Ashton, 1978; Wartman et al., 2013). Researchers have yet to definitively connect any of Cascadia’s abundant landslides to a megathrust rupture despite thorough surveys (Perkins et al., 2018; Hill et al., 2020, Struble et al., 2020; LaHusen et al., in press). The paucity of megathrust-triggered deep-seated landslides along the Cascadia margin may suggest that onshore ground shaking from past great earthquakes was not sufficiently strong. However, recent work suggests landslides from crustal earthquakes or major rainfall events overprints prior potential megathrust-generated landslides (Struble et al., 2020; LaHusen et al., in press). Candidate megathrust-generated landslides include rock slides near Newport, OR, where modern observations of landslide reactivation rates suggest that it began moving around 1700 CE and continues to move today (Schulz et al., 2012). On the Olympic Peninsula, a terrace formed from a breached rockslide-dammed lake containing buried trees in growth position (Leithold et al., 2018) and a landslide-buried Makah fishing village (Kirk, 2015) may correlate to the 1700 CE event. Confirming seismic triggers for these sites requires robust age control. 3. Contemporary deformation: Constraints from instrumental and geophysical datasets 3. Determining whether geological boundaries are present and their impact on rupture propagation and megathrust behavior is a major challenge that requires integrating paleoseismic and contemporary geophysical data and comparing the CSZ to other subduction zones. In this section, we review evidence of interplate coupling and contemporary indications of seismic activity in the forearc and discuss what we can infer about earthquake behavior from seismic and geodetic observations. We use several terms to describe portions of the subduction zone exhibiting common slip behavior, and noting that some studies use these terms differently, we define them here as follows. The seismogenic zone is the part of the plate boundary where dynamic friction is less than the static friction and exhibits stick-slip behavior. This behavior is a prerequisite for generating an earthquake. The coupled zone is a proxy for the seismogenic zone and is the part of the plate boundary that has geodetically inferred slip deficit and appears to be storing elastic energy. We define a rupture patch as the area on the megathrust that slips during a particular earthquake. We discuss evidence for and against geologically controlled rupture boundaries on the megathrust that may define persistent, recurrent rupture patches. Accurate CSZ megathrust earthquake scenarios hinge on our understanding of the existence and persistence of rupture boundaries, both along-strike and down-dip, and the structural or rheologic properties that modulate these boundaries. Heterogeneities evident in proxies for megathrust behavior may sometimes indicate spatially persistent rupture characteristics like slip or rupture boundaries. We note that potential boundaries do not necessarily inhibit all ruptures, depending on the physics of rupture propagation (Bilek & Lay, 2018). Rupture boundaries may be persistent, frequent, or ephemeral (rarely traversed, occasionally traversed, or always changing, respectively; Philibosian & Meltzner, 2020). For example, the Kii Peninsula in Japan is a boundary along the Nankai-Suruga Trough that impeded throughgoing rupture of the 1944 Tonankai and 1946 Nankai earthquakes, but the 1707 Hoei earthquake ruptured the entire margin (Garrett et al., 2016). While the 1700 CE event in Cascadia was likely an ~M9 earthquake that ruptured the entire length of the CSZ (Atwater et al., 2005), the geologic record likely also preserves smaller earthquakes that only rupture a portion of the subduction zone (Wells et al., 2003; Goldfinger et al., 2012). The long-term persistence of rupture boundaries in Cascadia and elsewhere is an ongoing question (Victor et al., 2011; Meltzner et al., 2012). 3.1. Depth-dependent seismic behavior and frictional properties All subduction zones exhibit depth-dependent slip behaviors along the plate interface (Lay et al., 2012; Bilek & Lay, 2018). In the upper coupled zone, at depths less than ~15 km, strain release generally occurs either largely aseismically or in earthquakes with relatively low amounts of short-period energy radiation and low stress drop (Newman & Okal, 1998; Ye et al., 2016; Sahakian et al., 2019), often termed tsunami earthquakes as they generate tsunamis that are anomalously large for the corresponding earthquake magnitude (Hill et al., 2012; Lay et al., 2012). This zone can rupture co-seismically during megathrust earthquakes (e.g., the 2011 MW 9.0 Tohoku-Oki and 2010 MW 8.8 Maule events). From ~15-35 km depths, earthquakes can produce large slip and emit broadband seismic waves, although the size of individual rupture patches and amount of slip in each event vary in space and time (Lay et al., 2012; Bilek & Lay, 2018). A transitional zone below ~35 km depth exhibits various types of slow-slip behaviors, including slow-slip events (SSEs) in which several cm of slip occurs over a large area over a period of days-to-years (Obara & Kato, 2016; Bilek & Lay, 2018; Bartlow, 2020). These events occur near where the downgoing plate meets the hydrated mantle wedge (Obara & Kato, 2016; Gao & Wang, 2017). Debates persist over the exact relationships between and physical controls on these depth zones in Cascadia and elsewhere (Obara & Kato, 2016; Wang & Tréhu, 2016; Gao & Wang, 2017). g ) Limited seafloor geodetic observations and an exceptionally low rate of low-magnitude background interplate seismicity in the CSZ blurs our understanding of the geometry and depth of the seismogenic zone and the degree of interseismic coupling (Wang & Tréhu, 2016). The relative lack of seismicity, along with inversion of geodetic datasets, suggests that the CSZ seismogenic zone is nearly fully coupled along much of its length, although the width and degree of coupling may vary along strike; notably, central Cascadia has been modeled as both an anomalously narrow zone of coupling or a wide zone of partial coupling (Fig. 5; McCaffrey et al., 2013; Schmalzle et al., 2014; Pollitz & Evans, 2017; Li et al., 2018; Michel et al., 2018). Calculated Holocene vertical land motion most closely matches models that include a fully locked CSZ at shallow (<30 km) depths (Fig. 5; Yousefi et al., 2020). 3. Contemporary deformation: Constraints from instrumental and geophysical datasets The geologic record is necessary to verify interpretations of rupture boundaries gleaned from geophysical data, but conversely, along-strike and downdip patterns evident in instrumental datasets may also help distinguish between conflicting interpretations of rupture boundaries the geologic record. Below we summarize the three- dimensional variations in the CSZ environment and megathrust slip behaviors that we can observe with modern geophysical instrumentation. 3.1. Depth-dependent seismic behavior and frictional properties In general, the width of the inferred seismogenic zone in Cascadia decreases to the south, potentially impacting megathrust earthquake slip magnitude, an interpretation that is consistent with the apparent increase in megathrust event frequency from the geologic record (Scholz, 2014; Tréhu, 2016). The recent and planned installation of offshore GNSS-Acoustic (GNSS-A) instrumentation should reduce the non-uniqueness of coupling models by helping to constrain offshore strain accumulation (Bürgmann & Chadwell, 2014; Heesemann et al., 2017; Chadwell et al., 2018; Fig. 5). Initial data from these GNSS-A sites indicates a high degree of near-trench coupling (Chadwell et al., 2018). Direct observations of earthquakes in other subduction zones inform our understanding of CSZ rupture processes. Ground motion observations from the 2011 MW 9.0 Tohoku-Oki and 2010 MW 8.8 Maule events suggest that the frequency content of the radiated seismic energy varies with depth within the seismogenic zone. Ground motions from these two events can be explained by incorporating high-stress-drop subevents, which are M8-size rupture patches at 20- 30 km depths superimposed on the lower-stress-drop background slip (Wang & Mori, 2011; Frankel, 2013). Recent CSZ ~M9 rupture models include such subevents (Frankel et al., 2018; Wirth et al., 2018) and are compatible with variability in 1700 CE coseismic subsidence estimates (Wirth & Frankel, 2019). Inclusion of modeled high-stress-drop subevents impacts slip patterns, ground motions, upper plate structure, and interpretation of ground shaking proxies in the geologic record, although their full impact requires further investigation. Shallow (depths less than ~10-15 km) tsunami earthquakes typically exhibit much weaker shaking (Sahakian et al., 2019). The resulting slip distribution and seafloor deformation from shallow earthquakes is also a critical control on coseismic hazards, specifically tsunami inundation (Priest et al., 2014; Melgar et al., 2016). Seismically and geodetically measured slow-slip and tremor phenomena, termed episodic tremor and slip (ETS), occurs with remarkable regularity along the CSZ (Fig. 1E; Dragert et al. 2001; Rogers & Dragert, 2003; Brudzinski & Allen, 2007; Gomberg, 2010; Boyarko et al., 2015; Wells et al., 2017; Bartlow, 2020). ETS occurs at ~30-40 km depths below the seismically coupled zone, with a creeping gap between the base of the coupled zone and the slow-slip zone (Hyndman et al., 2015; Bruhat & Segall, 2016; Bartlow, 2020; Fig. 1E). Slow slip and tremor phenomena migrate together, suggesting that these phenomena are different manifestations of the same seismic process (Bartlow et al., 2011). 3.1. Depth-dependent seismic behavior and frictional properties Although we currently do not fully understand the exact physical controls on slow slip and its relationship to geodetic coupling, high pore fluid pressures near the mantle wedge may be responsible for generating slow slip here (Hyndman et al., 2015; Wang & Tréhu, 2016; Gao & Wang, 2017). Globally, SSEs generally occur along megathrust interfaces that have relatively young downgoing oceanic lithosphere (Lay et al., 2012). SSEs do not accommodate the full slip budget along most of the subduction zone, implying significant inter-SSE creep may occur on the interface within the SSE zone (Bartlow, 2020). Whether any slip deficit in this depth range will contribute to slip during a future CSZ megathrust earthquake remains a mystery, and the degree to which stresses from slow-slip events may be important in triggering the next great earthquake in Cascadia is a matter of current debate (Mazzotti & Adams, 2004; Beeler et al., 2014; Bartlow, 2020). 3.2. Along-strike variability in slip behavior and structure Many geophysical imaging studies in Cascadia indicate that along-strike heterogeneity exists in forearc upper plate crustal structure. For example, the early Eocene-age Siletz/Crescent terrane that forms the crystalline basement throughout much of the Cascadia forearc (Fig. 5) is unusually thick and extends offshore between ~43°and 46°N. The unique composition of this terrane and other crystalline terranes within Cascadia has been correlated with along-strike variations in upper plate seismicity, ETS periodicity and slip, degree of coupling, and other factors (Fig. 5; Tréhu et al., 1994; 2012; Wells et al., 1998, 2003; Brudzinski & Allen, 2007; Porritt et al., 2011; Li & Liu, 2016; Delph et al., 2019; Egbert et al., 2019; Bartlow, 2020). The Siletz terrane exists along the stretch of central Cascadia where geodetic models show a narrow, fully coupled zone or a wide, partially coupled zone (Fig. 5; Schmalzle et al., 2014). Wells et al. (2017) speculated that upper-plate faults in the brittle Siletz terrane reduce fluid overpressure and de-optimize tremor conditions. In a comprehensive examination of the tectonic geomorphology, outer wedge taper, and seaward and landward structural vergence along the accretionary complex, Watt & Brothers (2020) concluded that along-strike variations in shallow megathrust behavior correlate with upper plate structural boundaries and suggested that the thickened Siletz terrane acts as a backstop influencing the frictional properties of the megathrust through modulation of wedge strength (Figs. 4 & 5). In the seismogenic zone, model results for 1700 CE slip distribution constrained by land- level change data (Wang et al., 2013) show possible low-slip regions that correlate with structural boundaries located roughly near 42-43°N, 44.5°N, and 46°N (Figs. 4 & 5). The degree of coupling along strike may relate to variation in buoyant asthenosphere beneath the downgoing plate; Bodmer et al. (2018) used seismic tomography to argue for decreased buoyancy of the subducting Juan de Fuca plate between ~43° and 46°N, relating it to decreased interplate coupling and non-volcanic tremor at these latitudes (Figs. 4 & 5). Wells et al. (2003) argued that forearc basins represent basal erosion of the upper plate due to increased frictional strength of the plate boundary, forming potentially recurrent high-slip patches over multiple earthquake cycles (Fig. 5). Stone et al. (2018) found generally higher rates of forearc seismicity south of 46°N and correlate this with incoming plate roughness and sediment thickness (Fig. 5). 3.2. Along-strike variability in slip behavior and structure Persistent clusters of seismicity during the past several decades on or near the plate boundary within the seismogenic zone near 44.3°N and 44.6°N also correlate with subducted seamounts inferred from potential field and seismic imaging data (Figs. 4 & 5; Tréhu et al., 2012, 2015; Morton et al., 2018; Stone et al., 2018). Tréhu et al. (2012) attributed these clusters to interactions between subducted seamounts and the Siletz terrane. While numerous geophysical and instrumental datasets reveal along-strike variation of the CSZ, the relevance of these observations for understanding the dynamic behavior of past and future CSZ earthquakes is complex and controversial (Philibosian & Meltzner, 2020). Along- strike variations in paleoseismic data (Goldfinger et al., 2017) remain the most direct proxies for past earthquake behavior and to verify boundaries hypothesized from geophysical data. Given the lack of coseismic observations, we cannot immediately resolve the causes for along- strike correlations in geophysical data, and we have limited ability to link inferred changes in frictional properties along the megathrust to slip behavior and long-term strain accumulation patterns in Cascadia. Well-resolved preseismic, coseismic, and postseismic observations on other subduction zones provide a framework for interpreting geophysical and instrumental records in Cascadia. Many studies have modeled and interpreted activity in subduction zone earthquakes in the context of geologic structure (Davis et al., 1983; von Huene & Scholl, 1991; Saffer & Bekins, 2002; Lamb, 2006; Fujie et al., 2013; Cubas et al., 2013; McNeill & Henstock, 2014; Henstock et al., 2016; Bassett et al., 2016; Saillard et al., 2017; Tréhu et al., 2019; Olsen et al., 2020). Comparative studies can help to reconcile geophysical observations with the geologic record to best understand CSZ recurrence. 4. Recurrence models and implications for seismic hazard A fundamental aim of CSZ paleoseismic studies is to determine a recurrence model that fits our understanding of past CSZ earthquakes. A well-constrained recurrence model is particularly relevant for Probabilistic Seismic Hazard Assessment (PSHA) models, which form the basis for the US National Seismic Hazard Maps (NSHM; Petersen et al., 2019). PSHA models estimate the probability of ground motion exceedance, termed hazard (Cornell, 1968), using input earthquake scenarios describing the slip distribution, fault location, fault geometry, and recurrence. Earthquake recurrence models typically considered for subduction zone margins and other major fault systems are categorized as either time-independent or time-dependent (Table 1). The time-independent model is a common choice for PSHA models, especially when applied to broad regions with multiple fault systems because it requires minimal information, namely mean recurrence rate. Often described as a Poisson process, time-independence assumes that events occur at a certain mean rate but with random event timing. The time-independent recurrence implies that occurrence is memoryless, hazard is constant, and may suggest that accumulated far-field stress on the fault system does not define earthquake rupture timing (Fig. 6; Table 1). The aggregate behavior of a region may appear Poissonian, even if composed of faults with individually time-dependent earthquake recurrence (Cornell & Winterstein, 1988). y p q Time-dependent recurrence assumes that earthquakes rupture with a regularity defined by accumulated stress levels on the fault system. In a periodic model, both the interevent time and slip during each event are predictable and earthquake hazard probabilities increase proximal to the mean recurrence time (Fig. 6; Table 1; Shimazaki & Nakata, 1980). Idealization of the periodic model suggests common slip magnitude (Fig. 6; Schwartz & Coppersmith, 1984); however, observations suggest a more flexible definition of the periodic model, with quasi- periodic large ruptures in addition to less periodic moderate events with variable rupture characteristics (Zielke, 2018). The clustered model is a subcategory of time-dependent models in which strain energy balances over multiple seismic events followed by a period of seismic quiescence (Fig. 6; Table 1). Slip rate averaged over multiple earthquake cycles is constant, but fault slip for each event can be variable (Fig. 6). Nested clusters of subduction zone earthquakes are termed supercycles (Sieh et al., 2008; Goldfinger et al., 2013b; Herrendörfer et al., 2015; Philibosian & Meltzner, 2020). 4. Recurrence models and implications for seismic hazard In this section, we summarize the methodology and underlying assumptions that differentiate between various recurrence models and, as a thought experiment, we explore the range of recurrence models compatible with interpretations of the paleoseismic record. We highlight the difficulty in distinguishing full-margin from serial ruptures in the geologic record, and discuss the implications for seismic hazard assessment. 4.1. The Coefficient of Variance and its application to the CSZ An outstanding controversy remains, in which some argue all events in the paleoseismic record are full-margin M9s and others argue that a portion of those events may be a series of smaller M8s that occur in quick succession irresolvable by geochronologic uncertainties (Atwater et al., 2014; Frankel et al., 2015). Additional uncertainty remains about potential rupture barriers and how to handle partial ruptures along the margin, particularly the more frequent ruptures interpreted in southern Cascadia. Below, we explore how these two outstanding uncertainties may affect the Coefficient of Variation (CV), a simple statistical metric that researchers commonly use to evaluate proposed recurrence models. While not always inclusive of nuanced detail in long paleoseismic records, CV values inform hazard analyses on possible recurrence scenarios and thus provide a basis from which to construct hazard models. The equation for CV is as follows: 𝐶𝑉 = 𝜎𝐼𝑇 𝜇𝐼𝑇 where 𝜎𝐼𝑇and 𝜇𝐼𝑇are the standard deviation and mean of interevent times, τ, respectively (Cramer et al., 2000; Field, 2015; Table 1). In the time-independent model, random processes lead to similar means and standard deviations, thus the 𝐶𝑉≈1. In the time-dependent periodic model, consistent interevent times result in a small standard deviation and 𝐶𝑉≤1. A 𝐶𝑉≥1 indicates variable interevent times and suggests clustered behavior (Table 1). Application of CV assumes a well-sampled seismic catalog that is long enough to capture typical recurrence behavior. Petersen et al. (2002) evaluated a CV between 0.1 and 0.4 for the Pacific Northwest but included crustal and intraplate events; here we focus on the megathrust to discuss the CSZ earthquake cycle model. Recurrence models and the CV apply to a catalog of significant events, which are fault slip events that release enough stress to permit statistical renewal of the recurrence process. This generally requires a rupture of the full fault system, or a large enough rupture to relieve sufficient accumulated stress (Herrendörfer et al., 2015). 4.2. Full-margin ruptures Geoscientists infer 19 - 20 full-margin ~M9 CSZ earthquakes over the past 10 kyr from marine and onshore geologic datasets (Goldfinger et al., 2012; 2017; Enkin et al., 2013, Hamilton et al., 2015; Fig. 4). Using this catalog, CV calculations imply time-dependent quasi- periodic recurrence in Cascadia (CV = 0.51; Tables S3-S4). If partial-margin ruptures longer than 660 km (Table S4) are significant and renew the recurrence process, CV reduces to 0.39 (Table S3). These CV estimates vary insignificantly regardless of whether we include events with weak onshore geologic support (e.g., T2; Table S3). These basic CV calculations strongly suggest a quasi-periodic recurrence model for the CSZ (Table S3), assuming correlated events are single ~M9 ruptures. If correct, the quasi-periodic recurrence model would suggest that the CSZ is currently in the late stages of the earthquake deformation model. y g q Goldfinger et al. (2012) and Kulkarni et al., (2013) identify temporal gaps after T5, T10, and T15 in the marine record to argue for clustered full-margin event recurrence; however, some onshore events along the margin may fill in these temporal gaps along the margin (e.g., John’s River to Lagoon Creek between T5 and T6; Fig. 4). The potential for clustered CSZ megathrust earthquakes has important hazard implications (Kulkarni et al., 2013), and therefore merits attention. 4.3. Serial and partial ruptures h i i C d i The uncertainty in 14C dating techniques (10s to 100s of years) allows for the possibility of interpreting some of the 19 - 20 correlated events as serial ruptures, in which time intervals smaller than dating uncertainties separate multiple ~M8 earthquakes (Fig. 2). Currently, little evidence supports serial rupture as a common seismic occurrence along the CSZ, however two events captured in the Bradley Lake record are separated by >22 years (Kelsey et al., 2005) correlate to a possible T5 turbidite doublet in Rogue Canyon marine cores (Goldfinger et al., 2012), suggesting serial ruptures may occur occasionally. If we assume one third to one half of the full-margin events interpreted by Goldfinger et al. (2012) are actually 3 - 4 serial ruptures separated by 10 - 100 years (Table S4), the resulting CVs suggest Poisson and clustered recurrence models, respectively (Table S3). We only consider up to half of events as possible serial ~M8 ruptures, as a majority of ~M9 ruptures are required to accommodate incoming plate convergence rate seismically (Frankel et al., 2015). These hypothetical rupture scenarios indicate that CV estimates for non-quasi-periodic recurrence are attainable only if a large portion of the geologic record has been misinterpreted as full-margin M9 ruptures. In addition to uncertainty in full-margin rupture regularity, portions of the CSZ seem to rupture more frequently and may have an earthquake cycle independent of the full-margin cycle. Some geologic data south of Cape Blanco show a striking increase in the number of events recorded and a corresponding decrease in the interevent time (Fig. 4; Table S3). The marine core record includes 17 additional events, many from mud turbidites, limited to southern Cascadia (Table S3; Goldfinger et al., 2012). Whether these events represent CSZ or crustal earthquakes remains an open question (Goldfinger & Gutierrez, 2019). Onshore records indicate 11 events limited to south of Cape Blanco and two limited to northern Cascadia (Fig. 4; Blais- Stevens et al., 2011; Williams et al., 2005; Nelson et al., 2006). Assuming these smaller ruptures represent CSZ earthquakes, the CV applied to southern Cascadia ruptures implies a time- dependent, quasi-periodic recurrence model (Tables S3-S4). The recurrence interval for ruptures limited to northern Cascadia remains elusive (Petersen et al., 2014). 4.4. Implications for the CSZ earthquake cycle model Various rupture scenarios discussed above lead to CV values consistent with interpretation of Poisson, quasi-periodic, and clustered recurrence models for the CSZ. This highlights how current dating uncertainties and debates on rupture variability along the CSZ render an evaluation of the earthquake cycle model in Cascadia premature. PSHA offers a means of quantifying the intrinsic variability of the system, termed aleatoric variability, and addressing uncertainties that stem from limited knowledge, termed epistemic uncertainty. The current U.S. NSHM uses extensive logic trees that weigh various M8 and M9 rupture scenarios to define two additive CSZ earthquake scenarios: (1) full margin ~M9 that recur every ~500 years and (2) partial M8.0-8.7 rupture of the CSZ (Frankel et al., 2015). The recurrence rates for partial ruptures in northern and southern Cascadia, which strongly influence hazard, are averaged between different possible scenarios supported by onshore or offshore evidence (Petersen et al., 2014; Frankel et al., 2015). Future updates to the U.S. NSHM may include the possibility of serial rupture (Frankel et al., 2015). Accurate hazard analyses can improve by reducing epistemic uncertainty (Sykes & Menke, 2006), which can only be addressed with further geologic and geophysical research. 5. Future research directions Decades of research have led to enviable geologic datasets that record past megathrust earthquakes in Cascadia as well as diverse geophysical observations along the margin. However, major outstanding questions on earthquake occurrence and rupture characteristics remain. In this section, we highlight knowledge gaps, discrepancies between datasets, and uncertainties in earthquake recurrence that may be addressed through collection of new data, careful integration of available datasets, and consideration of the processes that created the records we observe today in Cascadia. 5.2. Future research directions in CSZ science Geologic records at the CSZ still present multiple opportunities for advancement. New paleoseismic sites that capitalize on potential for longer temporal records will allow for further exploration of the extent of past megathrust rupture and help identify variability in rupture characteristics. Filling latitudinal spatial gaps in land-level change records may improve recurrence and rupture models (Fig S1). In addition to study of new locales, modern methodology and statistical analyses can help to reduce uncertainty in available datasets. New Bayesian transfer functions that can incorporate multiple microfossil proxies reduce uncertainties on subsidence estimates (Kemp et al., 2018), and applying this method downcore can resolve slip over multiple earthquake cycles, improving our knowledge of slip along the megathrust through time and space (Padgett et al., in review). Microfossil-based analyses also have the potential to quantify interseismic (Shennan et al., 1999) and postseismic (Horton et al., 2017) deformation, but constraining the age of the inorganic tidal mud that accumulates in the postseismic and interseismic periods remains a challenge. At previously investigated locales along the coast (Fig. S1), widespread, precise quantitative microfossil-based estimates of coseismic subsidence in 1700 CE have informed heterogenous rupture models; however, limited and imprecise subsidence estimates for older events do not resolve slip along the megathrust at a high-enough resolution to differentiate uniform and heterogenous model solutions (Leonard et al., 2010; Milker et al., 2016). Existing uncertainties in dating earthquake events remains one of the largest barriers to reducing the nonuniqueness of geologic correlations and interpretations (Hutchinson & Clague, 2017). Dendrochronology offers sub-annual temporal resolution of land-level changes, and while such resolution still cannot discriminate between serial partial-margin ruptures separated by days or months from single full-margin earthquakes, confidence in the interpretation could improve significantly. Modern dendrochronology methods utilize changes in wood chemistry that may accompany sudden coseismic subsidence (Pearl et al., 2020a) and known spikes in the radiocarbon record as chronologic tie points (Pearl et al., 2020b, Pearson et al., 2020). Dendrochronology could also assist with dating landslide-dammed lakes (Struble et al., 2020). Bayesian age-modeling of detrital macrofossil radiocarbon dates provides another promising approach to reduce uncertainties that has only been newly applied in Cascadia (Nelson et al., 2020; Padgett et al., in review). Offshore, turbidite ages may improve by using more standardized calibrations and reservoir corrections (Clark et al., 2019). 5.1. Outstanding knowledge gaps in CSZ earthquake characteristics and recurrence Discrepancies in onshore and offshore geologic evidence for megathrust rupture currently fuel ambiguity in records of megathrust recurrence. Paleoseismic events recorded in the marine record do not all share a corresponding record on land (Fig. 4). Mismatch between the datasets is at least partly due to variable evidence thresholds and analytical uncertainties inherent in geochronology (Nelson et al., 2006), but additionally, the geochronologic age corrections applied to onshore and offshore datasets differ, causing difficulty in correlation. The magnitude of past earthquake events is also difficult to resolve from geologic datasets. Current dating methods and models for CSZ events recorded at individual sites along the margin also have enough uncertainty that experts continue to debate whether full-margin events are always single ~M9 events or if some small portion might be multiple successive M8 events (Fig. 2C; Petersen et al., 2014). Without Japanese tsunami records and modeling, it is difficult to distinguish the 1700 CE earthquake as a single ~M9 or multiple ~M8s. Both paleoseismic and geophysical datasets hint at potentially persistent rupture barriers along the CSZ margin, but it is unclear which barrier proxies are most relevant for understanding coseismic rupture processes. The presence and persistence of rupture barriers may also cause the earthquake cycle model to vary along the megathrust, and the possibility that some past earthquakes were shallow tsunami earthquakes also contributes to uncertainty (Tréhu, 2016). Other aspects of coseismic rupture processes remain elusive. For instance, current geodetic coverage does not uniquely resolve coupling on the subduction zone interface. Without an instrumental record of a great CSZ megathrust earthquake, estimating coseismic onshore and offshore ground motion and secondary hazards, such as liquefaction, landslides, and turbidites, often relies on comparison to other subduction zone margins. The limited liquefaction and landslide evidence for the 1700 CE earthquake inhibits accurately estimating local and regional ground motion for future events. Additionally, numerous assumptions underpin current understanding of shaking-initiated sediment transport processes in the CSZ; we currently lack clarity on how, and under what conditions, the geologic record archives various shaking proxies. Due to the gaps in knowledge, there is currently no consensus on an appropriate recurrence model for the CSZ. For recurrence estimates, questions remain about the magnitude threshold required to constitute a significant event, and whether CSZ geologic records capture all significant earthquakes. 5.1. Outstanding knowledge gaps in CSZ earthquake characteristics and recurrence Some geologic records may record events <M8, or record events caused by other earthquake sources, such as the northern San Andreas fault. Defining a recurrence model and understanding the physical processes influencing recurrence also requires that the geologic record spans enough time to statistically capture potential variability. 5.2. Future research directions in CSZ science Geodetic models and the near absence of seismicity on the megathrust since the 1700 CE earthquake are consistent with coupling of the CSZ plate boundary to at least some degree (Schmalzle et al. 2014; Wang & Tréhu, 2016), but offshore geodetic data are critical for obtaining high-resolution spatial constraints on the degree of coupling and reducing the number of viable coupling models (Bürgmann & Chadwell, 2014). Twelve seafloor GNSS-A stations have been deployed on the Juan de Fuca and North American plates since 1991, most in the past few years. Sites on the North American plate near the trench measure shallow coupling (Fig. 5; Bürgmann & Chadwell, 2014; Heesemann et al., 2017; Chadwell et al., 2018). Researchers plan to deploy at least two more sites on and near the Gorda plate (Fig. 5), which features significant internal deformation that is currently poorly constrained (Bartlow, 2020). Comparison of the CSZ with other instrumentally monitored subduction zones, such as Nankai (Kano & Kato, 2020), can offer clues to the state of coupling, unusual paucity of interplate CSZ seismicity, and the role of slow slip in the accommodation of convergence (Wang & Tréhu, 2016; Bartlow, 2020). ) New structural imaging will also improve definition of potential along-strike rupture boundaries, allowing for better correlations between structure and dynamic behavior of the CSZ. Acquisition of high-resolution offshore imagery and sediment cores across Cascadia’s deformation front, combined with quantitative modeling of tsunami generation and sediment transport, will better inform interpretations of tsunami deposits left behind from past earthquakes. Future efforts may also focus on determining whether or not there is on-fault marine geologic evidence of near-trench rupture along the Cascadia deformation front and the role of splay faults in tsunamigenic rupture (Fig. 1D; Beeson et al., 2017). Broadening the spatial extent of shaking proxy datasets, such as landslides, liquefaction, lacustrine turbidites, and marine turbidites could substantially improve estimates of past earthquake ground motion. Repeat high-resolution bathymetric mapping and subsurface imaging offer promising techniques to test assumptions made in interpretation of mass-transport deposits (Mountjoy et al. 2018; Hill et al., 2020). Shaking from earthquakes along non- megathrust crustal faults can complicate interpretation of the geologic record (Clark et al., 2019), though systematic examination of this process along the CSZ has yet to happen and may be an important avenue for future investigation. 5.3. An integrative concept for CSZ science To address and potentially resolve discrepancies and uncertainties in the geologic data, we suggest that future work applies an integrative approach that considers different evidence thresholds of geologic datasets, proxies for megathrust behavior, and potential rupture barriers gleaned from geophysical and instrumental datasets to provide more accurate estimates of past earthquake rupture characteristics. q p We can leverage differences in evidence thresholds to learn more about the preservation of earthquake processes in the geologic record. An example from southern Oregon illustrates these thresholds, where Bradley Lake preserves evidence for 12 megathrust-generated tsunami deposits in the past 5000 years (Kelsey et al., 2005), while nearby subsidence records only show 9 or 10 events in the same time period (Kelsey et al., 2002; Witter et al., 2003). Similarly, while onshore records also suggest a greater number of earthquakes in southern Cascadia (Nelson et al., 2006), not all turbidite events have a corresponding record on land (Fig. 4). These records may suggest that for some CSZ ruptures, turbidite and/or tsunami deposits are more likely to be created and preserved in southern Cascadia compared to land-level change (Nelson et al., 2006). Rupture patch location, extent, and slip magnitude likely bear on evidence threshold, as different rupture properties can generate particular secondary effects. For instance, shallow rupture near the trench may cause sufficient seafloor deformation and ground shaking of the accretionary wedge to create tsunamis and turbidites, respectively. The potential for tsunami earthquakes can alter our interpretation of the geologic record and are relevant to consider for structural interpretation. A shallow tsunami earthquake can produce tsunami deposits in a large region indicative of a M8-M9 event, but in fact come from a smaller M7-M8 event (Hill et al., 2012). Tsunami earthquakes also emit limited high-frequency energy and thus may produce We can leverage differences in evidence thresholds to learn more about the preservation of earthquake processes in the geologic record. An example from southern Oregon illustrates these thresholds, where Bradley Lake preserves evidence for 12 megathrust-generated tsunami deposits in the past 5000 years (Kelsey et al., 2005), while nearby subsidence records only show 9 or 10 events in the same time period (Kelsey et al., 2002; Witter et al., 2003). Similarly, while onshore records also suggest a greater number of earthquakes in southern Cascadia (Nelson et al., 2006), not all turbidite events have a corresponding record on land (Fig. 4). 5.2. Future research directions in CSZ science To this end, lacustrine paleoseismology offers exciting new research avenues to address onshore ground motions for past megathrust events (Morey et al., 2013, Goldfinger et al., 2016), as well as to improve crustal and intraplate earthquake catalogs (van Daele et al., 2019). 5.3. An integrative concept for CSZ science These records may suggest that for some CSZ ruptures, turbidite and/or tsunami deposits are more likely to be created and preserved in southern Cascadia compared to land-level change (Nelson et al., 2006). Rupture patch location, extent, and slip magnitude likely bear on evidence threshold, as different rupture properties can generate particular secondary effects. For instance, shallow rupture near the trench may cause sufficient seafloor deformation and ground shaking of the accretionary wedge to create tsunamis and turbidites, respectively. The potential for tsunami earthquakes can alter our interpretation of the geologic record and are relevant to consider for structural interpretation. A shallow tsunami earthquake can produce tsunami deposits in a large region indicative of a M8-M9 event, but in fact come from a smaller M7-M8 event (Hill et al., 2012). Tsunami earthquakes also emit limited high-frequency energy and thus may produce little to no shaking proxies in the geologic record (Newman & Okal, 1998; Ye et al., 2016; Sahakian et al., 2019). Integration and careful consideration of the available geologic datasets may therefore enable better mapping of past earthquake extent and estimates of rupture characteristics. Numerous geophysical datasets provide information about the state of coupling, seismicity, and structure along the CSZ, but interpretations disagree, and models provide non-unique solutions. Systematic and analytical comparisons between geophysical, structural, and modeling datasets both within the CSZ and with other subduction zone margins could assist with better understanding the likelihood of potential rupture barriers and other rupture processes. For example, Wang & Tréhu (2016) note the potential for comparing the offshore morphology and structure of the CSZ accretionary complex to other subduction zone margins that have undergone trench-breaching slip (e.g., 2011 Mw 9.0 Tohoku event; Fujiwara et al., 2011). Inferred relationships between ground motions and shaking-induced sediment transport require rigorous testing, particularly with respect to submarine and terrestrial slope stability, the shear strength of slope sediments, and turbidity flow triggering. New monitoring systems offer in situ observations of shaking and how the sediment structure affects site-specific response to ground motion (Gomberg et al., 2019; Jibson et al., 2004). The distributions of landslides across the landscape in response to ground shaking is often complex and thus difficult to characterize and link to earthquake triggers (Struble et al., 2020; LaHusen et al., in press). 5.3. An integrative concept for CSZ science With improved understanding of the relationship between seismic shaking and site properties, there is potential to identify the influence of megathrust earthquake shaking on terrestrial landslides (Meunier et al., 2007, 2013) by comparing landslide catalogs (Jones et al., 2019) with modeled ~M9 seismic ground-motions (Frankel et al., 2018; Wirth et al., 2018). Compilation of liquefaction data along the CSZ can also improve shaking estimates in areas with sparse geologic proxies. The CSZ margin is primed for quantitative and inclusive comparisons of proposed rupture boundaries and characteristics with geologic datasets (Figs. 4 & 5). Clark et al. (2019) integrated complex and disparate datasets to identify the sources and extents of paleoearthquakes along the Hikurangi margin in New Zealand. Given the extensive geologic data in Cascadia, much of which is more clearly associated with megathrust rupture, a similar approach may be explored along the CSZ. Integration of onshore and offshore geologic records requires uniform treatment of geochronologic datasets, possibly using a Bayesian framework (Clark et al., 2019) that builds upon the recent use and testing of local-scale Bayesian age models (Goldfinger et al., 2014; Nelson et al., 2020; Padgett et al., in review), as well as identification, and ideally quantification, of evidence thresholds for different record types, with the overarching goal of reducing non- unique fit of past rupture scenarios to the geologic record. The addition of abundant geophysical and instrumental records in Cascadia provides prior knowledge of along-strike heterogeneity that will frame the integration of geologic datasets with constraints from numerical and theoretical modeling (Kemp et al., 2018). A comprehensive catalog of past CSZ megathrust rupture scenarios would provide concrete input for PSHA and may identify specific regions most susceptible to subduction zone earthquakes and associated hazards. Figure Captions Figure 1. (A) Oblique view of the northwest margin of North American, where the Juan de Fuca and Explorer oceanic plates subduct beneath the North American plate. On the side view, the thin red line between the two tectonic plates represents the region where great earthquakes occur. On the map overlay, the toothed blue line represents the surface trace of the where the subducting plate begins its descent. Major cities are shown as green dots. The purple swath shows the general region where episodic tremor and slip (ETS) occurs and the pink swath shows the general region considered to be the coupled zone. To the left and below (A), small diagrams illustrate various earthquake-related processes labeled beneath each diagram: (B) coseismic turbidite generation, (C) coseismic subsidence with dotted green line showing the pre-event coastal land-level, dead brown trees represent marine incursion onto a formerly terrestrial environment (D) and tsunami generation, (E) the relationship between the coupled fault and the ETS zone, with an ETS swarm depicted as blue circles, with a possible gap between the coupled zone and updip extent of ETS shown as a gradational area (F) coseismic landslide hazards, with schematic seismograms (in blue) showing the potential for topographic effects on ground motion amplification, and (G) how geologic features, such as sedimentary basins, can amplify seismic waves. Figure 2. (A) Block diagram of the interseismic period, when convergence along the coupled subduction zone interface (red zone) typically causes gradual uplift in the onshore overriding plate, and gradual subsidence offshore. (B) Diagram of the coseismic period, when earthquake rupture along the subduction zone interface relieves accumulated strain and generally causes sudden subsidence in the onshore overriding place and sudden uplift in the offshore overriding plate. Shallow rupture may generate a tsunami. (C) Possible scenarios for an ~M9 (orange) and ~M8 (blue) events that rupture the CSZ plate interface. (D) Schematic diagrams of stratigraphic evidence for the earthquake deformation cycle. Left column shows the effect of coseismic subsidence on wetland stratigraphy and coastal forests and their preservation in the stratigraphic record (see also Fig. 1C). Right column shows stratigraphic preservation of coseismic tsunami deposits and liquefaction injectites. Figure 3. (A) Evidence for coseismic subsidence and tsunami inundation from a sedimentary exposure of subaerial dune sand and prehistoric settlements overlain by tsunami sand and tidal mud along the Salmon River, Oregon (Atwater et al., 2005). Figure Captions (B) Coseismic subsidence evidence from a drowned tree stump surrounded by tidal mud in the Naselle River near Willapa Bay, Washington (Atwater et al., 2005). (C) Marine sediment core showing dark bands of sandy sediment, interpreted as coseismic turbidite deposits, interbedded with lighter colored hemipelagic clay (photograph by C. Goldfinger). (D) An example of a coseismic landslide that dammed a river to produce a “quake lake,” from the 1976 Guatemala earthquake (Espinosa, 1976). While not an example of coseismic landsliding in Cascadia, this photo demonstrates secondary hazard potential to the Pacific Northwest. Figure 4. (Left) Onshore and offshore geologic evidence for CSZ megathrust rupture. Semi- transparent green and blue horizontal bars indicate the temporal length of each record. Onshore and offshore event age range estimates are color-coded with site location (map figure to the right). Offshore age ranges are from turbidites analyzed by Goldfinger et al., 2012. Thick horizontal age ranges are sandy marine turbidite ages estimated from 14C dating of hemipelagic sediments, estimated basal erosion, and inferred sedimentation rate for each geographic locale. Thin horizontal age ranges are calculated ages of interbedded hemipelagic sediment. All age ranges are 2σ uncertainty propagated through 14C age calibration and correction. Vertical grey bars are interpreted event ages from a land-marine age compilation, which takes several onshore geology sites into account with some, but not all, of the geochronologic and stratigraphic information from marine sediment cores (Goldfinger et al., 2012; Table S1). The shade of grey reflects the number of onshore sites plotted here that are consistent with this interpretation (darker = more overlapping onshore data). Age ranges for onshore geologic evidence shown with hatched fill. Age ranges for subsidence (white arrow) and/or tsunami deposit (cute little wave) events are calibrated 14C dates or from OxCal modeling. GF2012 refers to Goldfinger et al. (2012). (Middle) Map shows the locations of onshore and offshore study sites, colored location markers correlate with the age-range panel to the left. Offshore canyons labeled using white text with colored outlines that correlate with turbidite age range bars determined for turbidites associated with that canyon. Black text outlines denote canyon data lacking or not used. Core ID numbers are available in Figure S1. Marine cores shown are only those used for age dating or stratigraphic correlation; additional marine core locations are in Goldfinger et al., 2012. Nearshore geographic features labeled in purple. Onshore geographic features labeled in blue. Figure Captions Bathymetric contours are 100 meter spacing in light grey, 500 meter interval in dark grey (derived from Wong & Grim, 2015). (Right) North-south evidence for possible rupture boundaries inferred from geophysical datasets, adapted from Watt & Brothers (2020). Circles denote locations of observations of along-strike heterogeneities. Latitudes correspond to the map. Figure 5. Maps of geophysical and geologic datasets used to infer along-strike heterogeneities along the CSZ. Left map shows heterogeneities on the incoming plate and plate interface. Time- averaged ETS slip rates from Bartlow (2020) are shown as contours with values from 30, 10, and 1 mm/yr. Seismicity from Stone et al. (2018) shows events associated with the CSZ, though note that earthquake depths are poorly constrained and some events may be located within the upper plate. Dense clusters of seismicity near latitudes 44.3° and 44.6° are coincident with subducted seamounts interpreted from magnetic and gravity anomalies (Trèhu et al., 2012). Right map shows heterogeneities on the overriding plate. Morphotectonic zones inferred do not necessarily have sharp boundaries (Watt & Brothers, 2020). VLM: Vertical Land Motion. Fault and lineament names: LR—Leech River fault; S—Seattlefault; SWI—South Whidbey Island fault; LCBC—Lake Creek Boundary Creek fault; DO—Doty fault; CR—Columbia River fault; GC—Gales Creek fault; TY—Tillamook-Yamhill fault; CO—Corvallis fault; WS—Wildlife Safari fault; CV—Canyonville fault; KR—Klamath River lineament; PH–Portland Hills fault; BC—Battle Creek fault. Bathymetric baselayer from Wong & Grim, 2015). Figure 6. Schematic depiction of recurrence models often proposed for subduction zone settings. (A) Time-dependent model suggests periodic earthquake occurrence is dependent on steady long-term strain accumulation and failure at a critical stress level (i.e., from σO to σF). This model suggests predictable slip magnitude. (B) Clustered time-dependent model suggests earthquake recurrence is variable, with clustered occurrence earthquakes punctuated by longer intervals, τB, of seismic quiescence. Within a cluster, the probability of recurrence at return- interval of τA is high. Following a cluster, probability of recurrence decreases until the onset of the next cluster at return interval of τB. This model suggests long-term strain accumulation and slip rate may be similar to the periodic model but that slip and timing is less predictable. (C) Time-independent models suggest that earthquake occurrence is unpredictable and may indicate that the displacement rate at the fault trace averaged over several consecutive earthquakes is non-linear. 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Broadband Synthetic Seismograms for Magnitude 9 Earthquakes on the Cascadia Megathrust Based on 3D Simulations and Stochastic Synthetics, Part 2: Rupture Parameters and Variability. Bull. Seismol. Soc. Am. 108(5A):2370–88. Witter RC, Kelsey HM, Hemphill-Haley E. 2003. Great Cascadia earthquakes and tsunamis of the past 6700 years, Coquille River estuary, southern coastal Oregon. Bull. Geol. Soc. Am. 115(10):1289–1306. Witter RC, Zhang Y, Wang K, Goldfinger C, Priest GR, Allan JC. 2012a. Coseismic slip on the southern Cascadia megathrust implied by tsunami deposits in an Oregon lake and earthquake- triggered marine turbidites. J. Geophys. Res. Solid Earth. 117(B10). Witter RC, Jaffe B, Zhang Y, Priest G, 2012b. Reconstructing hydrodynamic flow parameters of the 1700 tsunami at Cannon Beach, Oregon, USA. Natural Hazards. 63(1):223-40. Witter RC, Zhang YJ, Wang K, Priest GR, Goldfinger C, et al., 2013. Simulated tsunami inundation for a range of Cascadia megathrust earthquake scenarios at Bandon, Oregon, USA. Geosphere. 1;9(6):1783-803. Wong FL, Grim MS, 2015. Depth-to-Basement, Sediment-Thickness, and Bathymetry Data for the Deep-Sea Basins Offshore of Washington, Oregon, and California. United States Geological Survey Open File Report 2015-1118. Yamaguchi DK, Atwater BF, Bunker DE, Benson BE, Reid MS. 1997. Tree-ring dating the 1700 Cascadia earthquake. Nature. 389(6654):922–23. Ye L, Lay T, Kanamori H, Rivera L. 2016. Rupture characteristics of major and great (Mw≥ 7.0) megathrust earthquakes from 1990 to 2015: 2. Depth dependence. J. Geophys. Res. 121(2):845-63. Yousefi M, Milne G, Li S, Wang K, Bartholet A. 2020. Constraining Interseismic Deformation of the Cascadia Subduction Zone: New Insights From Estimates of Vertical Land Motion Over Different Timescales. J. Geophys. Res. Solid Earth. 125(3):e2019JB018248. Zielke O. 2018. Earthquake Recurrence and the Resolution Potential of Tectono‐Geomorphic Records. Bull. Seismol. Soc. Am. 108(3A):1399–1413. Terms and definitions Recurrence interval: The average time span between significant earthquake occurrences on a fault or in an earthquake source zone. Evidence threshold: Criteria that must be exceeded in order to create and preserve a geologic signature of an event (earthquake). Literature Cited volcanic arc Juan de Fuca Plate Explorer Plate Pacific Plate North American Plate mantle upwelling Portland OR Seattle WA Vancouver BC (A) (G) Site effects Seattle Basin amplification high low low-frequency low-frequency waves amplified waves amplified in sedimentary basins in sedimentary basins low-frequency waves amplified in sedimentary basins C ASC ADI A SU B DUC T I ON Z O N E (F) Landslides landslide dam strong ground motions strong ground motions result in slope failures result in slope failures strong ground motions result in slope failures (E) Episodic tremor & slip stable slip ETS coupled stable slip ETS coupled max. max. rupture rupture depth? depth? max. rupture depth? Juan de Fuca Plate (D) Tsunamis & splays upward wave splay fault flooded shoreline continental crust continental crust (C) Land-level change coastal subsidence coastal subsidence (B) Turbidites shelf abyssal plain slope turbidite deposit Figure 1. (A) Oblique view of the northwest margin of North American, where the Juan de Fuca and Explorer oceanic plates subduct beneath the North American Plate. On the side view, the thin red line between the two tectonic plates represents the region where great earthquakes occur. On the map overlay, the toothed blue line represents the surface trace of the where the subducting plate begins its descent. Major cities are shown as green dots. The purple swath shows the general region where episodic tremor and slip (ETS) occurs and the pink swath shows the general region considered to be the coupled zone. To the left and below (A), small diagrams illustrate various earthquake-related processes labeled beneath each diagram: (B) coseismic turbidite generation, (C) coseismic subsidence with dotted green line showing the pre-event coastal land-level, dead brown trees represent marine incursion onto a formerly terrestrial environment (D) and tsunami generation, (E) the relationship between the coupled fault and the ETS zone, with an ETS swarm depicted as blue circles, with a possible gap between the coupled zone and updip extent of ETS shown as a gradational area (F) coseismic landslide hazards, with schematic seismograms (in blue) showing the potential for topographic effects on ground motion amplification, and (G) how geologic features, such as sedimentary basins, can amplify seismic waves. (A) (B) Turbidites North American Plate mantle upwelling landslide dam (D) Tsunamis & splays p y (G) Site effects (F) Landslides Figure 1. Literature Cited Turbidites: Disturbance layers and evidence for turbidity currents (density flows) in marine or lacustrine environments. Turbidites: Disturbance layers and evidence for turbidity currents (density flows) in marine or lacustrine environments. ud turbidites: Turbidite deposits that lack a sandy component. Mud turbidites: Turbidite deposits that lack a sandy component. Confluence test: A physiographic criterion used to correlate turbidites across a margin by comparing deposits in tributary and master channels. Confluence test: A physiographic criterion used to correlate turbidites across a margin by comparing deposits in tributary and master channels. Seismogenic zone: The part of the plate boundary located from ~15-35 km depths that tends to rupture in large earthquakes. Seismogenic zone: The part of the plate boundary located from ~15-35 km depths that tends to rupture in large earthquakes. Stick-slip behavior: The frictional behavior required to generate an earthquake; where dynamic friction is less than the static friction. Stick-slip behavior: The frictional behavior required to generate an earthquake; where dynamic friction is less than the static friction. Coupled zone: A geodetically inferred proxy for the seismogenic zone. Rupture patch: The area on the megathrust that slips during a particular earthquake. Rupture boundaries: Heterogeneities in physical properties that may inhibit earthquake rupture propagation over multiple earthquake cycles. Rupture boundaries: Heterogeneities in physical properties that may inhibit earthquake rupture propagation over multiple earthquake cycles. Tsunami earthquakes: Earthquakes in which shallow (depths less than ~15 km) slip occurs offshore, generating tsunamis. Tsunami earthquakes: Earthquakes in which shallow (depths less than ~15 km) slip occurs offshore, generating tsunamis. Episodic tremor and slip: The phenomenon of seismically measured tremor that accompanies geodetically observed slip along a plate interface. Supercycles: Nested clusters of subduction zone earthquakes. Significant events: Fault slip events that relieve enough stress to permit statistical renewal of the recurrence process. Serial ruptures: A series of adjacent earthquakes along a margin separated by short time intervals (days to decades). Aleatoric variability: The irreducible intrinsic natural variability of a system. Epistemic uncertainty: Reducible uncertainties that stem from limited knowledge. ET S z on e co upl ed z o ne Vancouver Is. Literature Cited (A) Oblique view of the northwest margin of North American, where the Juan de Fuca and Explorer oceanic plates subduct beneath the North American Plate. On the side view, the thin red line between the two tectonic plates represents the region where great earthquakes occur. On the map overlay, the toothed blue line represents the surface trace of the where the subducting plate begins its descent. Major cities are shown as green dots. The purple swath shows the general region where episodic tremor and slip (ETS) occurs and the pink swath shows the general region considered to be the coupled zone. To the left and below (A), small diagrams illustrate various earthquake-related processes labeled beneath each diagram: (B) coseismic turbidite generation, (C) coseismic subsidence with dotted green line showing the pre-event coastal land-level, dead brown trees represent marine incursion onto a formerly terrestrial environment (D) and tsunami generation, (E) the relationship between the coupled fault and the ETS zone, with an ETS swarm depicted as blue circles, with a possible gap between the coupled zone and updip extent of ETS shown as a gradational area (F) coseismic landslide hazards, with schematic seismograms (in blue) showing the potential for topographic effects on ground motion amplification, and (G) how geologic features, such as sedimentary basins, can amplify seismic waves. A Uplift Subsidence continental crust oceanic plate B Uplift Subsidence continental crust oceanic plate possible tsunami A Uplift Subsidence continental crust oceanic plate B Uplift Subsidence continental crust oceanic plate possible tsunami Record sand mud soil injectite mud soil tree stump Coseismic tsunami liquefaction tide Interseismic tidal marsh D Juan de Fuca Explorer Plate Pacific Plate Juan de Fuca Explorer Plate Pacific Plate Ju Ju Exp Exp Expp PlPlPla p P p fic Plate a fic Plate C ~M9 rupture Spreading ridge Subduction zone S bd i ~M8 rupture ~M9 ruptur Spreading r Subduction an de Fuca an de Fu an de Fucaa lolo a re re orer er ore o e orrerr ate atee M9 ruptur ~M8 ~ ruptur Figure 2. (A) Block diagram of the interseismic period, when convergence along the coupled subduction zone interface (red zone) typically causes gradual uplift in the onshore overriding plate, and gradual subsidence offshore. Literature Cited (B) Diagram of the coseismic period, when earthquake rupture along the subduction zone interface relieves accumulated strain and generally causes sudden subsidence in the onshore overriding place and sudden uplift in the offshore overriding plate. Shallow rupture may generate a tsunami. (C) Possible scenarios for an ~M9 (orange) and ~M8 (blue) events that rupture the CSZ plate interface. (D) Schematic diagrams of stratigraphic evidence for the earthquake deformation cycle. Left column shows the effect of coseismic subsid- ence on wetland stratigraphy and coastal forests and their preservation in the stratigraphic record (see also Fig. 1C). Right column shows stratigraphic preservation of coseismic tsunami deposits and liquefaction injectites. Record sand mud soil injectite mud soil tree stump Coseismic tsunami liquefaction tide Interseismic tidal marsh D Juan de Fuca Explorer Plate Pacific Plate Juan de Fuca Explorer Plate Pacific Plate Ju Ju Exp Exp Expp PlPlPla p P p fic Plate a fic Plate C ~M9 rupture Spreading ridge Subduction zone S bd i ~M8 rupture ~M9 ruptur Spreading r Subduction an de Fuca an de Fu an de Fucaa lolo a re re orer er ore o e orrerr ate atee M9 ruptur ~M8 ~ ruptur A C D Interseismic B Record Figure 2. (A) Block diagram of the interseismic period, when convergence along the coupled subduction zone interface (red zone) typically causes gradual uplift in the onshore overriding plate, and gradual subsidence offshore. (B) Diagram of the coseismic period, when earthquake rupture along the subduction zone interface relieves accumulated strain and generally causes sudden subsidence in the onshore overriding place and sudden uplift in the offshore overriding plate. Shallow rupture may generate a tsunami. (C) Possible scenarios for an ~M9 (orange) and ~M8 (blue) events that rupture the CSZ plate interface. (D) Schematic diagrams of stratigraphic evidence for the earthquake deformation cycle. Left column shows the effect of coseismic subsid- ence on wetland stratigraphy and coastal forests and their preservation in the stratigraphic record (see also Fig. 1C). Right column shows stratigraphic preservation of coseismic tsunami deposits and liquefaction injectites. Marine sediment core Sand Hemipelagic clay C Coseismic landslide debris Dammed river Vegetated landscape D A Figure 3. (A) Evidence for coseismic subsidence and tsunami inundation from a sedimentary exposure of subaerial dune sand and prehistoric settlements overlain by tsunami sand and tidal mud along the Salmon River, Oregon (Atwater et al., 2005). Literature Cited (B) Coseismic subsidence evidence from a drowned tree stump surrounded by tidal mud in the Naselle River near Willapa Bay (Atwater et al., 2005). (C) Marine sediment core showing dark bands of sandy sediment, interpreted as coseismic turbidite deposits, interbedded with lighter colored hemi- pelagic clay. Photo by C. Goldfinger. (D) An example of a coseismic landslide that dammed a river to produce a “quake lake”, from the 1976 Guatemala earthquake (Espinosa, 1976). While not an example of coseismic landsliding in Cascadia, this photo demonstrates secondary hazard potential to the Pacific Northwest. B Buried forest floor Mud deposited by tides 0.5 m Stump Modern salt marsh Tsunami deposit Tidal mud Soil ameded by refuse Dune sand Hearths with charcoal and fire-modified rock A Tsunami deposit Tidal mud Soil ameded by refuse Dune sand Hearths with charcoal and fire-modified rock Marine sediment core Sand Hemipelagic clay C C A Coseismic landslide debris Dammed river Vegetated landscape D B Buried forest floor Mud deposited by tides 0.5 m Stump Modern salt marsh D B Figure 3. (A) Evidence for coseismic subsidence and tsunami inundation from a sedimentary exposure of subaerial dune sand and prehistoric settlements overlain by tsunami sand and tidal mud along the Salmon River, Oregon (Atwater et al., 2005). (B) Coseismic subsidence evidence from a drowned tree stump surrounded by tidal mud in the Naselle River near Willapa Bay (Atwater et al., 2005). (C) Marine sediment core showing dark bands of sandy sediment, interpreted as coseismic turbidite deposits, interbedded with lighter colored hemi- pelagic clay. Photo by C. Goldfinger. (D) An example of a coseismic landslide that dammed a river to produce a “quake lake”, from the 1976 Guatemala earthquake (Espinosa, 1976). While not an example of coseismic landsliding in Cascadia, this photo demonstrates secondary hazard potential to the Pacific Northwest. Literature Cited (Left) Onshore and offshore geologic evidence for CSZ megathrust rupture. Semi-transparent green and blue horizontal bars indicate the temporal length of each record. Onshore and offshore event age range estimates are color-coded with site location (map figure to the right). Offshore age ranges are from turbidites analyzed by Goldfinger et al., 2012. Thick horizontal age ranges are sandy marine turbidite ages estimated from 14C dating of hemipelagic sediments, estimated basal erosion, and inferred sedimentation rate for each geographic locale. Thin horizontal age ranges are calculated ages of interbedded hemipelagic sediment. All age ranges are 2σ uncertainty propagated through 14C age calibration and correction. Vertical grey bars are interpreted event ages from a land-marine age compilation, which takes several onshore geology sites into account with some, but not all, of the geochronologic and stratigraphic information from marine sediment cores (Goldfinger et al., 2012; Table S1). The shade of grey reflects the number of onshore sites plotted here that are consis- tent with this interpretation (darker = more overlapping onshore data). Age ranges for onshore geologic evidence shown with hatched fill. Age ranges for subsidence (white arrow) and/or tsunami deposit (cute little wave) events are calibrated 14C dates or from OxCal modeling. GF2012 refers to Goldfinger et al. (2012). (Middle) Map shows the locations of onshore and offshore study sites, colored location markers correlate with the age-range panel to the left. Offshore canyons labeled using white text with colored outlines that correlate with turbidite age range bars determined for turbidites associated with that canyon. Black text outlines denote canyon data lacking or not used. Core ID numbers are available in Figure S1. Marine cores shown are only those used for age dating or stratigraphic correlation; additional marine core locations are in Goldfinger et al., 2012. Nearshore geographic features labeled in purple. Onshore geographic features labeled in blue. Bathymetric contours are 100 meter spacing in light grey, 500 meter interval in dark grey (derived from Wong & Grim, 2015). (Right) North-south evidence for possible rupture boundaries inferred from geophysical datasets, adapted from Watt & Brothers (2020). Circles denote locations of observations of along-strike heterogeneities. Latitudes correspond to the map. Figure 4. (Left) Onshore and offshore geologic evidence for CSZ megathrust rupture. Semi-transparent green and blue horizontal bars indicate the temporal length of each record. Onshore and offshore event age range estimates are color-coded with site location (map figure to the right). Literature Cited Paleoseismic data Morphotectonic domains P-wave mantle velocity ETS recurrence patterns Geodetic locking model Near-plate- interface seismicity 1700 CE modeled slip distribution Forearc gravity anomalies 40° 44° 48° T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T17 T16 T18 T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T17 T16 T18 Southern Washington Southern Oregon Central Oregon Northern California Vancouver Island Northern Oregon 0 1000 2000 3000 4000 5000 6000 7000 8000 Calendar Years BP 1950 9000 10000 7+ 0 1-2 3-4 5-6 Horizontal bars age range of paleoseismic record Vertical bars full margin ruptures (GF 2012) # of overlapping onshore sites offshore onshore turbidite hemipelagic Barkley Canyon turbidite hemipelagic Juan de Fuca turbidite hemipelagic Hydrate Ridge turbidite hemipelagic Rogue Apron turbidite hemipelagic Smith Apron turbidite hemipelagic Klamath Canyon turbidite hemipelagic 1996 Astoria Channel turbidite Trinidad Plunge Pool turbidite Eel Channel turbidite 1996 Cascadia Channel hemipelagic tsunami evidence coseismic subsidence Coquille River Sixes River Alsea Bay Lagoon Creek Bradley Lake Talbot Creek Deserted Lake John’s River Willapa Bay Yaquina Bay Nehalem River South Vancouver Onshore evidence type Onshore Offshore 1700 CE WA BC CA OR Cascadia Channel Cascadia Channel Astoria Channel Astoria Channel Astoria Astoria Fan Fan Cascadia Channel Astoria Channel Astoria Fan Hydra Hydrate Ridg Ridge Barkle arkley Juan uan de de F Fuc uca Quinal uinalt Grarays Willap illapa Astori oria Rogu Rogue Smi mith Klama lamath Trinida rinidad Eel Mendocin Mendocino Guid uide Quillayu uillayute Noyo Noyo Hydrate Ridge Barkley Juan de Fuca Quinalt Grays Willapa Astoria Rogue Smith Klamath Trinidad Eel Mendocino Guide Quillayute Noyo Willapa Bay Willapa Bay Nehalem River Nehalem River Alsea Bay Alsea Bay Yaquina Bay Yaquina Bay Lagoon Creek Lagoon Creek Sixes River Sixes River Coquille River Coquille River Talbot Creek Talbot Creek Johns River Johns River Deserted Deserted Lake Lake Bradley Lake Bradley Lake Tofino Tofino Port Alberni Port Alberni Willapa Bay Nehalem River Alsea Bay Yaquina Bay Lagoon Creek Sixes River Coquille River Talbot Creek Johns River Deserted Lake Tofino Port Alberni Bradley Lake Heceta Heceta Bank Bank Cape Cape Blanco Blanco Cape Cape Mendocino Mendocino Nehalem Nehalem Bank Bank Olympic Olympic Peninsula Peninsula Puget Puget Sound Sound Vancouver Vancouver Island Island Columbia Columbia River River Salish Salish Sea Sea Heceta Bank Cape Blanco Cape Mendocino Nehalem Bank Olympic Peninsula Puget Sound Vancouver Island Columbia River Salish Sea Onshore paleoseismic site Turbidite core site Correlated age data and stratigraphy Correlation to core with ages Correlation in general Subsidence and tsunami record Tsunami record only -128° -126° -124° -128° -126° -124° 42° 44° 48° 40° 46° 42° 44° 48° 40° 46° Paleoseismic data Morphotectonic domains P-wave mantle velocity ETS recurrence patterns Geodetic locking model Near-plate- interface seismicity 1700 CE modeled slip distribution Forearc gravity anomalies 40° 44° 48° WA BC CA OR Cascadia Channel Cascadia Channel Astoria Channel Astoria Channel Astoria Astoria Fan Fan Cascadia Channel Astoria Channel Astoria Fan Hydra Hydrate Ridg Ridge Barkle arkley Juan uan de de F Fuc uca Quinal uinalt Grarays Willap illapa Astori oria Rogu Rogue Smi mith Klama lamath Trinida rinidad Eel Mendocin Mendocino Guid uide Quillayu uillayute Noyo Noyo Hydrate Ridge Barkley Juan de Fuca Quinalt Grays Willapa Astoria Rogue Smith Klamath Trinidad Eel Mendocino Guide Quillayute Noyo Willapa Bay Willapa Bay Nehalem River Nehalem River Alsea Bay Alsea Bay Yaquina Bay Yaquina Bay Lagoon Creek Lagoon Creek Sixes River Sixes River Coquille River Coquille River Talbot Creek Talbot Creek Johns River Johns River Deserted Deserted Lake Lake Bradley Lake Bradley Lake Tofino Tofino Port Alberni Port Alberni Willapa Bay Nehalem River Alsea Bay Yaquina Bay Lagoon Creek Sixes River Coquille River Talbot Creek Johns River Deserted Lake Tofino Port Alberni Bradley Lake Heceta Heceta Bank Bank Cape Cape Blanco Blanco Cape Cape Mendocino Mendocino Nehalem Nehalem Bank Bank Olympic Olympic Peninsula Peninsula Puget Puget Sound Sound Vancouver Vancouver Island Island Columbia Columbia River River Salish Salish Sea Sea Heceta Bank Cape Blanco Cape Mendocino Nehalem Bank Olympic Peninsula Puget Sound Vancouver Island Columbia River Salish Sea Onshore paleoseismic site Turbidite core site Correlated age data and stratigraphy Correlation to core with ages Correlation in general Subsidence and tsunami record Tsunami record only -128° -126° -124° -128° -126° -124° 42° 44° 48° 40° 46° 42° 44° 48° 40° 46° Figure 4. Literature Cited Offshore age ranges are from turbidites analyzed by Goldfinger et al., 2012. Thick horizontal age ranges are sandy marine turbidite ages estimated from 14C dating of hemipelagic sediments, estimated basal erosion, and inferred sedimentation rate for each geographic locale. Thin horizontal age ranges are calculated ages of interbedded hemipelagic sediment. All age ranges are 2σ uncertainty propagated through 14C age calibration and correction. Vertical grey bars are interpreted event ages from a land-marine age compilation, which takes several onshore geology sites into account with some, but not all, of the geochronologic and stratigraphic information from marine sediment cores (Goldfinger et al., 2012; Table S1). The shade of grey reflects the number of onshore sites plotted here that are consis- tent with this interpretation (darker = more overlapping onshore data). Age ranges for onshore geologic evidence shown with hatched fill. Age ranges for subsidence (white arrow) and/or tsunami deposit (cute little wave) events are calibrated 14C dates or from OxCal modeling. GF2012 refers to Goldfinger et al. (2012). (Middle) Map shows the locations of onshore and offshore study sites, colored location markers correlate with the age-range panel to the left. Offshore canyons labeled using white text with colored outlines that correlate with turbidite age range bars determined for turbidites associated with that canyon. Black text outlines denote canyon data lacking or not used. Core ID numbers are available in Figure S1. Marine cores shown are only those used for age dating or stratigraphic correlation; additional marine core locations are in Goldfinger et al., 2012. Nearshore geographic features labeled in purple. Onshore geographic features labeled in blue. Bathymetric contours are 100 meter spacing in light grey, 500 meter interval in dark grey (derived from Wong & Grim, 2015). (Right) North-south evidence for possible rupture boundaries inferred from geophysical datasets, adapted from Watt & Brothers (2020). Circles denote locations of observations of along-strike heterogeneities. Latitudes correspond to the map. Literature Cited WA WA BC CA OR 30 10 3 30 10 3 30 30 10 10 3 -5 -80 -60 -40 -20 -10 -100 -5 -80 -60 -40 -20 -10 -100 -126° -128° -124° -122° 42° 44° 48° 50° 40° 46° Blanco Fracture Zone Blanco Fracture Zone Sovanco Sovanco Fracture Zone Fracture Zone Explore Explore Plate Plate Juan de Fuca Juan de Fuca Plate Plate Gorda Plate Gorda Plate Pacific Plate Pacific Plate Blanco Fracture Zone Sovanco Fracture Zone Exploer Plate Juan de Fuca Plate Gorda Plate Pacific Plate incoming plate/plate interface 2 1 0 -1 -1 -1 LR S DO PH GC CO WS CV KR BC LCBCF SWI TY LR S DO PH GC CO WS CV KR BC LCBCF SWI TY Wrangellia Wrangellia Pleistocene Pleistocene wedge wedge Olympic Olympic Franciscan Franciscan Klamath Klamath Siletz Siletz Siletz Siletz (thickened) (thickened) Wrangellia Pleistocene wedge Olympic Franciscan Klamath Siletz Siletz (thickened) -128° -126° -124° -122° interpreted along- strike extents of correlated turbidites overriding plate Crustal faults (Wells et al., 2017) Change in margin strike Turbidite event extents (Goldfinger et al., 2012) Interseismic VLM (mm/yr) (Yousefi et al., 2020) Gravity lows (Wells et al., 2003) Terranes (Watt & Brothers, in review) Morphotectonic boundary (Watt & Brothers, in review) Offshore GNSS sites (active) (planned) 2.0 0.5 1.0 3.0 10.0 2.5 25.0 45.0 Seismicity (Stone et al., 2018) depth M= Apparent ETS segments (Brudzinski & Allen. 2007) ETS slip rate (mm/yr) (Bartlow et al., 2020) AD 1700 8-m+ slip patch (Wang et al., 2013) Subducted seamounts (Trehu et al., 2012) Slab buoyancy (Bodmer et al., 2018) Slab contours (McCrory et al., 2012) Locking fraction (Schmalzle et al., 2014) 1.0 0.5 0.0 Figure 5. Maps of geophysical and geologic datasets used to infer along-strike heterogeneities along the CSZ. Left map shows heterogeneities on the incoming plate and plate interface. Time-averaged ETS slip rates from Bartlow (2020) are shown as contours with values from 30, 10, and 1 mm/yr. Seismicity from Stone et al. (2018) shows events associated with the CSZ, though note that earthquake depths are poorly constrained and some events may be located within the upper plate. Dense clusters of seismicity near latitudes 44.3° and 44.6° are coincident with subducted seamounts interpreted from magnetic and gravity anomalies (Trèhu et al., 2012). Right map shows heterogeneities on the overriding plate. Morphotectonic zones inferred do not necessarily have sharp boundaries (Watt & Brothers, 2020). Literature Cited VLM: Vertical Land Motion. Fault and lineament names: LR–Leech River fault; S–Seattlefault; SWI–South Whidbey Island fault; LCBC–Lake Creek Boundary Creek fault; DO–Doty fault; CR–Columbia River fault; GC–Gales Creek fault; TY–Tillamook-Yamhill fault; CO–Corvallis fault; WS–Wildlife Safari fault; CV–Canyonville fault; KR–Klamath River lineament; PH–Portland Hills fault; BC–Battle Creek fault. Bathymetric baselayer from Wong & Grim, 2015). 2 1 0 -1 -1 -1 LR S DO PH GC CO WS CV KR BC LCBCF SWI TY LR S DO PH GC CO WS CV KR BC LCBCF SWI TY Wrangellia Wrangellia Pleistocene Pleistocene wedge wedge Olympic Olympic Franciscan Franciscan Klamath Klamath Siletz Siletz Siletz Siletz (thickened) (thickened) Wrangellia Pleistocene wedge Olympic Franciscan Klamath Siletz Siletz (thickened) -128° -126° -124° -122° interpreted along- strike extents of correlated turbidites overriding plate Crustal faults (Wells et al., 2017) Change in margin strike Turbidite event extents (Goldfinger et al., 2012) Interseismic VLM (mm/yr) (Yousefi et al., 2020) Gravity lows (Wells et al., 2003) Terranes (Watt & Brothers, in review) Morphotectonic boundary (Watt & Brothers, in review) CA OR 30 10 3 30 10 3 -5 -80 -60 -40 -20 -10 -100 -5 -80 -60 -40 -20 -10 -100 42° 44° 40° Blanco Fracture Zone Blanco Fracture Zone Gorda Plate Gorda Plate Pacific Plate Pacific Plate Blanco Fracture Zone Gorda Plate Pacific Plate Change in margin strike ETS slip rate (mm/yr) (Bartlow et al., 2020) Figure 5. Maps of geophysical and geologic datasets used to infer along-strike heterogeneities along the CSZ. Left map shows heterogeneities on the incoming plate and plate interface. Time-averaged ETS slip rates from Bartlow (2020) are shown as contours with values from 30, 10, and 1 mm/yr. Seismicity from Stone et al. (2018) shows events associated with the CSZ, though note that earthquake depths are poorly constrained and some events may be located within the upper plate. Dense clusters of seismicity near latitudes 44.3° and 44.6° are coincident with subducted seamounts interpreted from magnetic and gravity anomalies (Trèhu et al., 2012). Right map shows heterogeneities on the overriding plate. Morphotectonic zones inferred do not necessarily have sharp boundaries (Watt & Brothers, 2020). VLM: Vertical Land Motion. Fault and lineament names: LR–Leech River fault; S–Seattlefault; SWI–South Whidbey Island fault; LCBC–Lake Creek Boundary Creek fault; DO–Doty fault; CR–Columbia River fault; GC–Gales Creek fault; TY–Tillamook-Yamhill fault; CO–Corvallis fault; WS–Wildlife Safari fault; CV–Canyonville fault; KR–Klamath River lineament; PH–Portland Hills fault; BC–Battle Creek fault. Literature Cited Bathymetric baselayer from Wong & Grim, 2015). (B) CLUSTERED TIME-DEPENDENT 0 σ0 σF 0 long-term slip rate cluster 1 cluster 2 STRESS CUMULATIVE SLIP TIME τA τB (A) TIME-DEPENDENT STRESS 0 τ1 τ2 τ3 CUMULATIVE SLIP 0 τ1 τ2 τ3 long-term slip rate TIME σ0 σF (C) TIME-INDEPENDENT 0 σ0 σF TIME 0 STRESS CUMULATIVE SLIP long-term slip rate Figure 6. Schematic depiction of recurrence models often proposed for subduction zone settings. (A) Time-de- pendent model suggests periodic earthquake occurrence is dependent on steady long-term strain accumula- tion and failure at a critical stress level (i.e., from σO to σF). This model suggests predictable slip magnitude. (B) Clustered time-dependent model suggests earthquake recurrence is variable, with clustered occurrence earth- quakes punctuated by longer intervals, τB, of seismic quiescence. Within a cluster, the probability of recurrence at return-interval of τA is high. Following a cluster, probability of recurrence decreases until the onset of the next cluster at return interval of τB. This model suggests long-term strain accumulation and slip rate may be similar to the periodic model, but that slip and timing is less predictable. (C) Time-independent models suggest that earthquake occurrence is unpredictable and may indicate that the displacement rate at the fault trace averaged over several consecutive earthquakes is non-linear. (B) CLUSTERED TIME-DEPENDENT (A) TIME-DEPENDENT STRESS CUMULATIVE SLIP Figure 6. Schematic depiction of recurrence models often proposed for subduction zone settings. (A) Time-de- pendent model suggests periodic earthquake occurrence is dependent on steady long-term strain accumula- tion and failure at a critical stress level (i.e., from σO to σF). This model suggests predictable slip magnitude. (B) Clustered time-dependent model suggests earthquake recurrence is variable, with clustered occurrence earth- quakes punctuated by longer intervals, τB, of seismic quiescence. Within a cluster, the probability of recurrence at return-interval of τA is high. Following a cluster, probability of recurrence decreases until the onset of the next cluster at return interval of τB. This model suggests long-term strain accumulation and slip rate may be similar to the periodic model, but that slip and timing is less predictable. (C) Time-independent models suggest that earthquake occurrence is unpredictable and may indicate that the displacement rate at the fault trace averaged over several consecutive earthquakes is non-linear. Table 1. Literature Cited Characteristics of different earthquake recurrence models Time-independent Poisson Quasi-periodic Clustered Event rate and periodicity There is a general rate of occurence (e.g., 2 events per millenia), but events are not periodic There is a rate of occurence and events are periodic There is a rate of occurence and cycles are periodic. However, an earthquake cycle includes multiple superimposed cycles Energy balance and stress release Events independent of accumulated/released stress Single-event cycle with characteristic magnitude releases sufcient accumulated stress to renew the statistical process Stress accumulation and release balances over and earthquake cluster, or "supercycle" Interevent time and probability Random and unpredictable. Interevent time does not depend on slip rates or accummulated stress. There is equal probability for a 2 year and 200 year interevent time. Consistent and predictable. Interevent time depends on strain accumulation rates. Probability of occurence increases as mean interevent time is approached Interevent time depends on whether cluster is complete or in progress. Probability of occurence increases as either the mean 'intracluster' or 'extracluster' event times are approached Hazard rate Constant, independent of last event (memoryless) Normal distribution around the expected event recurrence Complex distribution around more than one event recurrence COV ~1 <1 >1 Time-dependent Table 1. Characteristics of different earthquake recurrence models
https://openalex.org/W3120157308	https://mirtr.elpub.ru/jour/article/download/1920/2364	Russian	null	Theoretical Comprehension of the Role of Transport in Ensuring Long-Term Economic Development	Mir transporta	2,021	cc-by	8,569	Дмитрий МАЧЕРЕТ Исходя из разработанной модели, сформулиро- ваны рекомендации относительно желательных для обеспечения экономического роста направлений развития транспорта. В частности, раскрыта необхо- димость развития тяжеловесного движения на же- лезных дорогах с целью удешевления перевозок относительно недорогих товаров и расширения возможностей для их доставки на дальние расстоя- ния, а также высокоскоростных перевозок для доро- гостоящих товаров, чувствительных ко времени до- ставки. Сфокусировано внимание на важности соз- дания соответствующей транспортной инфраструк- туры и необходимости объединения в этих целях усилий государства и бизнеса, способствующих развитию транспортной инфраструктуры за счёт как институциональных инструментов, так и бюджетных инвестиций в рамках проектов государственно- частного партнёрства. Целью описанного в статье исследования явля- ется развитие теоретической базы для оценки дол- госрочного влияния транспорта на экономическое развитие в условиях последовательного снижения транспортных издержек. При этом транспортные издержки предлагается рассматривать с учётом рисков потерь товаров при перевозке и нарушений сроков доставки, а также ущерба товаровладельца от «замораживания» на время перевозки воплощён- ного в товаре капитала. С использованием экономической дедукции пред- ложена теоретическая модель влияния транспортных издержек на производство и сбыт товаров, на основе которой сделан вывод о том, что снижение транспорт- ных издержек является катализатором экономического роста, «запуская» долгосрочные взаимосвязанные процессы расширения географической зоны сбыта товаров, роста объёмов, масштаба и эффективности производства. Напротив, отсутствие существенного прогресса в развитии транспорта и сохранение высоких транспортных издержек способствуют консервации технико-технологического уровня и низкой эффектив- ности производства. На основе примеров из экономи- ческой истории и современной практики, с использо- ванием методов статистического анализа и технико- экономических расчётов, показано, что предложенная модель согласуется с эмпирическими данными. Сделан вывод о том, что товарообменная деятельность (торговля и транспорт) – значимый фактор экономического развития, стимулирую- щий увеличение объёмов и повышение эффек- тивности производства. Поэтому экономическая наука должна строиться не «вокруг производства» или «вокруг обмена», а исходить из их взаимодей- ствия и активной роли обеих этих сфер в процес- се развития экономики. Ключевые слова: транспорт, экономический рост, межрегиональный товарообмен, транспортные издержк дальность транспортировки, масштаб производства, инновации. Информация об авторе: Мачерет Дмитрий Александрович – доктор экономических наук, профессор, первый заместитель председателя Объединённого учёного совета ОАО «РЖД», заведующий кафедрой экономики транспортной инфраструктуры и управления строительным бизнесом Российского университета транспорта, Москва, Россия, macheretda@rambler.ru. Статья поступила в редакцию 24.01.2020, принята к публикации 18.05.2020. ВОПРОСЫ ТЕОРИИ Теоретическое осмысление роли транспорта в обеспечении долгосрочного экономического развития УДК 656.2:330.12 DOI: https://doi.org/10.30932/1992-3252-2020-18-06-33 Мачерет Дмитрий Александрович – ОАО «РЖД», Российский университет транспорта, Москва, Россия. ВОПРОСЫ ТЕОРИИ Теоретическое осмысление роли транспорта в обеспечении долгосрочного экономического развития УДК 656.2:330.12 DOI: https://doi.org/10.30932/1992-3252-2020-18-06-33 Мачерет Дмитрий Александрович – ОАО «РЖД», Российский университет транспорта, Москва, Россия. 6 6 ВОПРОСЫ ТЕОРИИ УДК 656.2:330.12 DOI: https://doi.org/10.30932/1992-3252-2020-18-06-33 Дмитрий МАЧЕРЕТ Мачерет Дмитрий Александрович – ОАО «РЖД», Российский университет транспорта, Москва, Россия. Дмитрий МАЧЕРЕТ Информация об авторе: Мачерет Дмитрий Александрович – доктор экономических наук, профессор, первый заместитель председателя Объединённого учёного совета ОАО «РЖД», заведующий кафедрой экономики транспортной инфраструктуры и управления строительным бизнесом Российского университета транспорта, Москва, Россия, macheretda@rambler.ru. For the English text of the article please see р. 20. For the English text of the article please see р. 20. • МИР ТРАНСПОРТА, том 18, № 4, С. 6–33 (2020) ВВЕДЕНИЕ • риски, связанные с возможностью его потери или порчи в процессе транспорти‑ ровки, а также риски более поздней достав‑ ки товара получателю; Значение экономического обмена, ос‑ нованного на использовании преимуществ в производстве тех или иных товаров, для специализации и роста эффективности экономики показал ещё Адам Смит [1, с. 69–82; 443–445]. Дж. Мокир не случайно называет составляющую (тип) экономиче‑ ского роста, основанную на расширении обмена, выгодного для всех участвующих сторон, «смитианским ростом» [2]. К. Мен‑ гер [3] и Ф. А. фон Хайек [4] подчёркивали производительный характер обмена: уве‑ личение ценности благ в результате обмена равносильно созданию нового веществен‑ ного блага, а потому обмен имеет для эко‑ номического роста не меньшее значение, чем производство. • неявные затраты товаровладельца, связанные с «омертвлением» капитала, воплощённого в перевозимом товаре, в те‑ чение срока его доставки, зависящие от скорости и расстояния транспортировки. Во-вторых, провозные способности и качественные характеристики путей со‑ общения определяют как потенциальные общие объёмы перевозимых товаров, так и их возможный ассортимент. В теории международной торговли транспортные издержки учитываются при расчёте коэффициентов так называемого «гравитационного уравнения», предложен‑ ного Я. Тинбергеном [9] и позволяющего оценить объём торговли между двумя стра‑ нами в зависимости от ряда факторов. В данной модели транспортные издержки играют роль препятствий для международ‑ ной торговли. C повышением транспорт‑ ных издержек спектр неторгуемых товаров, производящихся исключительно для мест‑ ного потребления, расширяется [5, с. 49], а, значит, ограничиваются возможности для региональной специализации и ис‑ пользования сравнительных преимуществ. Наиболее значительное увеличение цен‑ ности благ зачастую достигается при между‑ народном (межрегиональном) обмене, когда его участников разделяют значитель‑ ные расстояния, а экономические характе‑ ристики производства, сбыта и потребления товаров существенно различаются. По мне‑ нию Э. Хелпмана, подтверждаемому анали‑ зом экономической истории с глубокой древности до наших дней, «исторические данные показывают, что торговля между отдалёнными торговыми партнёрами и эко‑ номическое развитие были сложным обра‑ зом взаимосвязаны и что эта торговля играла основную роль в историческом развитии мировой экономики» [5, с. 12]. Представляется, что рассмотрение роли транспорта в товарообменных процессах лишь как «генератора издержек», формирую‑ щего барьеры на пути торговли и ограничи‑ вающего возможности для экономической специализации, не позволяет адекватно осмыслить его действительное значение для развития экономики. Хотя транспорт‑ ные издержки, безусловно, можно рас сматривать как своеобразное «трение» в системе товарообмена, транспорт явля‑ ется не барьером, а материальной основой для его реализации. Отмеченный выше производительный характер обмена при сколь-нибудь значимых расстояниях может быть реализован только при посредстве магистрального транспорта. Мачерет Д. А. Теоретическое осмысление роли транспорта в обеспечении долгосрочного экономического развития ВВЕДЕНИЕ Активная роль транспорта в стимулировании экономиче‑ ского развития, формировании эпохи со‑ временного экономического роста отмече‑ на, на основе эмпирического анализа, в ряде работ, в частности, в [10–15]. В теории международной торговли, основы которой заложил в начале XIX века Д. Рикардо [6], а развитие обеспечили в на‑ чале XX века Э. Хекшер [7] и Б. Олин [8], определяющую роль играет характеристи‑ ка производства в регионах, между кото‑ рыми осуществляется товарообмен: срав‑ нительные затраты труда на производство тех или иных товаров (у Рикардо), относи‑ тельная обеспеченность факторами произ‑ водства (у Хекшера и Олина). Но в цепи товарообмена очень важны и экономиче‑ ские характеристики транспортно-логис тических артерий движения товаров. Во-первых, эти характеристики определя‑ ют издержки транспортировки, в которых можно выделить следующие компоненты: • непосредственные затраты на транс‑ портировку товара (провозные и прочие ) 77 • непосредственные затраты на транс‑ портировку товара (провозные и прочие платежи); • непосредственные затраты на транс‑ портировку товара (провозные и прочие платежи); Теоретическое осмысление роли транс‑ порта в повышении благосостояния обще‑ • МИР ТРАНСПОРТА, том 18, № 4, С. 6–33 (2020) Мачерет Д. А. Теоретическое осмысление роли транспорта в обеспечении долгосрочного экономического развития в работе [21], это свидетельствует не о росте «транспортной нагрузки» на экономику, а о том, что транспортные издержки, заме‑ щая за счёт перекомбинирования произ‑ водственных ресурсов в процессе регио‑ нальной специализации и межрегиональ‑ ного обмена более значительную величину других видов издержек, способствуют росту эффективности экономики. ства, создании «потребительского выигры‑ ша» представлено в классической работе Жюля Дюпюи [16]. Подходы Дюпюи полу‑ чили развитие в работе [17], где показано, как создание межрегионального транс‑ портного сообщения, позволяющее спе‑ циализировать производство в каждом регионе, формирует не только потреби‑ тельский выигрыш, но и выигрыш произ‑ водителей. 8 8 Для демонстрации согласованности сформированной теоретической модели с экономической практикой используются методы исторического и статистического анализа и технико-экономических расчё‑ тов. Среди эффектов торговли выделяется эффект разнообразия, связанный с воз‑ можностью потребительского выбора из более широкого ассортимента товаров [18]. В работе [19] на основе теоретической мо‑ дели показано, как расширение ассорти‑ мента товаров, достигаемое благодаря от‑ крытию транспортного сообщения, повы‑ шает объёмы производства и выигрыш потребителей. Мачерет Д. А. Теоретическое осмысление роли транспорта в обеспечении долгосрочного экономического развития • МИР ТРАНСПОРТА, том 18, № 4, С. 6–33 (2020) ТЕОРЕТИЧЕСКАЯ МОДЕЛЬ ВЛИЯНИЯ ТРАНСПОРТНЫХ ИЗДЕРЖЕК НА ПРОИЗВОДСТВО И СБЫТ ТОВАРОВ Изменение рыночных транспортных издержек в краткосрочном Рис. 1. Влияние транспортных издержек на сбыт товаров в удалённые регионы. Рис. 2. Изменение рыночных характеристик под влиянием снижения транспортных издержек в краткосрочном периоде. Рис. 2. Изменение рыночных транспортных издержек в краткосрочном Рис 1 Влияние транспортных издержек на сбыт Рис. 2. Изменение рыночных характеристик под влиянием снижения транспортных издержек в краткосрочном периоде. Рис. 2. Изменение рыночных транспортных издержек в краткосрочном Рис. 1. Влияние транспортных издержек на сбыт товаров в удалённые регионы. оценки которого раскрыта в работе [22]. Таким образом, на снижение транспортных издержек влияет не только непосредствен‑ ное удешевление перевозок, но и повыше‑ ние их надёжности и скорости). в «крутизне» кривой предложения уже в регионе производства. Ещё более впечатляющи д Существенное сокращение транспорт‑ ных издержек смещает вниз кривые пред‑ ложения товара в удалённых регионах (рис. 2). В результате расширяется геогра‑ фическая зона сбыта товара, а также увели‑ чивается объём сбыта в тех регионах, где он продавался и ранее. Соответственно, общий объём производства увеличивается, как и суммарная величина прибыли производи‑ телей и эффектов, получаемых потребите‑ лями («потребительского излишка»). Дру‑ гими словами, снижение транспортных издержек способствует экономическому росту и повышению благосостояния потре‑ бителей. Ещё более впечатляющи д транспортных издержек. Вызванное и производства стимулирует рост масш совершенствованием техники и тех издержек производства. Графически э кривых предложения (рис. 3). В географическая зона сбыта товара, сни тех регионах, где товар продавался производства. Кривые предложения в различных ре‑ гионах смещаются вверх по мере удаления от региона производства, объёмы сбыта при этом, соответственно, оказываются ниже, чем в регионе производства, а цены выше. При высоком уровне транспортных издержек (рис. 1) цены предложения в уда‑ лённых регионах существенно выше, чем в регионе производства. Географическая зона сбыта при принятом сочетании харак‑ теристик предложения и спроса будет ограничена Регионом 1. В Регионе 2 и бо‑ лее удалённых товар уже не будет востре‑ бован: хотя спрос на него есть и доставка технически возможна, однако кривые предложения лежат выше кривой спроса. Таким образом, высокий уровень транс‑ портных издержек ограничивает и геогра‑ фическую зону сбыта товаров, и объём сбыта. Соответственно, ограничивается и объём производства (в данном случае он составит q0 + q1). В условиях ограниченно‑ го сбыта организация массового производ‑ ства невозможна, что является тормозом для внедрения инноваций, а издержки производства высоки, что отражается Ещё более впечатляющи долгосрочные последствия снижения транспортных издержек. Вызванное ими увеличение зоны сбыта и объёмов производства сти‑ мулирует рост масштаба производства, сопровождающийся совершенствованием техники и технологий, что приводит к снижению издержек производства. Мачерет Д. А. Теоретическое осмысление роли транспорта в обеспечении долгосрочного экономического развития ТЕОРЕТИЧЕСКАЯ МОДЕЛЬ ВЛИЯНИЯ ТРАНСПОРТНЫХ ИЗДЕРЖЕК НА ПРОИЗВОДСТВО И СБЫТ ТОВАРОВ Влияние надёжности транспортных сообщений и величины транспортных из‑ держек на характеристики производства и сбыта товаров можно раскрыть с помо‑ щью следующей модели. Несмотря на наличие значительного корпуса работ, раскрывающих те или иные аспекты влияния транспорта на экономи‑ ческий рост и общественное благосостоя‑ ние, роль транспорта в обеспечении дол‑ госрочного экономического развития нуждается в углубленном теоретическом осмыслении. Цель настоящего исследова‑ ния – выявить, с использованием эконо‑ мической дедукции, сущность влияния поступательного развития транспорта и снижения транспортных издержек на изменение объёмов производства и цен товаров, а также на повышение масштабов и технико-технологического уровня про‑ изводства, и, тем самым, на снижение производственных издержек. Такой подход позволяет показать активную роль транс‑ порта в долгосрочном стимулировании и поддержании темпов экономического роста, а динамический характер рассмотре‑ ния транспортных издержек даёт возмож‑ ность взглянуть на них в новом ракурсе: если в статике транспортные издержки – это просто барьер для товарообмена и эко‑ номического роста, то в динамике снижаю‑ щиеся транспортные издержки – стимул повышения объёмов производства и рас‑ ширения географии сбыта товаров, сниже‑ ния рыночных цен и роста благосостояния потребителей. Важно отметить, что при сокращении удельных издержек на пере‑ возку товаров их общая величина в долго‑ срочном периоде растёт [20]. Как показано Пусть какой-либо товар производится в определённом регионе (Регион 0) и тех‑ нически может доставляться в иные регио‑ ны (1, 2, 3 и т.д.). Кривая спроса на товар (D) во всех ре‑ гионах одинакова (это условие не является существенным и принято для более нагляд‑ ной визуализации; при различиях кривых спроса в регионах выводы принципиально не изменятся). Производство в Регионе 0 используется, прежде всего, для удовлетворения спроса в данном регионе. При отсутствии транс‑ портных сообщений объём производства составит q0. При открытии транспортных сообщений производятся дополнительные объёмы товара для поставки в иные регио‑ ны. Этим условием, а также величиной транспортных издержек определяется ха‑ рактер кривых предложения в регионах 1, 2 и т.д. (Транспортные издержки, как ска‑ зано выше, учитывают не только стоимость перевозки товара, но и возможные потери, связанные с несохранностью и нарушения‑ ми сроков доставки товара, либо соответ‑ ствующие страховые платежи, а также ущерб товаровладельца от «заморажива‑ ния» на время перевозки оборотного капи‑ тала, воплощённого в товаре, методология • МИР ТРАНСПОРТА, том 18, № 4, С. 6–33 (2020) • МИР ТРАНСПОРТА, том 18, № 4, С. 6–33 (2020) Рис. 1. Влияние транспортных издержек на сбыт товаров в удалённые регионы. Рис. 2. Изменение рыночных характеристик под влиянием снижения транспортных издержек в краткосрочном периоде. Рис. 2. ТЕОРЕТИЧЕСКАЯ МОДЕЛЬ ВЛИЯНИЯ ТРАНСПОРТНЫХ ИЗДЕРЖЕК НА ПРОИЗВОДСТВО И СБЫТ ТОВАРОВ Гра‑ фически это выражается в уменьшении наклона кривых предложения (рис. 3). В результате ещё более расширяется гео‑ графическая зона сбыта товара, снижают‑ ся цены и растут объёмы сбыта в тех ре‑ гионах, где товар продавался и ранее, в том числе – в регионе производства. 9 • МИР ТРАНСПОРТА, том 18, № 4, С. 6–33 (2020) • МИР ТРАНСПОРТА, том 18, № 4, С. 6–33 (2020) Рис. 3. Изменение рыночных характеристик под влиянием снижения транспортных издержек в долгосрочном периоде. реализовать эффект масштаба и инновации и в сфере транспорта. В результате транс‑ портные издержки ещё более снизятся, что повлечёт за собой дальнейшее сближение кривых предложения в разных регионах и расширение географических масштабов сбыта, общий рост объёмов производства. Это сделает выгодным новый рост масшта‑ бов и технико-технологического уровня производства, что приведёт к продолже‑ нию «уположивания» кривых предложе‑ ния, даст новый импульс повышению объёмов производства, дальности сбыта, снижению цен. Другими словами, процес‑ сы, показанные на рисунках 2 и 3, будут итерационно повторяться, и каждый этап станет новым шагом на пути экономиче‑ ского развития и повышения благосостоя‑ ния людей. 10 Рис. 3. Изменение рыночных характеристик под влиянием снижения транспортных издержек в долгосрочном периоде. Рис. 3. Изменение рыночных характеристик под влиянием снижения транспортных издержек в долгосрочном периоде. На практике описанные процессы будут переплетаться между собой и хронологи‑ чески запараллеливаться, так как в каждый период времени какие-то производители будут реагировать на новые возможности, связанные с произошедшим снижением транспортных издержек и расширением географии сбыта, и заниматься увеличени‑ ем масштаба и технико-технологическим совершенствованием производства, а какие-то перевозчики – реагировать на уже свершившееся увеличение объёмов производства и перевозок, расширять мас‑ штаб своей деятельности и внедрять инно‑ вации, добиваясь снижения транспортных издержек. Поэтому результирующие гра‑ фики долгосрочных многоэтапных изме‑ нений транспортных издержек и объёмов производства могут быть представлены не в виде ломаных линий, а в виде плавных кривых (рис. 4). Поскольку рост географи‑ ческих масштабов сбыта и средней дально‑ сти транспортировки происходит под влиянием как снижения транспортных издержек, так и снижения издержек про‑ изводства, соответствующие графики могут быть представлены в качестве прямых ли‑ ний. (Это согласуется с эмпирическими данными о росте средней дальности пере‑ возок в крупнейших железнодорожных системах мира за более чем вековой пери‑ од [23, с. 148–149]). Рис. 3. Изменение рыночных р характеристик под влиянием снижения анспортных издержек в долгосрочном периоде. На последний момент надо обратить особое внимание. Мачерет Д. А. Теоретическое осмысление роли транспорта в обеспечении долгосрочного экономического развития • МИР ТРАНСПОРТА, том 18, № 4, С. 6–33 (2020) ТЕОРЕТИЧЕСКАЯ МОДЕЛЬ ВЛИЯНИЯ ТРАНСПОРТНЫХ ИЗДЕРЖЕК НА ПРОИЗВОДСТВО И СБЫТ ТОВАРОВ В краткосрочном перио‑ де расширение вывоза товара в другие ре‑ гионы может привести к росту цен и даже сокращению потребления в регионе про‑ изводства. Мы абстрагировались от такой возможности в данной модели, но её сле‑ дует отметить. Соответственно, сокраще‑ ние вывоза товара из-за возникших транс‑ портных или торговых барьеров в кратко‑ срочном периоде приведёт к снижению цен и увеличению потребления в регионе его производства. В связи с этим иногда воз‑ никает иллюзия, прежде всего – в массо‑ вом сознании, что вывоз товара невыгоден потребителям, находящимся в регионе его производства, а ограничения вывоза пой‑ дут им на пользу. Однако, подчёркнем ещё раз, подобные последствия могут возни‑ кать только в краткосрочном периоде. В долгосрочной перспективе расширение вывоза товаров, при условии повышения технико-технологического уровня произ‑ водства и реализации эффекта масштаба, приведёт к снижению цен и увеличению объёмов потребления также и в регионе производства. Поэтому поощрение вывоза товаров в долгосрочной перспективе может быть выгодно не только производителям, но и потребителям в регионе производства. С б ё Таким образом, снижение транспорт‑ ных издержек, учитывающее повышение надёжности и скорости перевозок, являет‑ Следует отметить, что увеличение объё‑ ма и дальности сбыта товаров позволяет • МИР ТРАНСПОРТА, том 18, № 4, С. 6–33 (2020) ся катализатором экономического роста, «запуская» долгосрочные взаимосвязанные процессы снижения производственных и транспортных издержек, роста объёмов, масштаба и эффективности производства и расширения географии сбыта товаров. ся катализатором экономического роста, «запуская» долгосрочные взаимосвязанные процессы снижения производственных и транспортных издержек, роста объёмов, масштаба и эффективности производства и расширения географии сбыта товаров. Рис. 4. Долгосрочная взаимосвязь многоэтапных изменений издержек, объёмов производства и дальности транспортировки. Но что будет происходить в условиях долговременного сохранения высоких транс‑ портных издержек, т.е. ситуации, показанной на рис. 1? В этом случае, в удалённых от места производства регионах откроется воз‑ можность организации прибыльного вы пуска данного товара или его заменителя более низкого качества («эрзац-продукта») даже при значительно более высоком уровне производственных издержек, чем в регионе изначального производства, но при более низкой, по сравнению с привозным товаром, цене. В результате потребители в удалённых регионах получат стимул переключиться на потребление товаров-заменителей местного производства, конкурентоспособность кото‑ рых обеспечивается единственным факто‑ ром – дороговизной привозного товара. При этом экономические ресурсы, неэффектив‑ но затрачиваемые на производство товаров- заменителей, будут отвлекаться от производ‑ ства тех товаров, в которых данные регионы имеют сравнительное преимущество, и ко‑ торые было бы целесообразно производить не только для внутреннего потребления, но и для вывоза в другие регионы. ТЕОРЕТИЧЕСКАЯ МОДЕЛЬ ВЛИЯНИЯ ТРАНСПОРТНЫХ ИЗДЕРЖЕК НА ПРОИЗВОДСТВО И СБЫТ ТОВАРОВ Следователь‑ но, будет сдерживаться региональная спе‑ циализация, являющаяся весьма значимым фактором экономического роста. В таких условиях не будет достаточных стимулов для роста масштабов и технико-технологического уровня производства в «Регионе 0», а в отда‑ лённых регионах производство может закон‑ сервироваться на ещё более низком уровне технологии и эффективности. Рис. 4. Долгосрочная взаимосвязь многоэтапных изменений издержек, объёмов производства и дальности транспортировки. лённых регионах, с другой стороны (иными словами, чем выше транспортная состав‑ ляющая – отношение транспортных издер‑ жек к издержкам производства или цене товара), тем меньше будут географическая зона сбыта товара и экономически оправ‑ данная дальность его транспортировки. Поэтому при высоких транспортных из‑ держках «дешёвые» товары являются не‑ торгуемыми, а для более «дорогих» товаров дальность и объёмы транспортировки и сбыта ограничиваются. лённых регионах, с другой стороны (иными словами, чем выше транспортная состав‑ ляющая – отношение транспортных издер‑ жек к издержкам производства или цене товара), тем меньше будут географическая зона сбыта товара и экономически оправ‑ данная дальность его транспортировки. Поэтому при высоких транспортных из‑ держках «дешёвые» товары являются не‑ торгуемыми, а для более «дорогих» товаров дальность и объёмы транспортировки и сбыта ограничиваются. Чем ближе кривые предложения товара в регионе производства и в удалённых ре‑ гионах сбыта, тем более высоким требова‑ ниям должно соответствовать местное производство этого товара или производ‑ ство товаров-заменителей в удалённых регионах, чтобы быть конкурентоспособ‑ ным. Итак, если снижение транспортных издержек является мощным стимулом тех‑ нико-технологического развития и роста экономической эффективности, то высо‑ кие транспортные издержки создают усло‑ вия для консервации техники, технологий и низкой эффективности. (Следует заме‑ тить, что подобным образом действуют и иные барьеры – таможенные и др., пре‑ пятствующие развитию товарообмена). Мачерет Д. А. Теоретическое осмысление роли транспорта в обеспечении долгосрочного экономического развития ЭМПИРИЧЕСКИЕ ДАННЫЕ Дальняя торговля и, соответственно, транспортировка товаров на значительные расстояния существовали уже в глубокой древности [4; 24]. Однако, в силу высоких транспортных издержек, связанных, в том Чем выше транспортные издержки с одной стороны и ниже издержки произ‑ водства товара или его заменителей в уда‑ • МИР ТРАНСПОРТА, том 18, № 4, С. 6–33 (2020) • МИР ТРАНСПОРТА, том 18, № 4, С. 6–33 (2020) Мачерет Д. А. Теоретическое осмысление роли транспорта в обеспечении долгосрочного экономического развития 12 12 в XIII веке и продолжавшейся до начала XVIII века. Коммерческая революция обес‑ печила не только увеличение объёмов и расширение географии торговли, но и стимулировала развитие новых форм и инструментов коммерческой деятельно‑ сти (бухгалтерского учёта, банковской и кредитной систем и др.), увеличение денежного обращения. Она справедливо рассматривается как начало экономиче‑ ского возрождения Европы, качественно новый этап в развитии западноевропей‑ ской экономики, ставший основой изме‑ нения структуры европейского рынка и общества [30]. Стала меняться и структу‑ ра торговли. Так, к XVI веку значительную часть международного товарооборота со‑ ставляли зерно, лес, рыба, вино, соль, ме‑ таллы, ткани и сырьё для текстильной промышленности [11]. числе, с ненадёжностью и даже опасно‑ стью, дальняя торговля осуществлялась преимущественно дорогими (шёлк, пряно‑ сти, драгоценности) и так называемыми неконкурирующими товарами – то есть товарами, дефицитными или вовсе не про‑ изводящимися в импортирующих регио‑ нах, производство которых (или их заме‑ нителей) в достаточном для удовлетворе‑ ния спроса количестве было сложно или невозможно по причине отсутствия необ‑ ходимых ресурсов. 12 12 В тех случаях, когда возможно было наладить местное производство товаров- заменителей, прежде всего – ремесленных изделий, это зачастую происходило, даже если такое производство было мелким и примитивным, а потому – дорогим, а ка‑ чество продукции – ниже, чем привозной [25, с. 164–168]. Разрывы между кривыми предложения в разных регионах, обуслов‑ ленные высокими транспортными издерж‑ ками (как показано на рис. 1), создавали условия для реализации такого подхода. При отсутствии впечатляющего прогресса на транспорте, существенного снижения транспортных издержек не происходило. Об этом свидетельствует сохранение в Рим‑ ской империи на протяжении нескольких веков значительных межрегиональных ценовых различий без заметных изменений [26]. Высокие транспортные издержки стали там существенным препятствием для укрупнения производства и внедрения инноваций, что в итоге заблокировало возможность осуществления в тот период промышленной революции [15, с. 160– 162]. Мачерет Д. А. Теоретическое осмысление роли транспорта в обеспечении долгосрочного экономического развития • МИР ТРАНСПОРТА, том 18, № 4, С. 6–33 (2020) ЭМПИРИЧЕСКИЕ ДАННЫЕ Это логически следует из разработанной модели и соответствует последовательности событий экономиче‑ ской истории: коммерческая революция и Великие географические открытия пред‑ шествовали революции промышленной. (Более подробно влияние развития транс‑ порта на осуществление индустриализации и вхождение человечества в эпоху совре‑ менного экономического роста описано в работе [15]). [товаров] во всех направлениях, и только тогда производство в свою очередь может начать строиться на основе этих новых условий…» [10, с. 43]. Сближение кривых предложения това‑ ра в разных регионах, описанное в разра‑ ботанной модели, проявляется в снижении ценовых различий между региональными рынками. Например, появление в XIX веке железных дорог и дальнейшее развитие железнодорожной сети в Пруссии суще‑ ственно сократили различия цен на зерно в сельскохозяйственных и промышленных регионах (табл. 2). Это особенно наглядно проявлялось в неурожайные годы: при отсутствии железных дорог соответствую‑ щие различия были очень велики, с разви‑ тием железнодорожного сообщения, кото‑ рое, по сравнению с гужевым транспортом, и удешевляло, и ускоряло доставку товаров в несколько раз, а, кроме того, существен‑ но повышало её надёжность, ценовые разницы последовательно сокращались. А снижение стоимости доставки зерна из США в Англию (на 80 % с 1868 по 1895 гг. [10, с. 59]), благодаря одновременному удешевлению железнодорожных и морских перевозок, привело к почти двукратному удешевлению пшеницы в Англии (табл. 3). При этом в сельскохозяйственных штатах США тенденции к росту цен на пшеницу не наблюдалось. В связи с этим, соглашаясь с тезисом Хелпмана о том, «что торговля между от‑ далёнными партнёрами влияла на эконо‑ мическое развитие, а экономическое раз‑ витие – на торговлю» [5, с. 21], хотелось бы уточнить другое его высказывание: «изме‑ нения в сферах производства и потребле‑ ния существенно влияли на объём торгов‑ ли, изначально низкий, и его последующий рост» [5, с. 21]. Безусловно, торгово- транспортная деятельность зависит от производства, ведь, в конце концов, про‑ дать и перевезти можно только то, что произведено. Но именно возможности выгодного сбыта товаров в удалённые ре‑ гионы, открывающиеся благодаря усовер‑ шенствованию транспорта и снижению транспортных издержек, стимулируют рост объёмов и масштабов производства и меж‑ региональную специализацию, позволяю‑ щую произвести из тех же ресурсов боль‑ ший объём товаров. Это следует из теоре‑ тической модели и подтверждается ходом экономической истории. Как отмечал К. Я. Загорский, «сначала создаются новые пути и средства транспорта, которые от‑ крывают возможности получения и сбыта Из представленной модели следует, что в долгосрочном периоде должно происхо‑ дить общее снижение уровня цен на торгуе‑ мые товары. Этот вывод также подтвержда‑ ется практикой. ЭМПИРИЧЕСКИЕ ДАННЫЕ Рост объёмов и дальности транспорти‑ ровки товаров в ходе коммерческой рево‑ люции и последующей эпохи Великих географических открытий способствовал ускорению экономического роста в XVI– XVIII веках и, прежде всего, в странах, наиболее преуспевавших в международной торговле – Нидерландах и Англии (табл. 1). Тем не менее до создания сети благо‑ устроенных дорог и каналов и появления в начале XIX века парового транспорта прогресс средств сообщения был недоста‑ точен для кардинального снижения транс‑ портных издержек в масштабах мировой экономики. Поэтому, вплоть до XVIII сто‑ летия, торговля между отдалёнными регио‑ нами всё ещё «состояла по большей части из неконкурирующих продуктов» [5, с. 19]. Появление железных дорог и пароходов открыло качественно новые возможности для развития товарообмена – перевозки стали массовыми, регулярными, надёжны‑ ми и относительно дешёвыми. Поэтому «на протяжении XIX в. торговля быстро разви‑ валась отчасти из-за значительного сниже‑ ния транспортных расходов, отчасти из-за подъёма промышленного производства» [5, с. 19]. Разработанная модель свидетель‑ ствует, что это были не просто дополняю‑ щие друг друга, а взаимоподдерживающие факторы, причём снижение транспортных издержек и расширение обмена в резуль‑ тате совершенствования средств транс‑ порта можно в совокупности считать не В Средневековье высокий уровень транспортных издержек сохранялся, что предопределило и сохранение характера дальней торговли, где по-прежнему доми‑ нировали дорогие и неконкурирующие товары: меха, пряности, шёлк, фарфор, чай, серебро, медь и пр. [27]. В позднем Средневековье (в XIV– XV веках), благодаря совершенствованию морского транспорта, началось удешевле‑ ние перевозок, в том числе – за счёт роста их надёжности, что выразилось в суще‑ ственном снижении страховых тарифов [28]. Это способствовало осуществлению в Европе так называемой «коммерческой революции» [29], начавшейся примерно • МИР ТРАНСПОРТА, том 18, № 4, С. 6–33 (2020) • МИР ТРАНСПОРТА, том 18, № 4, С. 6–33 (2020) еоретическое осмысление роли транспорта в обеспечении долгосрочного о развития Таблица 1 Ускорение экономического роста в результате коммерческой революции и Великих географических открытий 1000–1500 гг. 1500–1820 гг. Среднегодовой темп прироста ВВП, % Среднегодовой темп прироста по‑ душевого ВВП, % Среднегодовой темп прироста ВВП, % Среднегодовой темп прироста по‑ душевого ВВП, % Весь мир 0,15 0,05 0,32 0,05 Западная Европа 0,28 0,12 0,40 0,14 Нидерланды 0,35 0,12 0,56 0,28 Великобритания 0,25 0,12 0,80 0,27 Источник: Maddison, 2007 [20]. Ускорение экономического роста в результате коммерческой революции и Великих географических открытий Ускорение экономического роста в результате коммерческой просто катализатором, а «спусковым крюч‑ ком» промышленной революции и форми‑ рования эпохи современного экономиче‑ ского роста. Мачерет Д. А. Теоретическое осмысление роли транспорта в обеспечении долгосрочного экономического развития Таблица 2 Таблица 2 Влияние развития транспортных сообщений на цены зерна в регионах Пруссии в неурожайные годы XIX века (цена ржи в сельскохозяйственных регионах в 1817 году = 100) Неуро‑ жайные годы Цена ржи Цена пшеницы Характеристика железных дорог Сельско‑ хозяй‑ ственные регионы Промыш‑ ленные регионы Разница Сельско‑ хозяй‑ ственные регионы Промыш‑ ленные регионы Разница 1817 100 236 136 171 296 125 Железные дороги отсутствуют 1847 131 178 47 175 227 52 Начальный этап строи‑ тельства железных дорог 1855 141 183 42 201 232 31 Значимая железнодо‑ рожная сеть 1867 128 148 20 180 209 29 Основные экономиче‑ ские центры соединены железными дорогами Источник: расчёты автора по данным [54] 14 Влияние развития транспортных сообщений на цены зерна в регионах Пруссии в неурожайные годы XIX века ц Влияние снижения транспортных издержек на цены пшеницы в Миннесоте (США) и Англии в конце XIX века (цена пшеницы в Миннесоте в 1891–1894 годах = 100) Период времени Миннесота Англия Разница 1871–1874 гг. 118 271 153 1875–1878 гг. 117 231 114 1879–1882 гг. 149 212 63 1883–1886 гг. 104 168 64 1887–1890 гг. 118 150 32 1891–1894 гг. 100 138 38 Влияние снижения транспортных издержек на цены пшеницы в Миннесоте (США) и Англии в конце XIX века (цена пшеницы в Миннесоте в 1891–1894 годах = 100) Таким образом, разработанная теорети‑ ческая модель согласуется с эмпирически‑ ми данными и позволяет лучше понять роль транспорта в экономической истории и обеспечении долгосрочного роста эконо‑ мики. ственное снижение цен на потребитель‑ ские товары длительного использования (доставляемые в значительной степени из Китая) при росте цен на услуги [31]. Ещё одной наглядной иллюстрацией согласованности теоретической модели с эмпирическими данными служит долго‑ срочная динамика объёмов добычи, сред‑ ней дальности транспортировки и реаль‑ ной доходной ставки на железнодорожные перевозки угля 1 в Российской Федерации (рис. 5). Примечательно, что во время гло‑ бального кризиса снижение дальности перевозки угля (в 2008 году) предшество‑ вало падению его добычи (в 2009 году). Это показательный пример воздействия това‑ рообменных процессов, в частности – дальности сбыта товара, на объём произ‑ водства. Мачерет Д. А. Теоретическое осмысление роли транспорта в обеспечении долгосрочного экономического развития ЭМПИРИЧЕСКИЕ ДАННЫЕ Так, в США в течение последних пятидесяти лет индекс цен на товары промышленного производства устойчиво отставал от индекса цен на услу‑ ги. А с начала XXI века наблюдалось суще‑ 13 13 • МИР ТРАНСПОРТА, том 18, № 4, С. 6–33 (2020) • МИР ТРАНСПОРТА, том 18, № 4, С. 6–33 (2020) Мачерет Д. А. Теоретическое осмысление роли транспорта в обеспечении долгосрочного экономического развития 14 ВЫВОДЫ И РЕКОМЕНДАЦИИ Убедившись в «объяснительной силе» разработанной теоретической модели, следует остановиться на тех выводах, кото‑ рые можно сделать на её основе относи‑ тельно желательных для роста экономики направлений развития транспорта. В условиях современной глобальной экономики дальность транспортировки товаров достигла весьма значительного уровня. Тем не менее, существуют резервы дальнейшего роста эффективности эконо‑ мики за счёт повышения как максималь‑ ной, так и средней дальности транспорти‑ ровки. При этом относительно дешёвые товары более чувствительны к явной со‑ ставляющей транспортных издержек – 1 Выбор в качестве примера железнодорожных пере‑ возок угля обусловлен тем, что почти весь добывае‑ мый в России уголь перевозится железнодорожным транспортом, и по показателям железнодорожных перевозок можно судить о сбыте угля в целом. • МИР ТРАНСПОРТА, том 18, № 4, С. 6–33 (2020) • МИР ТРАНСПОРТА, том 18, № 4, С. 6–33 (2020) еоретическое осмысление роли транспорта в обеспечении долгосрочного о развития 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 Добыча угля 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 20 Добыча угля Добыча угля Средняя дальность перевозки угля железнодорожным транспортом Реальная доходная ставка за железнодрожные перевозки угля Рис. 5. Индексы показателей добычи и железнодорожных перевозок угля в Российской Федерации, 2004–2018 гг. (%), 2004 г. = 100 %. Источник: расчёты автора по данным Росстата, ОАО «РЖД». . Индексы показателей добычи и железнодорожных перевозок угля в Российско Рис. 5. Индексы показателей добычи и железнодорожных перевозок угля в Российской Федерации, 2004–2018 гг. (%), 2004 г. = 100 %. Источник: расчёты автора по данным Росстата, ОАО «РЖД». Индексы показателей добычи и железнодорожных перевозок угля в Российско ми для общей величины эксплуатационных затрат факторами [33]. Движение тяжело‑ весных поездов с целью увеличения объё‑ мов и удешевления перевозок массовых грузов (угля, руды и т.п.) активно развива‑ ется на железных дорогах в ряде стран мира (Австралии, Бразилии, Канаде, Ки‑ тае, США, Швеции, ЮАР), в том числе и в России [34]. Модернизация инфра‑ структуры Восточного полигона сети рос‑ сийских железных дорог с целью развития движения тяжеловесных поездов – важная составляющая долгосрочной программы развития ОАО «РЖД» до 2025 года. Её реа‑ лизация позволит улучшить транспортные возможности для отечественных экспортё‑ ров. Источник: расчёты автора по данным О «РЖД». оретическая модель согласуется с лучше понять роль транспорта в олгосрочного роста экономики. ВЫВОДЫ И РЕКОМЕНДАЦИИ иле» разработанной теоретической водах, которые можно сделать на её ля роста экономики направлений провозной плате, т.е. к уровню транспорт‑ ных тарифов. Если морской и трубопро‑ водный транспорт, в силу технологических особенностей, обеспечивают весьма низ‑ кий уровень себестоимости перевозок и тарифов, который позволяет эффективно осуществлять межконтинентальные и трансконтинентальные перевозки даже сырьевых грузов, то уровень себестоимости перевозок и тарифов на железнодорожном и автомобильном транспорте гораздо вы‑ ше. Их снижение позволило бы расширить области сбыта наиболее дешёвого и каче‑ ственного сырья в тех случаях, когда пере‑ возки необходимо осуществлять по суше. Как следует из представленной модели, это имело бы позитивные долгосрочные по‑ следствия. дерации, 2004 2018 гг. (%), 2004 г. 100 % Росстата, ОА Таким образом, разработанная те рическими данными и позволяет омической истории и обеспечении д ВОДЫ И РЕКОМЕНДАЦИИ Убедившись в «объяснительной ли, следует остановиться на тех вы ве относительно желательных дл провозной плате, т.е. к уровню транспорт‑ ных тарифов. Если морской и трубопро‑ водный транспорт, в силу технологических особенностей, обеспечивают весьма низ‑ кий уровень себестоимости перевозок и тарифов, который позволяет эффективно осуществлять межконтинентальные и трансконтинентальные перевозки даже сырьевых грузов, то уровень себестоимости перевозок и тарифов на железнодорожном и автомобильном транспорте гораздо вы‑ ше. Их снижение позволило бы расширить области сбыта наиболее дешёвого и каче‑ ственного сырья в тех случаях, когда пере‑ возки необходимо осуществлять по суше. Как следует из представленной модели, это имело бы позитивные долгосрочные по‑ следствия. дерации, 004 0 8 гг. (%), 004 г. 00 % Росстата, ОА Таким образом, разработанная т рическими данными и позволяет омической истории и обеспечении д ВОДЫ И РЕКОМЕНДАЦИИ Убедившись в «объяснительной ли, следует остановиться на тех вы ве относительно желательных д Чем дороже перевозимый товар, тем выше его чувствительность к неявной со‑ ставляющей транспортных издержек – ущербу от «омертвления» капитала, вопло‑ щённого в перевозимом товаре. Снизить его возможно за счёт кардинального повы‑ шения скорости доставки. Одним из вари‑ антов реализации этой задачи является использование для доставки дорогостоя‑ обальной экономики дальность ьма значительного уровня. Тем не роста эффективности экономики за Ключевым направлением для удешев‑ ления перевозок является повышение веса транспортных средств, так как себестои‑ мость перевозок и вес транспортного сред‑ ства связаны обратной зависимостью [32, с. 253–269]. На железнодорожном транс‑ порте средний вес поезда и доля тяжело‑ весных поездов являются весьма значимы‑ ития транспорта. В условиях современной гл спортировки товаров достигла вес е, существуют резервы дальнейшего 15 ь е а • МИР ТРАНСПОРТА, том 18, № 4, С. Мачерет Д. А. Теоретическое осмысление роли транспорта в обеспечении долгосрочного экономического развития ВЫВОДЫ И РЕКОМЕНДАЦИИ 6–33 (2020) • МИР ТРАНСПОРТА, том 18, № 4, С. 6–33 (2020) Мачерет Д. А. Теоретическое осмысление роли транспорта в обеспечении долгосрочного экономического развития [37, с. 12]. «Постоянный рост расходов на развитие транспортной инфраструктуры… позволит повысить эффективность произ- водства в долгосрочном периоде» [38, с. 52]. Такие расходы «следует рассматривать как вложения в долгосрочный экономический рост» [39, с. 14]. щих товаров высокоскоростного железно‑ дорожного сообщения (со скоростью свыше 200 км/ч), обычно применяемого только для пассажирских перевозок. В настоящее время уже прорабатываются варианты организации трансконтинен‑ тальных высокоскоростных железнодо‑ рожных перевозок товаров, в т.ч. товаров электронной торговли [35]. 16 16 В связи с этим целесообразным пред‑ ставляется участие государства в развитии не только автодорожной, но и железнодо‑ рожной инфраструктуры как для тяжело‑ весного движения, так и для высокоско‑ ростных перевозок. В то же время мировой и российский опыт свидетельствует о том, что наибольший динамизм транспортного строительства обеспечивается при задей‑ ствовании частной инициативы [14; 15; 40–42]. Поэтому ещё более важной задачей является создание привлекательных усло‑ вий для частных инвестиций в транспорт‑ ную инфраструктуру. Таким образом, реализация тяжеловес‑ ных перевозок для относительно дешёвых товаров и высокоскоростных – для доро‑ гостоящих – будет способствовать сниже‑ нию издержек доставки тех и других и, тем самым, росту объёмов мирового производ‑ ства. Осуществление таких перевозок, яв‑ ляясь, безусловно, предпринимательским решением, требует наличия соответствую‑ щей инфраструктуры. Между тем развитие российской транспортной инфраструктуры отстаёт от требований бизнеса. «В настоя‑ щее время очевидна потребность россий‑ ской экономики в развитии и улучшении инфраструктуры», которая может оказать «долгосрочное позитивное влияние на экономический рост», «стать основой для возникновения новых направлений эконо‑ мической деятельности… а также создать предпосылки для активного вовлечения российской экономики в международную торговлю» [36, с. 23]. Таким образом, задачи развития транс‑ портной инфраструктуры должны решать‑ ся государством и бизнесом совместно. Государство за счёт прямого бюджетного и льготного заёмного финансирования, совершенствования тарифной системы, создания условий для привлечения частно‑ го капитала, снятия излишних ограниче‑ ний на использование имущества органи‑ заций транспорта в хозяйственном оборо‑ те должно формировать «каркас» транс‑ портной системы, гармонизировать развитие разных видов транспорта и транс‑ портную обеспеченность регионов, созда‑ вать благоприятные возможности как для внутренних, так и для международных экономических связей. Бизнес должен участвовать в инвестициях в развитие и мо‑ дернизацию инфраструктуры, обеспечи‑ вать обновление подвижного состава за счёт собственного и привлечённого финан‑ сирования [43, с. 20]. Интегрирующим частное и государственное участие вариан‑ том развития транспортной инфраструк‑ туры является модель государственно- частного партнёрства, неплохо зарекомен‑ довавшая себя в мировой практике [44, с. Мачерет Д. А. Теоретическое осмысление роли транспорта в обеспечении долгосрочного экономического развития ВЫВОДЫ И РЕКОМЕНДАЦИИ Проблема воздействия транспорта на окру‑ жающую среду должна решаться системно, с созданием государством соответствую‑ щих экономических, предпочтительно – рыночных, механизмов, позволяющих интернализировать отрицательные экстер‑ налии [45; 47]. Таким образом, ускорение поездок, способствуя как повышению конкуренто‑ способности рынка услуг (а, значит, и сни‑ жению их цен, росту качества и разнооб‑ разия), так и расширению предложения трудовых ресурсов, является важным фактором экономического роста и повы‑ шения благосостояния людей. Правиль‑ ный учёт этих эффектов позволит сделать более объективной экономическую оценку эффективности транспортных проектов в области пассажирских перевозок, а, зна‑ чит, повысить качество инвестиционной деятельности в этой сфере. При этом важ‑ ное значение имеет гармонизация инвести‑ ционной и инновационной активности на транспорте [51]. Инновационные транс‑ портные технологии, открывая новые возможности для удешевления и повыше‑ ния дальности доставки товаров и поездок пассажиров, являются значимым фактором экономического развития. Поэтому эконо‑ мическая политика, поддерживающая их реализацию за счёт как институциональ‑ ных инструментов, так и бюджетных инве‑ стиций в сооружение инновационной транспортной инфраструктуры, будет способствовать повышению динамики и устойчивости роста экономики. Как уже отмечалось выше, развитие транспортных систем и снижение издер‑ жек транспортировки позволили превра‑ тить практически все товары в торгуемые и глобализировать товарные рынки, ре‑ зультатом чего стала устойчивая тенден‑ ция реального удешевления товаров. Ло‑ гично, что в отношении цен услуг наблю‑ дается совершенно иная тенденция, ведь услуги «привязаны» к месту их оказания, и они либо могут перемещаться вместе с производителями услуг (как правило, на относительно небольшие расстояния), либо к местам оказания услуг могут пере‑ мещаться сами клиенты (масштаб таких перемещений также ограничен). Поэтому рынки услуг гораздо менее конкурентны, чем рынки товаров, а, следовательно, бо‑ лее консервативны. Однако и на рынках важнейших, наиболее ценных услуг с раз‑ витием транспорта, прежде всего с повы‑ шением скорости пассажирских перево‑ зок, происходят качественные изменения. Возникает «глобализация предоставления услуг и международная конкуренция за клиентов, когда образовательные и лечеб‑ ные учреждения конкурируют не с сосед‑ ними школами и больницами и даже не с соответствующими заведениями в своей стране, а во всём мире» [48, с. 10]. Ещё ранее глобализировался рынок рекреаци‑ онных услуг. ВЫВОДЫ И РЕКОМЕНДАЦИИ 304–356]. Строительство транспортной инфра‑ структуры отличается высокой капитало‑ ёмкостью и медленной окупаемостью, что делает его малопривлекательным и труд‑ нореализуемым для частного бизнеса. Учитывая, что такая инфраструктура, с од‑ ной стороны, стимулирует экономический рост, а с другой – может использоваться для перевозок множества разных товаров, про‑ изводимых различными отраслями, то есть не оказывает искажающего влияния на структуру производства, государственные инвестиции в её сооружение более оправ‑ даны, чем в какие-либо другие отрасли экономики. Уместно вспомнить, что ещё Адам Смит относил сооружение и поддер‑ жание транспортной инфраструктуры к обязанностям государства [1, с. 675–676]. Следует согласиться с мнением, что в со‑ временных российских условиях «на транс‑ порте… тактически и стратегически оправданы меры государственной поддерж‑ ки инвестиционной деятельности» Обсуждая проблематику дальнейшего роста дальности транспортировки товаров в интересах повышения эффективности экономики, следует отметить, что некото‑ рые уже реализованные варианты встраи‑ • МИР ТРАНСПОРТА, том 18, № 4, С. 6–33 (2020) • МИР ТРАНСПОРТА, том 18, № 4, С. 6–33 (2020) еоретическое осмысление роли транспорта в обеспечении долгосрочного о развития нии скоростей пассажирских перевозок [49]. Рост скоростей пассажирских сооб‑ щений будет также способствовать повы‑ шению мобильности трудовых ресурсов, давая возможность ежедневно преодоле‑ вать «в оба конца» уже не десятки, а сотни километров. Повышение мобильности человеческого капитала, а это – самый ценный ресурс современной экономики [50, с. 44], позволит повысить эффектив‑ ность его использования, способствуя экономическому росту и сглаживанию межрегиональных диспропорций спроса и предложения труда, а также, возможно, смягчению проблемы структурной безра‑ ботицы. вания транспорта в производственные цепочки протяжённостью несколько тысяч километров вызывают критическую реак‑ цию в связи с ростом экологической на‑ грузки [45, с. 204–205]. Не исключено, что она может порождать общественный за‑ прос на запретительные меры со стороны государства в духе борьбы с так называе‑ мыми «излишне дальними» перевозками, которые советские экономисты относили к нерациональным [46, с. 227–233]. При‑ нятие таких мер весьма нежелательно. Проблема воздействия транспорта на окру‑ жающую среду должна решаться системно, с созданием государством соответствую‑ щих экономических, предпочтительно – рыночных, механизмов, позволяющих интернализировать отрицательные экстер‑ налии [45; 47]. вания транспорта в производственные цепочки протяжённостью несколько тысяч километров вызывают критическую реак‑ цию в связи с ростом экологической на‑ грузки [45, с. 204–205]. Не исключено, что она может порождать общественный за‑ прос на запретительные меры со стороны государства в духе борьбы с так называе‑ мыми «излишне дальними» перевозками, которые советские экономисты относили к нерациональным [46, с. 227–233]. При‑ нятие таких мер весьма нежелательно. Мачерет Д. А. Теоретическое осмысление роли транспорта в обеспечении долгосрочного экономического развития ЛИТЕРАТУРА 1. Смит А. Исследование о природе и принципах богатства народов / Пер. с англ. – М.: Эксмо, 2009. – 960 с. 2. Mokyr, J. The Lever of Riches. Technological Creativity and Economic Progress. N.Y., Oxford University Press, 1990, 368 p. Это представляется важным для того, чтобы показать необоснованность проти‑ вопоставления производства и обмена, имеющего глубокие исторические корни [4], и фетишизации производства (прежде всего тяжёлой промышленности), харак‑ терной для марксистского направления экономической мысли, особенно в её со‑ ветском варианте, а сейчас возрождаемой в рамках противопоставляемого «стандарт‑ ной» экономической теории так называе‑ мого «Другого канона экономической на‑ уки». Один из ярких представителей «Другого канона», Эрик Райнерт, напри‑ мер, отмечает, что экономисты «мейнстри‑ ма» «обращаются к теориям, основанным на обмене и торговле, которые не оставля‑ ют места технологиям и новым знаниям», и «путают носитель прогресса (торговлю) с причиной прогресса (технологией)», не‑ обходимо же, чтобы экономическая наука строилась «вокруг производства, а не об‑ мена» [52, с. 81, 88]. 3. Менгер К. Основания политической экономии // В кн.: К. Менгер. Избранные работы / Пер. с нем. – М.: Издательский дом «Территория будущего», 2005. – С. 57–286. 4. Hayek, F. A. The Fatal Conceit. The Errors’ of Socialism. Chicago, The University of Chicago Press, 1988, 194 p. p 5. Хелпман Э. Понимание мировой торговли / Пер. с англ. – М.: Изд-во Института Гайдара, 2017. – 312 с. 5. Хелпман Э. Понимание мировой торговли / Пер. с англ. – М.: Изд-во Института Гайдара, 2017. – 312 с. 6. Рикардо Д. Начала политической экономии и налогового обложения. Избранное / Пер. с англ. – М.: Эксмо, 2009. – 960 с. , 7. Heckscher, E. The Effect of Foreign Trade on the Distribution of Income. Readings in the Theory of International Trade. Philadelphia, Blakiston, 1949, pp. 272–300. 8. Ohlin, B. Interregional and International Trade. The Economic Journal, March 1934, Vol. 44, pp. 95–102. 9. Tinbergen, Y. Shaping the World Economy. N.Y., Twentieth Century Fund, 1962, хviii + 330 p. 10. Загорский К. Я. 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Как Запад стал богатым: экономическое преобразование индустри‑ ального мира / Пер. с англ. ной роли обеих этих сфер в процессе роста экономики, формируя соответствующие рекомендации для экономической поли‑ тики. ной роли обеих этих сфер в процессе роста экономики, формируя соответствующие рекомендации для экономической поли‑ тики. роли транспорта в обеспечении долгосроч‑ ного экономического роста, показывает приоритетность развития транспорта для поступательного увеличения объёмов и эф‑ фективности производства на основе взаи‑ мостимулирующих процессов расширения товарообмена и масштабов производства, повышения технологического уровня про‑ изводственной и транспортной деятельно‑ сти. 18 18 ЗАКЛЮЧЕНИЕ Разработанная в статье теоретическая модель влияния транспортных издержек на производство и сбыт товаров раскрывает экономическую взаимосвязь развития то‑ варообменной и производственной дея‑ тельности, включая его инновационный аспект. 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https://openalex.org/W1968448345	https://europepmc.org/articles/pmc2826780?pdf=render	English	null	Primary Epithelioid Angiosarcoma of the Breast Masquerading as Carcinoma	Current oncology	2,010	cc-by	3,864	C A s E R E P O R T C A s E R E P O R T C A s E R E P O R T 1. INTRODUCTION Sarcomas represent fewer than 1% of primary breast malignancies 1,2. The most commonly reported primary non-phyllodes sarcomas of the breast are angiosarcoma, fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, and leiomyosarcoma 2. Angiosarcomas account for fewer than 0.05% of all malignant mammary tumours 3. ABSTRACT infiltrate the skin only in advanced cases. The grading of the tumour is thought to affect prognosis more in primary angiosarcomas; it has questionable value in prognosticating secondary angiosarcomas 4. Here we report a case of primary epithelioid angiosar- coma (eas) of the breast occurring in a 30-year-old woman. Following fine-needle asspiration cytology (fnac) and tru-cut biopsy, the patient was initially diagnosed with mammary carcinoma and thereafter underwent modified radical mastectomy. Postop- erative histopathologic examination and immunohis- tochemistry revealed a diagnosis of primary epithe- lioid angiosarcoma of the breast. The patient received postoperative radiotherapy to the chest wall and was started on adjuvant thalidomide. Preoperatively, eas can be mistaken for carcinoma because it is difficult to appreciate the typical morphology on fnac or tru-cut biopsy. Indeed, this is an area of potential diagnostic error because, nowadays, neoadjuvant therapy is often instituted after core biopsy of a breast mass. This case is being reported not only for its diagnostic difficulty, but also because of its rarity in English literature. p g g y g Primary epithelioid angiosarcoma (eas) is distinc- tively rare in the breast and was first reported by Weiss and Enzinger in 1982 5. It has been more frequently reported in other sites such as the skin 6, uterus 7, small intestine 8, lung 9, thyroid 10, and central nervous system and orbit 11. About 100 cases of secondary angiosarcoma of the breast after breast-conserving therapy have been reported, but primary eas of the breast is rare 12. After an extensive literature search using key words such as “eas,” “breast,” and “pri- mary” in PubMed, Scopus, Ovid, and IngentaCon- nect, we could identify only 9 cases of primary eas of the breast 13–16.i The striking preoperative difficulty in diagnos- ing these lesions may be attributable to a limitation in cells or tissue yielded by fine-needle aspiration cytology (fnac) and tru-cut biopsy. A misdiagnosis of carcinoma may lead to delay in initiating the aggres- sive management needed, given that angiosarcomas are the most malignant of all breast tumours 17. Breast, primary epithelioid angiosarcoma We were recently confronted with a case of nonmetastatic primary eas of the breast that was diagnosed preoperatively as infiltrating ductal carci- noma. Here we discuss the clinical, pathologic, and immunohistochemical features of the case with an intent to convey the diagnostic dilemmas involved. Primary epithelioid angiosarcoma of the breast masquerading as carcinoma S. Muzumder md,* P. Das md,† M. Kumar md,* S. Bhasker md,* C. Sarkar md,† K. Medhi dm,‡ V.K. Iyer md,† and G.K. Rath md* 2. CASE DESCRIPTION A 30-year-old premenopausal woman presented to a peripheral hospital with a 15-day history of a painless lump in the right breast in which fnac was suggestive of carcinoma. This patient was then referred to the breast cancer clinic at our institute. On physical ex- amination, a lump measuring 6×5 cm was detectable centrally in the right breast. This lump was hard in consistency and not fixed to the underlying structure. Overlying skin was unremarkable. No axillary or Angiosarcomas of the breast are commonly seen secondary to radiation therapy; they are also associ- ated with postoperative lymphedema (Stewart–Treves syndrome). In comparison, primary angiosarcomas are relatively rarer. Secondary angiosarcomas arise from skin and show a pattern of infiltration into breast from skin and subcutaneous tissues. In contrast, primary angiosarcomas arise from mammary tissues and 64 Copyright © 2010 Multimed Inc. Current Oncology—Volume 17, Number 1 MUZUMDER et al. supraclavicular lymph nodes were palpable. The contralateral breast and axilla were normal. major muscle was therefore excised along with tu- mour. The axillary dissection proceeded to the level 3 lymph node, because multiple lymph nodes, the larg- est being 1.5 cm, were present at levels 1 and 2. As initial screening, a fnac was advised, followed by tru-cut biopsy of the lump. A diagnosis of high-grade carcinoma was suggested in both tests. Immunohis- tochemical staining for cytokeratin was positive in tu- mour cells; staining for vimentin was negative. The cells were negative for the estrogen and progesterone receptor immunostains, but 2+ positivity for the human epidermal growth factor receptor [her2/neu (ErbB2)] was noted. Chest radiography, abdominal ultrasonography, and bone scan did not reveal any metastatic disease. 2.1.1 FNAC O i i On aspiration of the lesion, cellular yield was moderate, comprising predominantly single cells with occasional cell clusters. The tumour cells were noted in a back- ground of blood, together with scattered histiocytes and neutrophils. The tumour cells were epithelioid with eccentric nuclei, coarse chromatin. and moderate- to-abundant cytoplasm with an indistinct margin. The nuclear margin was irregular with identifiable nuclear indentation. Fine cytoplasmic vacuolations were identi- fied. Based on morphology, a possibility of high-grade malignancy was considered [Figure 1(a,b)]. Based on the overall preoperative workup, a diagnosis of carcinoma of the right breast T3N0M0 was made. A preoperative mammogram was not done, because right modified radical mastectomy (mrm) followed by adjuvant therapy was planned for the patient and performed in July 2008. Intraoperatively, the lump was close to deep fascia; a cuff of pectoralis figure 1 (a,b) Fine-needle aspiration cytology of the breast lesion showed singly-lying epithelioid cells with irregular pleomorphic nuclei and moderate-to-abundant bluish cytoplasm [Giemsa stain: (a) 200×, (b) 400×]. (c,d) Tru-cut biopsy of the breast showed irregular cords and singly-lying pleomorphic cells with moderate cytoplasm and focal necrosis [hematoxylin and eosin stain: (c) 40×, (d) 100×]. (d, inset) The cells were immunopositive for cytokeratin (40×). howed singly-lying epithelioid cells with irregular pleomorphic nuclei (b) 400×]. (c,d) Tru-cut biopsy of the breast showed irregular cords and crosis [hematoxylin and eosin stain: (c) 40× (d) 100×] (d inset) The figure 1 (a,b) Fine-needle aspiration cytology of the breast lesion showed singly-lying epithelioid cells with irregular pleomorphic nuclei and moderate-to-abundant bluish cytoplasm [Giemsa stain: (a) 200×, (b) 400×]. (c,d) Tru-cut biopsy of the breast showed irregular cords and singly-lying pleomorphic cells with moderate cytoplasm and focal necrosis [hematoxylin and eosin stain: (c) 40×, (d) 100×]. (d, inset) The cells were immunopositive for cytokeratin (40×). 65 Current Oncology—Volume 17, Number 1 2.1.2 Tru-Cut Biopsy 2.1.3 MRM Specimen The gross resected mrm specimen measured 10×11×6 cm. On sectioning, a spongy hemorrhagic tumour measuring 7×6×2.8 cm was seen centrally and in the upper outer quadrant. The tumour was close to the pectoralis fascia. Based on a suggestion from the fnac report, a tru-cut biopsy was planned. The biopsy comprised one core showing high-grade malignancy with epithelioid cell morphology. The cells were raggedly infiltrating the fibrocollagenous stroma. Focal hemorrhage was noted, but no definite identifiable vascular lumen was seen. The cells had moderate eosinophilic cytoplasm. Few mitotic figures and areas of focal necrosis were seen. No definite ductal pattern or residual benign ducts were noted in this biopsy [Figure 1(c,d)]. An immunohistochemical panel including cytokeratin (ck); vimentin; estrogen (er), progesterone (pr), and her2/neu receptor proteins; and thyroid transcrip- tion factor was applied. Among these, ck and her2/ neu were positive in tumour cells, and a diagnosis of primary breast carcinoma was given. p Multiple sections showed features of a poorly dif- ferentiated malignant tumour with entrapped benign ductules at the tumour periphery [Figure 2(a)]. The tumour was composed of pleomorphic epithelioid cells, multinucleated giant cells, slit-like vascular spaces, fresh and old hemorrhage, necrosis, and scattered inflammatory cells [Figure 2(b)]. The epi- thelioid cells showed indented hyperchromatic nuclei and a moderate amount of eosinophilic cytoplasm [Figure 2(c)]. Mitotic activity was identified. These cells were seen lining the vascular spaces. figure 2 (a) Low-power photomicrograph of the resected tissue shows an infiltrating malignant tumour entrapping the residual ductules (hematoxylin and eosin stain, 40×). (b) The tumour is highly vascular, comprising many large and slit-like vascular spaces (hematoxylin and eosin stain, 200×). (c) The slit-like spaces are lined by pleomorphic epithelioid malignant cells. Few giant cells are noted (hematoxylin and eosin stain, 400×). (d) The tumour cells are strongly immunopositive for CD31 (200×). (d, inset) Immunostaining for the human epidermal growth factor receptor (her2/neu) was positive in tumour cells (200×). figure 2 (a) Low-power photomicrograph of the resected tissue sho (hematoxylin and eosin stain, 40×). (b) The tumour is highly vascular, eosin stain, 200×). (c) The slit-like spaces are lined by pleomorphic e figure 2 (a) Low-power photomicrograph of the resected tissue shows an infiltrating malignant tumour entrapping the residual ductules (hematoxylin and eosin stain, 40×). (b) The tumour is highly vascular, comprising many large and slit-like vascular spaces (hematoxylin and eosin stain, 200×). MUZUMDER et al. previously 13–16, and 5 cases were mentioned in a clini- copathologic series reported by Nascimento et al. 3 of 49 cases of primary angiosarcoma of breast. The tumour is most common during the third and fourth decades of life. It may present as a small painless lump or even as a large hemorrhagic mass. In advanced cases, there may be skin involvement, ulceration, and bleeding. Table i enumerates the clinical, pathologic, and immunohistochemical features and management of the cases that have been described in detail in the English literature (excluding the cases mentioned by Nascimento et al.). The tumour cells were immunopositive for CD31, and 2+ immunopositivity for her2/neu was noted [Figure 2(d)]. The cells were negative for CD34 and the er and pr immunostains. Overall features of the tumour were compat- ible with an eas of the breast. The overlying skin, nipple, areola, and deep resected margins were free of tumour. All 13 lymph nodes dissected were free of tumour. Postoperative computed tomography imaging of the chest (for ruling out lung metastasis) was normal. Left mammogram showed a normal study. The patient was planned for adjuvant radiotherapy and chemotherapy. She received chest wall telecobalt radiotherapy, 50 Gy in 25 fractions over 5 weeks, us- ing two tangential fields. She tolerated radiotherapy well, with grade 1 skin reactions. Epithelioid angiosarcoma is composed predomi- nantly or exclusively of large, rounded “epithelioid” endothelial cells with abundant amphophilic or eosinophilic cytoplasm and large vesicular nuclei. Initial preoperative incisional biopsy can lead to a misdiagnosis of ductal carcinoma because of similar histopathology. Both diseases can show solid sheets of polygonal cells with intracytoplasmic clear spaces or vacuoles. Only primary eas shows slit-like vascular spaces, which are lined with pleomorphic epithelioid malignant cells. The differential diagnosis of primary eas of the breast includes ductal carcinoma and other poorly differentiated sarcomas 19. Because the role of chemotherapy is not well defined in this disease, the patient was started on thalidomide 100 mg daily based on previous experi- ence at our institute 18. She developed asymptomatic rashes over her thighs, which resolved spontaneously. She is clinically disease free at 11 months after sur- gery. This patient has been receiving thalidomide for 10 months and is tolerating it well. We are planning to continue thalidomide till disease recurrence or intolerance to thalidomide. MUZUMDER et al. In our case, the diagnosis was missed on the fnac and tru-cut biopsies, and eas of breast is known to be able to mimic a high-grade carcinoma in a fnac smear or tru-cut biopsy core. Although the cells in our case had nuclear indentation, the absence of cytoplasmic vacuolations, vesicular nuclei, and identifiable vas- cular channels in the tru-cut biopsy core led us away from the correct diagnosis 20,21. 2.1.2 Tru-Cut Biopsy (c) The slit-like spaces are lined by pleomorphic epithelioid malignant cells. Few giant cells are noted (hematoxylin and eosin stain, 400×). (d) The tumour cells are strongly immunopositive for CD31 (200×). (d, inset) Immunostaining for the human epidermal growth factor receptor (her2/neu) was positive in tumour cells (200×). figure 2 (a) Low-power photomicrograph of the resected tissue shows an infiltrating malignant tumour entrapping the residual ductules (hematoxylin and eosin stain, 40×). (b) The tumour is highly vascular, comprising many large and slit-like vascular spaces (hematoxylin and eosin stain, 200×). (c) The slit-like spaces are lined by pleomorphic epithelioid malignant cells. Few giant cells are noted (hematoxylin and eosin stain, 400×). (d) The tumour cells are strongly immunopositive for CD31 (200×). (d, inset) Immunostaining for the human epidermal growth factor receptor (her2/neu) was positive in tumour cells (200×). 66 Current Oncology—Volume 17, Number 1 a Excludes cases mentioned by Nascimento et al., 2008. 3 F = female; M = male; nr = not reported; her2/neu = human epidermal growth factor receptor; er = estrogen receptor; pr = progesterone receptor. PRIMARY EPITHELIOID ANGIOSARCOMA OF THE BREAST Immunohistochemistry is an important adjunc- tive procedure in the diagnosis of angiosarcoma— particularly for poorly differentiated forms in which vascular channel formation is difficult to identify. Angiosarcomas express (to a greater or lesser de- gree) the usual vascular antigens, including von Willebrand factor, CD31, and CD34. Although von Willebrand factor is the most specific of the vascular markers, it is also the least sensitive, often present in a few angiosarcomas as weak focal staining. On the other hand, CD31 combines both relative speci- ficity with excellent sensitivity, and it is positive in approximately 90% of angiosarcomas of all types. Cytokeratin is present in about one third of soft-tissue angiosarcomas, particularly the epithelioid subtype, reflecting the fact that ck cannot be used as an absolute discriminant between angiosarcoma and carcinoma. Epithelioid angiosarcoma is a variant that is positive for CD31, but it is classically negative for CD34, which is another marker of endothelial differentia- tion. In this case, the cells were negative for vimentin, er, pr, and Bcl2. Many cases express ck along with endothelial markers. The principal significance of those markers is the close resemblance they share with carcinoma. Immunohistochemical overexpres- sion of her2/neu in breast carcinomas is described as a predictor of response to alkylating agents or anthracycline therapies; however, its definite role in primary eas of breast remains to be explored 22. In our case, 2+ immunopositivity for her2/neu was seen in tumour cells from both the tru-cut biopsy and the postoperative specimen. recurrence rates 23. Axillary lymph node dissection is not recommended, because the involvement of lymph nodes is extremely rare 23. According to the series reported by Nascimento et al., primary angiosarcoma of breast showed a high rate of metastasis and mor- tality regardless of tumour grade 3. In that series, the authors reported no subset analysis with respect to the epithelioid variant of angiosarcoma, of which 5 cases were seen. The most common sites of metastasis are lung, bone, liver, and skin. The roles of adjuvant radiotherapy and chemotherapy are not well defined in primary angiosarcoma of breast. Primary eas of the breast, a rare variant of an- giosarcoma, is a highly aggressive malignancy as- sociated with poor survival. In the 4 reported cases, 1 developed local recurrence, 1 developed axillary recurrence, and the remaining 2 developed distant metastasis. 3. DISCUSSION Epithelioid angiosarcoma is a rare variant of angiosar- coma described in various sites. Only 4 individual cases of primary eas of breast have been described table i Cases of primary epithelioid angiosarcoma of the breasta table i Cases of primary epithelioid angiosarcoma of the breasta Reference Patient Treatment Immunohistochemistry Outcome Age Sex [positive (+), negative (–)] (years) Martinez et al., 1997 13 26 F Modified radical mastectomy, postoperative radiotherapy (4860 cGy), doxorubicin and dacarbazine Vimentin+, factor viii+, CD31+, cytokeratin– Alive at 7 months, with local recurrence Farina et al., 2003 14 49 F Modified radical mastectomy, no adjuvant Vimentin+, factor viii+, CD31+, CD34+, cytokeratin– Died at 15 months of metastasis Carter et al., 2005 15 33 F Simple mastectomy, no adjuvant nr Alive at 7 months, with axillary recurrence Wang et al., 2007 16 20 M Complete excision, no adjuvant Vimentin+, factor viii+, CD31+, CD34+, cytokeratin+ Died at 6 months of metastasis Muzumder et al. (present case) 30 F Modified radical mastectomy, postoperative radiotherapy (5000 cGy), thalidomide Vimentin+, CD31+, CD34–, her2/neu (erbB-2)+, er–, pr–, cytokeratin– Alive at 9 months, free of disease; on thalidomide a Excludes cases mentioned by Nascimento et al., 2008. 3 F = female; M = male; nr = not reported; her2/neu = human epidermal growth factor receptor; er = estrogen receptor; pr = progesterone receptor. 67 Current Oncology—Volume 17, Number 1 4. CONCLUSIONS We report a rare and aggressive case of primary eas of the breast—a disease that can be misdiagnosed as carcinoma because of similar histopathology. An immu- nohistochemical panel including ck, vimentin, CD31, CD34, er, pr, and her2/neu should be used to differenti- ate lesions with a similar histomorphology to reach a final diagnosis. Mastectomy with adjuvant radiotherapy and chemotherapy appears to be the best treatment modality. Thalidomide appears to be a promising drug in the management of angiosarcoma. Exploration of newer agents is warranted to improve survival. p p p An epithelioid morphology can also be found in other vascular tumours that vary considerably in their presentation and behaviour. Low-grade lesions such as epithelioid and spindle-cell hemangioendothelioma, and benign lesions such as epithelioid hemangioma, also appear in the differential diagnosis, as does a pleomorphic carcinoma 21. Epithelioid angiosarcoma is sometimes difficult to distinguish from epithelioid hemangioendothelioma. However, the presence of a solid growth pattern with necrosis and mitotic activity should generally be regarded as a diagnostic clue in favour of epithelioid angiosarcoma 22. The distinction from metastatic carcinoma, melanoma, and proximal- type epithelioid sarcoma is based particularly on immunohistochemistry and a relative rarity of such lesions in this primary site. Epithelioid angiosarcoma may mimic the angiomatous variant of epithelioid sarcoma both in morphology and by the occasional expression of ck. However, angiosarcoma is more pleomorphic and usually expresses CD31 together with factor viii. Differentiating the epithelioid variant of angiosarcoma from the usual angiosarcoma de- pends on typical cell morphology: nuclear indentation and immunonegativity for CD34 stain 22. PRIMARY EPITHELIOID ANGIOSARCOMA OF THE BREAST In our case, chest wall radiotherapy was given in view of the large size of the tumour and the intraoperative finding of deep fascia involvement. Adjuvant thalidomide 100 mg daily was started after surgery, because the benefits of other chemotherapy are doubtful, and a case of complete response to thalidomide in angiosarcoma of the breast has been reported from our institute 18. 5. REFERENCES 1. McGowan TS, Cummings BJ, O’Sullivan B, Catton CN, Miller N, Panzarella T. An analysis of 78 breast sarcoma patients without distant metastases at presentation. Int J Radiat Oncol Biol Phys 2000;46:383–90. 1. McGowan TS, Cummings BJ, O’Sullivan B, Catton CN, Miller N, Panzarella T. An analysis of 78 breast sarcoma patients without distant metastases at presentation. Int J Radiat Oncol Biol Phys 2000;46:383–90. 2. Blanchard DK, Reynolds CA, Grant CS, Donohue JH. Primary nonphylloides breast sarcomas. Am J Surg 2003;186:359–61. 2. Blanchard DK, Reynolds CA, Grant CS, Donohue JH. Primary nonphylloides breast sarcomas. Am J Surg 2003;186:359–61. 3. Nascimento AF, Raut CP, Fletcher CD. Primary angiosar- coma of the breast: clinicopathologic analysis of 49 cases, suggesting that grade is not prognostic. Am J Surg Pathol 2008;32:1896–904. 3. Nascimento AF, Raut CP, Fletcher CD. Primary angiosar- coma of the breast: clinicopathologic analysis of 49 cases, suggesting that grade is not prognostic. Am J Surg Pathol 2008;32:1896–904. 4. Billings SD, McKenney JK, Folpe AL, Hardacre MC, Weiss SW. Cutaneous angiosarcoma following breast-conserving surgery and radiation: an analysis of 27 cases. Am J Surg Pathol 2004;28:781–8. 4. Billings SD, McKenney JK, Folpe AL, Hardacre MC, Weiss SW. Cutaneous angiosarcoma following breast-conserving surgery and radiation: an analysis of 27 cases. Am J Surg Pathol 2004;28:781–8. g y The recommended therapy for primary angiosar- coma of the breast is simple mastectomy, because wide excision alone is associated with high local 5. Weiss SW, Enzinger FM. Epithelioid hemangioendothelioma: a vascular tumor often mistaken for a carcinoma. Cancer 1982;50:970–81. 5. Weiss SW, Enzinger FM. Epithelioid hemangioendothelioma: a vascular tumor often mistaken for a carcinoma. Cancer 1982;50:970–81. 68 Current Oncology—Volume 17, Number 1 MUZUMDER et al. 6. Mobini N. Cutaneous epithelioid angiosarcoma: a neo- plasm with potential pitfalls in diagnosis. J Cutan Pathol 2009;36:362–9. 17. McDivitt RW, Stewart FW, Berg JW. Tumors of the breast. In: Atlas of Tumor Pathology. Fascicle 2. 2nd series. Washington, DC: Armed Forces Institute of Pathology; 1966. 7. Olawaiye AB, Morgan JA, Goodman A, Fuller AF Jr, Penson RT. Epithelioid angiosarcoma of the uterus: a review of man- agement. Arch Gynecol Obstet 2008;278:401–4. 18. Raina V, Sengar M, Shukla NK, et al. Complete response from thalidomide in angiosarcoma after treatment of breast cancer. J Clin Oncol 2007;25:900–1. 19. Brodie C, Provenzano E. Vascular proliferations of the breast. Histopathology 2008;52:30–44. 8. Al Ali J, Ko HH, Owen D, Steinbrecher UP. 5. REFERENCES Epithelioid angiosarcoma of the small bowel. Gastrointest Endosc 2006;64:1018–21. 20. Siddaraju N, Soundararaghavan J, Bundele MM, Roy SK. Fine needle aspiration cytology of epithelioid angiosarcoma: a case report. Acta Cytol 2008;52:109–13. 9. Pandit SA, Fiedler PN, Westcott JL. Primary angiosarcoma of the lung. Ann Diagn Pathol 2005;9:302–4. 10. Fulciniti F, Di Mattia D, Bove P, et al. Fine needle aspiration of metastatic epithelioid angiosarcoma: a report of 2 cases. Acta Cytol 2008;52:612–18. 21. Gagner JP, Yim JH, Yang GC. Fine-needle aspiration cytology of epithelioid angiosarcoma: a diagnostic dilemma. Diagn Cytopathol 2005;33:429–33. 11. Fernandes AL, Ratilal B, Mafra M, Magalhaes C. Aggressive intracranial and extra-cranial epithelioid hemangioendothe- lioma: a case report and review of the literature. Neuropathol- ogy 2006;26:201–5. 22. Ellis IO, Schnitt SJ, Sastre–Garau X, et al. Pathology and genet- ics of tumors of breast and female genital organs. In: Tavassoli FA, Devilee P, eds. World Health Organization Classification of Tumors. Lyon, France: IARC Press; 2003: 58. 12. West JG, Qureshi A, West JE, et al. Risk of angiosarcoma following breast conservation: a clinical alert. Breast J 2005;11:115–23. 23. Chen KT, Kirkegaard DD, Bocian JJ. Angiosarcoma of the breast. Cancer 1980;46:368–71. 23. Chen KT, Kirkegaard DD, Bocian JJ. Angiosarcoma of the breast. Cancer 1980;46:368–71. 13. Macías–Martínez V, Murrieta–Tiburcio L, Molina–Cárdenas H, Domínguez–Malagón H. Epithelioid angiosarcoma of the breast. Clinicopathological, immunohistochemical, and ultrastructural study of a case. Am J Surg Pathol 1997;21:599–604. Correspondence to: Sandeep Muzumder, Department of Radiotherapy, Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi 110029 India. 14. Fariña MC, Casado V, Renedo G, Estévez L, Martín L, Requena L. Epithelioid angiosarcoma of the breast involving the skin: a highly aggressive neoplasm readily mistaken for mammary carcinoma. J Cutan Pathol 2003;30:152–6. E-mail: sandeepradonc@hotmail.com * Department of Radiotherapy, Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi, India. 15. Carter E, Ulusarac O, Dyess DL. Axillary lymph node involve- ment in primary epithelioid angiosarcoma of the breast. Breast J 2005;11:219–20. , , † Department of Pathology, All India Institute of Medical Sciences, New Delhi, India. 16. Wang ZS, Zhan N, Xiong CL, Li H. Primary epithelioid an- giosarcoma of the male breast: report of a case. Surg Today 2007;37:782–6. , , ‡ Department of Medical Oncology, All India In- stitute of Medical Sciences, New Delhi, India. ‡ Department of Medical Oncology, All India In- stitute of Medical Sciences, New Delhi, India. 5. REFERENCES 69 Current Oncology—Volume 17, Number 1
https://openalex.org/W3020843544	https://docusalut.com/bitstream/20.500.13003/10809/1/000539246000069.pdf	English	null	CUL4A, ERCC5, and ERCC1 as Predictive Factors for Trabectedin Efficacy in Advanced Soft Tissue Sarcomas (STS): A Spanish Group for Sarcoma Research (GEIS) Study	Cancers	2,020	cc-by	8,911	Article CUL4A, ERCC5, and ERCC1 as Predictive Factors for Trabectedin Eﬃcacy in Advanced Soft Tissue Sarcomas (STS): A Spanish Group for Sarcoma Research (GEIS) Study David S. Moura 1 , Paloma Sanchez-Bustos 1, Antonio Fernandez-Serra 2, María Lopez-Alvarez 1, José L. Mondaza-Hernandez 1, Elena Blanco-Alcaina 1, Angela Gavilan-Naranjo 1, Paula Martinez-Delgado 1, Serena Lacerenza 1, Paloma Santos-Fernandez 1,3, Irene Carrasco-Garcia 1,3 , Samuel Hidalgo-Rios 1, Antonio Gutierrez 4, Rafael Ramos 5, Nadia Hindi 1,3, Miguel Taron 1,6, Jose Antonio Lopez-Guerrero 2,7 and Javier Martin-Broto 1,3,* David S. Moura 1 , Paloma Sanchez-Bustos 1, Antonio Fernandez-Serra 2, María Lopez-Alvarez 1, José L. Mondaza-Hernandez 1, Elena Blanco-Alcaina 1, Angela Gavilan-Naranjo 1, Paula Martinez-Delgado 1, Serena Lacerenza 1, Paloma Santos-Fernandez 1,3, Irene Carrasco-Garcia 1,3 , Samuel Hidalgo-Rios 1, Antonio Gutierrez 4, Rafael Ramos 5, Nadia Hindi 1,3, Miguel Taron 1,6, Jose Antonio Lopez-Guerrero 2,7 and Javier Martin-Broto 1,3,* g Irene Carrasco-Garcia 1,3 , Samuel Hidalgo-Rios 1, Antonio Gutierrez 4, Rafael Ramos 5, Nadia Hindi 1,3, Miguel Taron 1,6, Jose Antonio Lopez-Guerrero 2,7 and Javier Martin-Bro 1 Institute of Biomedicine of Sevilla (IBIS, HUVR, CSIC, Universidad de Sevilla), 41013 Sevilla, Spain; david.moura@usal.es (D.S.M.); sanchezbustospaloma@gmail.com (P.S.-B.); marlopalv4@gmail.com (M.L.-A.); joseluciniomondaza@gmail.com (J.L.M.-H.); elena.blancoalcaina@gmail.com (E.B.-A.); amgavnar@gmail.com (A.G.-N.); paula.mrtnez.delgado@gmail.com (P.M.-D.); serelac@hotmail.it (S.L.); paloma.santos.fdez@gmail.com (P.S.-F.); irenecg1990@gmail.com (I.C.-G.); hidalgosamuel@hotmail.com (S.H.-R.); nhindi@mustbesevilla.org (N.H.); taron.miquel@gmail.com (M.T.) hidalgosamuel@hotmail.com (S.H. R.); nhindi@mustbesevilla.org (N.H.); taron.miquel@gmail.com (M 2 Laboratory of Molecular Biology, Fundación Instituto Valenciano de Oncología, 46009 Valencia, Spain; afernandez@ﬁvo.org (A.F.-S.); jalopez@ﬁvo.org (J.A.L.-G.) 3 Medical Oncology Department, University Hospital Virgen del Rocio, 41013 Sevilla, Spain 4 Hematology Department, University Hospital Son Espases/IdISBa, 07120 Mallorca, Spain; antoniom.gutierrez@ssib.es g 5 Pathology Department, University Hospital Son Espases, 07120 Mallorca, Spain; rafaelf.ramos@ssib.es 5 Pathology Department, University Hospital Son Espases, 07120 Mallorca, Spain; rafaelf.ramos@ssib.es 6 Synlab Diagnosticos Globales SAU, 28108 Madrid, Spain 6 Synlab Diagnosticos Globales SAU, 28108 Madrid, Spain 6 Synlab Diagnosticos Globales SAU, 28108 Madrid, Spain 7 Department of Basic Medical Sciences, School of Medicine, Catholic University of Valencia ‘San Vicent Martir’, 46001 Valencia, Spain * Correspondence: jmartin@mustbesevilla.org; Tel.: +34-95-592-3113 Received: 1 April 2020; Accepted: 27 April 2020; Published: 30 April 2020 cancers cancers cancers cancers cancers 1. Introduction Trabectedin is a tetrahydroisoquinoline alkaloid approved in 2007, for the treatment of adult patients with advanced soft-tissue sarcoma (STS), after failure of anthracyclines and ifosfamide, or patients unsuited to receive these agents. Trabectedin has a wide spectrum of mechanisms of action in the tumor and in the microenvironment. As a DNA-binding agent, it interferes with gene transcription and with the DNA repair machinery, leading to DNA damage accumulation and cell cycle perturbation, with a delay in S phase progression and accumulation of cells in G2 phase [1–3]. More importantly, trabectedin interacts with the nucleotide excision repair (NER) machinery to exert its antitumor activity [4]. Yet and although the mechanisms of action of trabectedin have been comprehensively described, there are only few potential predictive biomarkers of drug activity [5,6]. Few retrospective studies have shown that NER- and homologous recombination (HR)-associated genes could be potential predictive factors for trabectedin eﬃcacy in STS [5,7–9]. Trabectedin seems to bind to speciﬁc triplets of the DNA minor groove, projecting out of the DNA a part of its structure, which probably traps other proteins at the site of adduct such as XPG (ERCC5), forming large ternary complexes. Of note, trabectedin seems to be more active in the context of high levels of expression of NER genes (ERCC1 and ERCC5) and low expression levels of HR genes (BRCA1) [8–10]. NER-deﬁcient cells are generally more resistant to trabectedin treatment [11]. Noteworthy, trabectedin produces DNA double-strand breaks, which associated with the impaired damage repair present in some sarcomas, through HR deﬁciency or BRCAness-like phenotype, leads to the rapid cell death of cancer cells; in a concept known as synthetic lethality [12]. Trabectedin is a tetrahydroisoquinoline alkaloid approved in 2007, for the treatment of adult patients with advanced soft-tissue sarcoma (STS), after failure of anthracyclines and ifosfamide, or patients unsuited to receive these agents. Trabectedin has a wide spectrum of mechanisms of action in the tumor and in the microenvironment. As a DNA-binding agent, it interferes with gene transcription and with the DNA repair machinery, leading to DNA damage accumulation and cell cycle perturbation, with a delay in S phase progression and accumulation of cells in G2 phase [1–3]. More importantly, trabectedin interacts with the nucleotide excision repair (NER) machinery to exert its antitumor activity [4]. Yet and although the mechanisms of action of trabectedin have been comprehensively described, there are only few potential predictive biomarkers of drug activity [5,6]. Received: 1 April 2020; Accepted: 27 April 2020; Published: 30 April 2020 Abstract: A translational study was designed to analyze the expression of nucleotide excision repair (NER) and homologous recombination (HR) genes as potential predictive biomarkers for trabectedin in soft-tissue sarcoma (STS). This study is part of a randomized phase II trial comparing trabectedin plus doxorubicin versus doxorubicin in advanced STS. Gene expression levels were evaluated by qRT-PCR, while CUL4A protein levels were quantiﬁed by immunohistochemistry. Expression levels were correlated with patients’ progression-free survival (PFS) and overall survival (OS). Gene expression was also evaluated in cell lines and correlated with trabectedin sensitivity. In doxorubicin arm and in the whole series, which includes samples from both arms, no signiﬁcant diﬀerences in terms of PFS were observed amongst the analyzed genes. In the group treated with trabectedin plus doxorubicin, the median of PFS was signiﬁcantly longer in cases with CUL4A, ERCC1, or ERCC5 overexpression, while BRCA1 expression did not correlated with PFS. Gene expression had no prognostic inﬂuence in OS. CUL4A protein levels correlated with worse PFS in doxorubicin arm and in the whole series. In cell lines, only overexpression of ERCC1 was signiﬁcantly correlated with trabectedin sensitivity. In conclusion, CUL4A, ERCC5, and mainly ERCC1 acted as predictive factors for trabectedin eﬃcacy in advanced STS. Keywords: trabectedin; ERCC1; CUL4A; predictive biomarkers; soft-tissue sarcoma Keywords: trabectedin; ERCC1; CUL4A; predictive biomarkers; soft-tissue sarcom Cancers 2020, 12, 1128; doi:10.3390/cancers12051128 www.mdpi.com/journal/cancers Cancers 2020, 12, 1128 2 of 14 1. Introduction Few retrospective studies have shown that NER- and homologous recombination (HR)-associated genes could be potential predictive factors for trabectedin eﬃcacy in STS [5,7–9]. Trabectedin seems to bind to speciﬁc triplets of the DNA minor groove, projecting out of the DNA a part of its structure, which probably traps other proteins at the site of adduct such as XPG (ERCC5), forming large ternary complexes. Of note, trabectedin seems to be more active in the context of high levels of expression of NER genes (ERCC1 and ERCC5) and low expression levels of HR genes (BRCA1) [8–10]. NER-deﬁcient cells are generally more resistant to trabectedin treatment [11]. Noteworthy, trabectedin produces DNA double-strand breaks, which associated with the impaired damage repair present in some sarcomas, through HR deﬁciency or BRCAness-like phenotype, leads to the rapid cell death of cancer cells; in a concept known as synthetic lethality [12]. Moreover, DNA-damage binding proteins (i.e., DDB1 and DDB2), which are known components of NER pathway [13] have been described to be part of the CUL4A ubiquitin ligase complex, suggesting a link between NER and the ubiquitin–proteasome pathway (UPP). CUL4-DDB1 E3-ligase complex seems to regulate the proteolysis of key proteins responsible for DDR [13]. In fact, after DNA damage, DDBs proteins form a complex that targets histones towards their ubiquitination and degradation during NER. This degradation induces chromatin remodeling and the activation of NER pathway, by recruiting XPC-containing complex that recognizes DNA lesions [14,15]. Therefore, the expression of CUL4A could be an indicator of NER pathway integrity and trabectedin eﬃcacy. In line with this, high expression of CUL4A has been associated with trabectedin activity, suggesting that this gene/protein could be a predictive biomarker of drug eﬃcacy [7,16]. The aim of this study was to analyze the expression of NER and HR genes (i.e., ERCC1, ERCC5, and BRCA1), as well as CUL4A, as potential predictive factors of trabectedin eﬃcacy in STS. These genes were selected from bibliography, where they were described as potential predictive biomarkers for trabectedin [7,8,16–18]. This prospective translational analyses were performed as a correlative study within the comparative phase II trial that compared trabectedin plus doxorubicin versus doxorubicin alone as ﬁrst line of advanced STS [19]. 2.1. Demographic and Pathologic Features A cohort of 66 cases, derived from the randomized phase II study of trabectedin and doxorubicin compared with doxorubicin alone as ﬁrst-line treatment in patients with advanced STS, were included in the translational study associated with the clinical trial. All the patients included in the translational study had FFPE tumor samples available, with enough tissue (i.e., derived from surgery) for gene expression analysis. Of those initially included in the clinical trial (n = 113), 47 were removed from the translational study due to the lack of enough FFPE tumor tissue. All the patients were enrolled in the trial between November 2009 and October 2012, at the time of clinical cut-oﬀthe median of follow-up was 13 months. The median age of the subset of patients included in the translational research was 52 years (21–72); 31 (47%) being females and 35 (53%) males. Median tumor size was of 90 mm (2–300 mm). Primary 3 of 14 Cancers 2020, 12, 1128 tumor sites were: Extremities (38.4%), head and neck (3.1%), truck wall (4.6%), retroperitoneum (23.1%), and other sites (30.8%). This translational study includes several subtypes: Leiomyosarcoma (n = 22), liposarcoma (n = 12), undiﬀerentiated pleomorphic sarcoma (UPS; n = 12), ﬁbrosarcoma (n = 4), hemangiopericytoma (n = 3), malignant peripheral nerve sheath tumor (MPNST; n = 3), synovial sarcoma (n = 3), and others (n = 7). Within the clinical trial, 34 patients were included in the control arm (i.e., doxorubicin), whereas 32 were enrolled in the experimental arm (doxorubicin plus trabectedin). Of the 66 cases included in the translational cohort, 12 (18.2%) had speciﬁc chromosomal translocations. There were 62 events of progression and 39 patients eventually died, among the patients included in the translational research. The demographic and clinicopathologic characteristics are represented in Table 1. Table 1. Demographics and clinical-pathologic information (n = 66). 2.1. Demographic and Pathologic Features Median Age (Range) 52 (21–72) Sex: Female 31 (47%) Male 35 (53%) Median tumor Size (mm) (Range) 90 (2–300) Histological Grade: 1 10 (15.6%) 2 18 (28.1%) 3 36 (56.3%) Primary tumor site Extremity 25 (38.4%) Head and neck 2 (3.1%) Trunk wall 3 (4.6%) Retroperitoneum 15 (23.1%) Others 20 (30.8%) Disease type Localized 38 (62.3%) Metastatic 23 (37.7%) Sarcoma subtypes: Leiomyosarcoma 22 (33.3%) Liposarcoma 12 (18.1%) UPS * 12 (18.1%) Fibrosarcoma 4 (6.1%) Haemangiopericytoma 3 (4.6%) MPNST 3 (4.6%) Synovial Sarcoma 3 (4.6%) Others * 7 (10.6%) Experimental Arm Doxorubicin 34 (51.5%) Doxorubicin plus Trabectedin 32 (48.5%) * UPS: Undiﬀerentiated pleomorphic sarcoma; MPNST: Malignant peripheral nerve sheath tumor. * Others: Angiosarcoma (n = 1) and Unclassiﬁed sarcoma (n = 6). Table 1. Demographics and clinical-pathologic information (n = 66). 2.2. Expression of DDR-Associated Genes in STS Samples 2.2. Expression of DDR-Associated Genes in STS Samples Gene expression analysis of 4 genes (BRCA1, CUL4A, ERCC1, and ERCC5), involved in the DNA damage repair (DDR) machinery, was performed in 66 surgical tumor samples. The median absolute expression of BRCA1, CUL4A, ERCC1, and ERCC5 in the whole series and both individual cohorts are shown in Table 2. 4 of 14 Cancers 2020, 12, 1128 Table 2. Gene expression results. Gene Median Expression 1 in Whole Series (Range) Median Expression 1 in Control Arm (Range) Median Expression 1 in Experimental Arm (Range) BRCA1 (n = 64) 0.52 (0.04–3.75) 0.47 (0.08–2.97) 0.59 (0.04–3.75) CUL4A (n = 65) 1.31 (0.10–31.07) 1.20 (0.24–7.79) 1.46 (0.10–31.07) ERCC1 (n = 64) 1.18 (0.11–10.82) 1.14 (0.16–7.70) 1.22 (0.11–10.82) ERCC5 (n = 66) 0.37 (0.01–7.07) 0.37 (0.02–1.45) 0.39 (0.01–7.07) 1 2−∆∆CT, median relative expression. In the whole series, which included both arms of the clinical study, the expression of CUL4A signiﬁcantly correlated with the expression of ERCC1 (ρ = 0.668, p < 0.001) and ERCC5 (ρ = 0.703, p < 0.001). The expression of ERCC1 also signiﬁcantly correlated with ERCC5 expression; unexpectedly, the expression of BRCA1 was also positively correlated with the expression of the other 3 genes—Table S1. 2.3. Association of BRCA1, CUL4A, ERCC1, and ERCC5 with Clinical Outcome .3. Association of BRCA1, CUL4A, ERCC1, and ERCC5 with Clinical Outcome Sixty-six patients were included in the univariate analysis with a median follow-up of 13 months—Table 3. Table 3. Survival analysis in accordance to gene expression. Whole Series 1 Biomarker Median PFS (Months) (95% CI) p Median OS (Months) (95% CI) p BRCA1 (n = 64) 0.902 0.684 Below median (n = 32) 4.60 (0.00–9.22) 22.47 (4.43–40.51) Above median (n = 32) 5.70 (3.02–8.38) 17.47 (12.15–22.78) CUL4A (n = 65) 0.173 0.343 Below median (n = 33) 4.60 (0.25–8.95) 14.03 (4.68–23.39) Above median (n = 32) 5.50 (2.17–8.83) 21.83 (11.62–32.05) ERCC1 (n = 64) 0.696 0.406 Below median (n = 32) 3.73 (0.30–7.23) 17.47 (2.99–31.94) Above median (n = 32) 5.50 (2.87–8.13) 17.97 (10.75–25.18) ERCC5 (n = 66) 0.559 0.593 Below median (n = 33) 4.60 (1.15–8.05) 17.97 (6.89–29.04) Above median (n = 33) 5.97 (1.99–9.94) 17.47 (7.38–27.56) Control Group 2 Biomarker Median PFS (months) (95% CI) p Median OS (months) (95% CI) p BRCA1 (n = 34) 0.642 0.406 Below median (n = 17) 5.43 (1.18–9.69) 8.73 (-) Above median (n = 17) 6.03 (0.12–11.95) 17.97 (11.16–24.77) CUL4A (n = 33) 0.626 0.994 Below median (n = 16) 4.60 (0.00–12.70) - Above median (n = 17) 5.50 (0.97–10.03) 15.10 (7.41–22.79) ERCC1 (n = 32) 0.321 0.871 Below median (n = 16) 6.93 (3.80–10.07) 27.03 (0.00–61.26) Above median (n = 16) 2.53 (0.18–4.89) 13.73 (9.96–17.51) ERCC5 (n = 34) 0.515 0.746 Below median (n = 17) 6.93 (4.78–9.09) - Above median (n = 17) 2.60 (0.00–8.02) 13.73 (9.51–17.96) Table 3. Survival analysis in accordance to gene expression. 5 of 14 Cancers 2020, 12, 1128 Table 3. Cont. 2.3. Association of BRCA1, CUL4A, ERCC1, and ERCC5 with Clinical Outcome Experimental Group 3 Biomarker Median PFS (months) (95% CI) p Median OS (months) (95% CI) p BRCA1 (n = 30) 0.420 0.608 Below median (n = 15) 1.70 (0.00–4.02) 14.23 (13.22–15.24) Above median (n = 15) 5.70 (0.87–10.54) 21.07 (10.37–31.77) CUL4A (n = 32) 0.038 0.059 Below median (n = 16) 1.80 (0.00–3.63) 13.53 (6.25–20.81) Above median (n = 16) 6.53 (0.00–13.39) 22.63 (17.02–28.25) ERCC1 (n = 32) 0.006 0.295 Below median (n = 16) 2.63 (0.41–4.86) 14.03 (5.73–22.34) Above median (n = 16) 8.10 (4.77–11.43) 21.07 (11.32–30.81) ERCC5 (n = 32) 0.039 0.521 Below median (n = 16) 1.70 (1.05–2.35) 13.53 (5.94–21.13) Above median (n = 16) 7.67 (5.64–9.69) 21.07 (15.04–27.09) 1 Whole series: includes all the cases from both arms; 2 Control Group: Doxorubicin; 3 Experimental Group: Doxorubicin plus Trabectedin. The median values were calculated for each gene in the whole series and in each treatment group. Experimental Group 3 1 Whole series: includes all the cases from both arms; 2 Control Group: Doxorubicin; 3 Experimental Group: Doxorubicin plus Trabectedin. The median values were calculated for each gene in the whole series and in each treatment group. In the whole series, the expression of BRCA1, CUL4A, ERCC1, and ERCC5 did not achieve signiﬁcant correlation with PFS and OS. Similar results were observed regarding the control group, which included the patients treated with doxorubicin in monotherapy. In the whole series, the expression of BRCA1, CUL4A, ERCC1, and ERCC5 did not achieve signiﬁcant correlation with PFS and OS. Similar results were observed regarding the control group, which included the patients treated with doxorubicin in monotherapy. Nonetheless, in the experimental group overexpression of CUL4A, ERCC1, and ERCC5 signiﬁcantly correlated with better mPFS. Amongst the transcripts analyzed, high expression of ERCC1 was the most signiﬁcantly associated with longer PFS on trabectedin plus doxorubicin arm (8.10 months (95% CI: 4.77–11.43) vs 2.63 months (95% CI: 0.41–4.86) p = 0.006). Likewise, high expression of ERCC5 (7.67 months (95% CI: 5.64–9.69) vs 1.70 months (95% CI: 1.05–2.35); p = 0.039) and of CUL4A (6.53 months (95% CI: 0.00–13.39) vs 1.80 months (95% CI: 0.00–3.63); p = 0.038) were all associated with better PFS on the experimental group—Table 3 and Figure 1. BRCA1 did not correlate with PFS in this arm—Table 3. 2.3. Association of BRCA1, CUL4A, ERCC1, and ERCC5 with Clinical Outcome Considering these translational variables as continues variables using univariate and univariate COX regression, only ERCC1 was signiﬁcantly associated to PFS (HR: 0.76; 95%CI: 0.61-0.96; p = 0.021). None of these genes were statistically signiﬁcant correlated with OS in the trabectedin plus doxorubicin group; however, a trend was observed in the case of CUL4A, where high expression showed a tendency for better OS (22.63 months (95% CI: 17.02–28.25) vs 13.53 months (95% CI 6.25–20.81); p = 0.059)—As observed in Figure S1. Gene expression was also correlated with clinical variables that were reported to have prognostic value in this study [19]. In the whole series, the histologic grade negatively correlated with the expression of CUL4A (ρ = −0.298; p = 0.017), ERCC1 (ρ = −0.321; p = 0.011) or ERCC5 (ρ = −0.280; p = 0.025). This statistical signiﬁcant negative correlation between histologic grade and CUL4A (ρ = −0.423; p = 0.018) or ERCC1 (ρ = −0.423; p = 0.018) expression was maintained in the experimental group, whereas the negative correlation between histologic grade and ERCC5 (ρ = −0.393; p = 0.024) was only maintained in the doxorubicin arm—Table S2. 6 of 14 ues ed Cancers 2020, 12, 1128 correlate with P bl variables using univariate and univariate COX regression, only ERCC1 was significantly associated to PFS (HR: 0.76; 95%CI: 0.61-0.96; p = 0.021). Figure 1. Prognostic and predictive value of DNA-damage repair genes. Samples were grouped taking into account the median of gene expression. (A) high expression of CUL4A significantly correlated with better progression-free survival (PFS) on trabectedin plus doxorubicin arm (6.53 months (95% CI: 0.00–13.39) vs 1.80 months (95% CI: 0.00–3.63); p = 0.038); (B) high expression of Figure 1. Prognostic and predictive value of DNA-damage repair genes. Samples were grouped taking into account the median of gene expression. (A) high expression of CUL4A signiﬁcantly correlated with better progression-free survival (PFS) on trabectedin plus doxorubicin arm (6.53 months (95% CI: 0.00–13.39) vs 1.80 months (95% CI: 0.00–3.63); p = 0.038); (B) high expression of ERCC1 signiﬁcantly correlated with better (PFS) on trabectedin plus doxorubicin arm (8.10 months (95% CI: 4.77–11.43) vs 2.63 months (95% CI: 0.41–4.86) p = 0.006) and (C) high expression of ERCC5 signiﬁcantly correlated with better PFS on trabectedin plus doxorubicin arm (7.67 months (95% CI: 5.64–9.69) vs 1.70 months (95% CI: 1.05–2.35); p = 0.039). g PFS (HR: 0.76; 95%CI: 0.61-0.96; p = 0.021). Figure 1. 2.3. Association of BRCA1, CUL4A, ERCC1, and ERCC5 with Clinical Outcome Prognostic and predictive value of DNA-damage repair genes. Samples were grouped taking into account the median of gene expression. (A) high expression of CUL4A significantly correlated with better progression-free survival (PFS) on trabectedin plus doxorubicin arm (6.53 months (95% CI: 0.00–13.39) vs 1.80 months (95% CI: 0.00–3.63); p = 0.038); (B) high expression of Figure 1. Prognostic and predictive value of DNA-damage repair genes. Samples were grouped taking into account the median of gene expression. (A) high expression of CUL4A signiﬁcantly correlated with better progression-free survival (PFS) on trabectedin plus doxorubicin arm (6.53 months (95% CI: 0.00–13.39) vs 1.80 months (95% CI: 0.00–3.63); p = 0.038); (B) high expression of ERCC1 signiﬁcantly correlated with better (PFS) on trabectedin plus doxorubicin arm (8.10 months (95% CI: 4.77–11.43) vs 2.63 months (95% CI: 0.41–4.86) p = 0.006) and (C) high expression of ERCC5 signiﬁcantly correlated with better PFS on trabectedin plus doxorubicin arm (7.67 months (95% CI: 5.64–9.69) vs 1.70 months (95% CI: 1.05–2.35); p = 0.039). 2.4. CUL4A Protein Expression Analysis 2.4. CUL4A Protein Expression Analysis CUL4A protein expression was determined in a series of 85 patients, of which 41 cases were negative and 44 cases were positive for CUL4A immunostaining. Among the 85 cases, 44 samples were from patients treated with doxorubicin in monotherapy (n = 22 CUL4A positive and n = 22 CUL4A negative) and 41 samples derived from patients treated with the combination of trabectedin plus doxorubicin (n = 22 CUL4A positive and n = 19 CUL4A negative). An example of CUL4A immunostaining is represented in Figure S2. Demographic and clinicopathologic features of this subset of 85 cases are displayed in the Table S3. Regarding the univariate analysis, positive expression of nuclear CUL4A protein was associated with worse PFS in the whole series: 2.60 months (95% CI: 0.58–4.62) vs 7.03 months (95% CI: 5.03–9.04), p = 0.009; and with worse PFS in doxorubicin arm: 2.53 months (95% CI: 1.12–4.00) vs 7.4 months (95% CI: 4.45–10.35), p = 0.025. On the other hand, CUL4A protein expression did not signiﬁcantly correlate with PFS in the combination arm: 3.40 months (95% CI: 0.83–6.00) vs 5.77 months (95% CI: 4.25–7.28), p = 0.127—Figure 2 and Table S4. Likewise, positive expression of CUL4A protein was signiﬁcantly associated with worse OS, in the whole series: 10.57 months (95% CI: 5.95–15.18) vs 21.07 months (95% CI: 17.70–24.43), p = 0.001; and in doxorubicin arm: 8.73 months (95% CI: 4.62–12.84) vs 27.03 months (95% CI: 16.99–37.08), p = 0.004. Also, a trend was observed for worse OS in the combination arm, when CUL4A protein expression was positive: 14.23 months (95% CI: 5.68–22.79) vs 19.70 months (95% CI: 8.82–30.58), p = 0.176—Figure 2 7 of 14 7 of 14 Cancers 2020, 12, 1128 and Table S4. The expression of CUL4A did not correlate with CUL4A gene expression in our series (Spearman’s ρ = 0.109; p = 0.386). p g p pearman’s ρ = 0.109; p = 0.386). 1 Figure 2. Prognostic and predictive value of CUL4A protein expression. Samples were grouped as CUL4A positive or negative, taking into account the nuclear expression levels evaluated by immunohistochemistry. Antibody: anti-CUL4A polyclonal antibody (1:50, 2699s, Cell Signaling Technology, Danvers, MA, USA). 2.5. In Vitro Correlation between Gene Expression and Trabectedin Sensitivity 2.5. In Vitro Correlation between Gene Expression and Trabectedin Sensitivity 2.5. In Vitro Correlation between Gene Expression and Trabectedin Sensitivity In Vitro Correlation between Gene Expression and Trabectedin Sensitivity The expression of signiﬁcant genes (ERCC1, ERCC5, and CUL4A) and its correlation with trabectedin activity were also an object of study in the pre-clinical context, with the purpose of validating the translational results. In cell lines, the mean absolute levels of ERCC1 were 0.0338 (0.0179–0.0685), of ERCC5 0.0023 (0.0006–0.0068), and of CUL4A 0.0126 (0.046–0.126). The expression levels of each gene, by cell line, are represented in Figure S3. Noteworthy, high expression of ERCC1 was signiﬁcantly correlated with lower trabectedin IC50 values (ρ = −0.964; p < 0.001). Higher expression of CUL4A also showed a correlation trend for lower trabectedin IC50 values (ρ = −0.750; p = 0.052). The expression of ERCC5 was not correlated with trabectedin In Vitro activity in the selected STS cell line panel (ρ = −0.143; p = 0.760)—Table 4. Table 4. Trabectedin IC50 and gene expression levels in soft-tissue sarcoma (STS) cell lines. Table 4. Trabectedin IC50 and gene expression levels in soft-tissue sarcoma (STS) cell lines. Cell line IC50 (pM) CUL4A * ERCC1 * ERCC5 * 93T449 156 0.0046 0.0179 0.0006 AA 107 0.0106 0.0318 0.0007 CP0024 399 0.0099 0.0252 0.0019 HT-1080 148 0.0083 0.0263 0.0022 SK-UT-1 87 0.0323 0.0685 0.0015 SW872 142 0.0124 0.0270 0.0068 SW982 90 0.0103 0.0403 0.0022 Spearman's rank correlation coeﬃcient (ρ) ** CUL4A * ERCC1 * ERCC5 * Trabectedin IC50 −0.750 −0.964 −0.143 p = 0.052 p < 0.001 p = 0.760 * Absolute mean levels (∆CT); ** Spearman’s rank correlation coeﬃcient between trabectedin IC50 and gene expression values. 2.4. CUL4A Protein Expression Analysis In the whole series CUL4A protein expression was associated with worse PFS (A): 2.60 months (95% CI: 0.58–4.62) vs 7.03 months (95% CI: 5.03–9.04), p = 0.009; and (B) and with worse OS (B): 10.57 months (95% CI: 5.95–15.18) vs 21.07 months (95% CI: 17.70–24.43), p = 0.001. In the doxorubicin arm, CUL4A expression was also associated with worse PFS (C): 2.53 months (95% CI: 1.12–4.00) vs 7.4 months (95% CI: 4.45–10.35), p = 0.025 and worse OS (D): 8.73 months (95% CI: 4.62–12.84) vs 27.03 months (95% CI: 16.99–37.08), p = 0.004. In the combination series, CUL4A protein expression did not correlate with PFS (E): 3.40 months (95% CI: 0.83–6.00) vs 5.77 months (95% CI: 4.25–7.28), p = 0.127, nor OS (F): 14.23 months (95% CI: 5.68–22.79) 19.70 months (95% CI: 8.82–30.58), p = 0.176. 1 Figure 2. Prognostic and predictive value of CUL4A protein expression. Samples were grouped as CUL4A positive or negative, taking into account the nuclear expression levels evaluated by immunohistochemistry. Antibody: anti-CUL4A polyclonal antibody (1:50, 2699s, Cell Signaling Technology, Danvers, MA, USA). In the whole series CUL4A protein expression was associated with worse PFS (A): 2.60 months (95% CI: 0.58–4.62) vs 7.03 months (95% CI: 5.03–9.04), p = 0.009; and (B) and with worse OS (B): 10.57 months (95% CI: 5.95–15.18) vs 21.07 months (95% CI: 17.70–24.43), p = 0.001. In the doxorubicin arm, CUL4A expression was also associated with worse PFS (C): 2.53 months (95% CI: 1.12–4.00) vs 7.4 months (95% CI: 4.45–10.35), p = 0.025 and worse OS (D): 8.73 months (95% CI: 4.62–12.84) vs 27.03 months (95% CI: 16.99–37.08), p = 0.004. In the combination series, CUL4A protein expression did not correlate with PFS (E): 3.40 months (95% CI: 0.83–6.00) vs 5.77 months (95% CI: 4.25–7.28), p = 0.127, nor OS (F): 14.23 months (95% CI: 5.68–22.79) 19.70 months (95% CI: 8.82–30.58), p = 0.176. Cancers 2020, 12, 1128 8 of 14 3. Discussion In this prospective study, only the expression levels of ERCC5, CUL4A, and mainly of ERCC1 behaved as predictive biomarkers of trabectedin eﬃcacy, supporting the relationship between DDR-associated genes and trabectedin activity. Our data showed that high expression of ERCC5 and mainly ERCC1, which are key factors in NER pathway, were related with increased anti-tumoral activity of trabectedin (i.e., PFS). Additionally, cell lines with higher levels of ERCC1 were also more sensitive to trabectedin treatment. Meanwhile, ERCC5 levels were not signiﬁcantly correlated with trabectedin activity, in our pre-clinical studies. These results suggest that the overexpression of ERCC1 could be a reliable positive predictive biomarker of trabectedin activity, whereas its low expression may be related with trabectedin-resistance. Indeed, resistance to trabectedin has been reported in human NER-deﬁcient cell lines. Cells lacking functional ERCC1 had a 2- to 8-fold increase in trabectedin IC50 values as compared to the parental cell line. Moreover, the ectopic expression of ERCC1 in NER-deﬁcient cells sensitized them to trabectedin [11]. Besides, high expression levels of ERCC1 and ERCC5 had also been associated with improved PFS on the trabectedin line, in a retrospective series, whereas the levels of BRCA1 were not correlated with the clinical outcome. Similar data was observed in our prospective study [8,20–22]. Yet, another study reported that low levels of BRCA1 expression correlated with statistically signiﬁcant better response to trabectedin [9]. It is worth mentioning that in our series patients were treated with trabectedin at the ﬁrst-line of advance disease, and samples were collected at the time of diagnosis. Hence, this fact would indicate higher reliability since no systemic treatment that could induce changes in gene or protein expression, were given between diagnostic tumor biopsy time and at the baseline of the study. p p g g p y y The high expression of CUL4A was also signiﬁcantly associated with better outcome in patients treated with trabectedin. These results could be related to the fact that CUL4A forms proteolytic complexes with DDBs proteins, which are relevant in the activation of NER mechanism of DNA 9 of 14 Cancers 2020, 12, 1128 repair [13]. Accordingly, and taking into account that trabectedin activity seems to rely at least partially on NER-eﬃciency [7,23,24], our results support that high expression of CUL4A should be associated with improved trabectedin activity. 3. Discussion The potential predictive value of CUL4A has previously been reported in a panel of 10 breast cancer cell lines, where the expression of CUL4A was associated with higher trabectedin sensitivity [16]. Moreover, the downregulation of CUL4A in these cell lines increased resistance to trabectedin. In the same study, lower BRCA1/ERCC5, BRCA1/CUL4A, and XRCC3/CUL4A expression ratios were also associated with trabectedin activity; however, the ratios between BRCA1/ERCC5, BRCA1/CUL4A, or BRCA1/ERCC1 were not correlated with trabectedin activity (data not shown) in our series. Nonetheless, it is important to mention that, in our series, the results of CUL4A mRNA levels were not consistent with the results attained at protein level. High expression levels of CUL4A were associated with better PFS for trabectedin in nontranslocation-related sarcomas [16]; however, protein expression analysis showed an unexpected association between worse PFS and cases with high expression of CUL4A in our series. This result was signiﬁcant in the whole series as well as in the doxorubicin-treated cases, while in the combination group there was also a tendency for worse PFS in samples with high protein levels of CUL4A. Of note, CUL4A has been shown to regulate the expression of ABC eﬄux pumps, more precisely multidrug resistance-associated protein 1 (MRP-1) and P-glycoprotein (P-gp) [25]. These transporters confer doxorubicin-resistance in STS, which might explain the worse PFS in doxorubicin arm associated with high CUL4A protein expression [26,27]. Yet, it is important to note that accurate quantiﬁcation of CUL4A immunostaining was deemed to be diﬃcult, mainly due to the lack of unique epitope and the cross-positivity with CUL4B [28]. Accordingly, CUL4A protein expression data should be carefully interpreted, since the levels of protein expression may represent both cullin E3 ligase scaﬀolding proteins CUL4A and CUL4B. This issue could also justify the diﬀerent prognostic value of CUL4A protein and RNA. Contrariwise, NER pathway seems to be involved in the repair of doxorubicin-induced lesions [29,30], indicating that NER-deﬁcient tumors could be more sensitive to doxorubicin treatment. Nevertheless, our data did not show any association between the expression of NER-associated genes and the clinical outcome of patients treated with doxorubicin. These results could be justiﬁed by clinical and pre-clinical evidence, describing tissue-speciﬁc patterns of DNA repair, which in turn might be related to mutations in DDR-speciﬁc genes [31]. 3. Discussion Hence, the heterogeneity of STS subtypes and of tumor localizations in our series, associated with other genetic factors not explored (i.e., mutational analysis) may impact the reparation of doxorubicin-induced lesions and the correlations taken from this study. Our results also showed a statistically signiﬁcant correlation between histologic grade and gene expression. High expression of CUL4A, ERCC1, and ERCC5 correlated with low histologic grade in the wholes series and this association was also statistically signiﬁcant in the experimental arm, at least for CUL4A and ERCC1. Similar to our data, high expression of CUL4A or ERCC1 had been associated with better outcome in sarcomas [8,16,32,33]; however, and to our knowledge, no correlation between CUL4A or ERCC1 expression levels and histologic grade had been reported in STS. Pre-clinical studies should be performed in sarcomas to address if these genes may play an anti-tumoral role in sarcomas or if they are only relevant in the mechanisms of action of doxorubicin and trabectedin. Moreover, it could be relevant to perform multivariate analysis in series with a higher number of cases and including both clinical (e.g., histologic grade and others) and translational (e.g., CUL4A and ERCC1) variables. This analysis could help validate the predictive value of these genes. In this study, only 2 variables could be considered for multivariate analysis, taking into account the number of cases included in the trabectedin plus doxorubicin arm. Our study has however some limitations that should be taken into account. The most important is the lack of a cohort of cases treated with trabectedin in monotherapy and in which the validation of predictive biomarkers could be performed. Still and to further explore the predictive value of these molecular factors, the expression of DDR-associated genes is being currently evaluated in a separate study, using a series of 301 cases treated with trabectedin in second or further lines of advance 10 of 14 Cancers 2020, 12, 1128 disease [34]; with the limitation that diagnostic specimens in some cases were collected far from the time in which the patients were treated with trabectedin. Results from this study will help understand and validate the data attained in this prospective analysis. Moreover, it is important to notice that the median cut-oﬀvalues used in our study to group expression data might represent a limitation. 4.2. Gene Expression of Tumour Samples One representative formalin-ﬁxed paraﬃn-embedded (FFPE) block was selected from each patient and three sections of 20 µm thick were cut. For the isolation of the mRNA, RecoverAll™Total Nucleic Acid Isolation Kit for FFPE (Ambion, Texas, TX, USA) was used according to manufacturer’s instructions. RNA concentration was measured in a NanoDrop-1000 spectrophotometer (Thermo scientiﬁc, Waltham, MA, USA). One µg of RNA obtained was used to test RNA integrity, by the presence of the 28S and 18S ribosomal bands in a 1% agarose gel electrophoresis stained with ethidium-bromide and visualized under ultraviolet light. Reverse transcription was performed from 200 ng of total RNA using the High Capacity cDNA Reverse Transcription Kit® (Applied Biosystems, Foster City, CA, USA), following manufacturers’ instructions and as described elsewhere [26]. Gene expression was measured by qRT-PCR using the following TaqMan assays on demand (Applied Biosystems, Foster City, CA, USA): BRCA1 (Hs01556190_m1), CUL4A (Hs00757716_m1), ERCC1 (Hs01012159_m1), ERCC5 (Hs01012159_m1) in a 7500 Fast thermocycler (Applied Biosystems). Furthermore, beta-2-microglobulin (Hs99999907_m1) and GAPDH (Hs00266705_m1) were used as housekeeping genes. Expression was then calibrated using a universal human RNA pool (Stratagene, La Jolla, CA, USA) to normalize the relative expression of the genes analyzed following the 2−∆∆Ct method [35]. 4.1. Patients The cases included in this translational study, for gene (n = 66) and protein expression (n = 85) analyses were collected prospectively within the randomized phase II trial of trabectedin and doxorubicin compared with doxorubicin alone as ﬁrst-line treatment in patients with advanced STS All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Illes Balears (EudraCT 2008-008922-55). Patients included in this trial had locally advanced non-resectable or metastatic STS, with measurable disease according to Response Evaluation Criteria in Solid Tumors (RECIST) 1.0. Additional inclusion and exclusion criteria have been previously described [19]. 3. Discussion Other statistical metrics as receiver operating characteristic (ROC) curve could be considered to group continuous variables in future biomarkers studies; mostly in big series of cases, where it will be more reliable to detect a high sensitive and speciﬁc cut-oﬀ, with a meaningful clinical value. For this reason, the data obtained in this study is an interesting exploratory observation, but that should be validated in a bigger independent sample, where a more robust cut-oﬀmay arise. Another limitation is the lack of reliable antibodies for protein expression analysis in paraﬃn tumor samples, which limits the validation of potential biomarkers (e.g., ERCC1 and CUL4A). 4.4. Cell Lines The following STS cell lines were used for ERCC1, ERCC5 and CUL4A gene expression analysis: Liposarcoma cell lines 93T449 (ATCC® CRL-3043™; ATCC, Old Town Manassas, VA, USA) and SW872 (ATCC® HTB-92™; ATCC, Manassas, VA, USA); leiomyosarcoma primary cell lines AA (kindly provided by Dr. Amancio Carnero of the Institute of Biomedicine of Seville, CSIC, US, HUVR; Seville, Spain) and CP0024 (established in Martin-Broto laboratory); SW982 (ATCC® HTB-93™; ATCC) synovial sarcoma cell line; ﬁbrosarcoma cell line HT-1080 (ATCC® CCL-121™; ATCC) and uterine leiomyosarcoma cell line SK-UT-1 (ATCC® HTB-114™; ATCC). HT-1080 and AA cell line were maintained in F-10 medium (GibcoTM, Thermo Fischer Scientiﬁc, Waltham, MA, USA), 93T449 and CP0024 were cultured in RPMI cell medium (GibcoTM), SK-UT-1 was maintained in DMEM cell culture medium (GibcoTM) and both SW872 and SW982 were cultured in Leibovitz’s L-15 Medium (GibcoTM). All the cell culture mediums were supplemented with 10% FBS, and 100 units/mL penicillin (PAA) and 100 µg/mL streptomycin. Cells were checked routinely and test for contamination by Mycoplasma or fungi. All the cells lines were discarded after 2 months and new lines obtained from frozen stocks. 4.6. Gene Expression Determination in Cell Lines Cells were cultured in 10 cm dishes for 48 h, time in which they were harvested for gene expression analysis. Total RNA was isolated by TRIzol® (Invitrogen Corp., Carlsbad, CA, USA)—chloroform method, from all the cell lines, according to the manufacturer’s protocol. One microgram of RNA was submitted to reverse transcription using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems™—Thermo Fischer Scientiﬁc), in the presence of MultiScribe™Reverse Transcriptase and a random primer scheme for initiating cDNA synthesis. The cDNA obtained was ampliﬁed and quantiﬁed by real-time quantitative PCR, using the GoTaq® qPCR Master Mix Kit (Promega). Individual quantiﬁcation of gene expression was performed using the comparative CT method (CT) and the relative expression will be calculated as 2−∆CT. The following assays were used for determine gene expression: ERCC1; ERCC5; CUL4A; and GAPDH. Three biological replicates with three technical replicas each, were performed. Cancers 2020, 12, 1128 Cancers 2020, 12, 1128 Technology, Danvers, MA, USA). Nuclear CUL4A expression was analyzed as negative and positive (positive cases were considered if they displayed staining in at least 5% of cells). CUL4A protein expression was determined in 85 tumor samples, collected at disease onset. Colorectal cancer tissue was used as positive control of CUL4A expression. 4.5. Determination of Trabectedin IC50 Values Cell lines were seeded in 96-well plates and treated separately with increasing concentrations (1 × 10−13 M to 1 × 10−7 M) of trabectedin for 72 h. Cell proliferation was evaluated by MTS assay (Promega, Madison, WI, USA) and the concentrations that inhibit 50% of cell growth (IC50) were determined using nonlinear regression in Prism 5.0 (GraphPad Software; San Diego, CA, USA). 4.3. Immunohistochemistry Two or three representative areas (1 mm in diameter) of each tumor were selected for tissue microarray (TMA) production by ﬁrst examining the hematoxylin and eosin-stained tumor slide and then sampling the tissue from the corresponding paraﬃn blocks. A TMA instrument (Beecher Instruments; Sun Prairie, WI, USA) was used for TMA assembly. Immunohistochemistry was performed in TMAs 4-µm sections, using an anti-CUL4A polyclonal antibody (1:50, 2699s, Cell Signaling 11 of 14 5. Conclusions High expression levels of ERCC1, CUL4A, and ERCC5 seem to be predictive biomarkers of trabectedin activity and they were associated in our series with longer PFS for trabectedin in advanced STS. The results showed in this study support the importance of NER-eﬃciency on the mechanism of action of trabectedin, while it opens new roads for further research on the role of CUL4A on the activity of other chemotherapeutic agents in the context of STS. CUL4A is activated, in the DDBs complexes, by NEDD8 and this latter protein seems to be critical in the activation of NER [36]; therefore, the combination of pevonidestat, an inhibitor of NEDD8-activating enzyme (NAE) that prevents activation of cullin-RING ligases, with doxorubicin, gemcitabine or other chemotherapeutic agents that are active in NER-deﬁcient conditions should be explored in pre-clinical experiments. Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6694/12/5/1128/s1, Table S1: Bivariate correlations of gene expression. (Spearman’s rank correlation coeﬃcient (ρ)), Table S2: Correlation between gene expression and clinical variables, Table S3: Demographics and clinical-pathologic information of the subset of cases included in protein expression analysis (n = 85), Table S4: Univariate analysis taking into account CUL4A protein expression, Figure S1: Overall Survival taking into account the median expression of CUL4A, Figure S2: CUL4A protein expression, Figure S3: ERCC1, ERCC5 and CUL4A expression in soft-tissue sarcoma cell lines. Author Contributions: Conceptualization, J.M.-B. and D.S.M.; Methodology, D.S.M. and J.M.-B.; Validation, D.S.M.; Formal Analysis, D.S.M., A.F., A.G., J.A.L.-G. and J.M.-B.; Investigation, All the authors; Resources, J.M.-B.; Data Curation, D.S.M. and J.M.-B.; Writing—Original Draft Preparation, D.S.M. and J.M.-B.; Writing—Review and Editing, All the authors; Visualization, D.S.M. and J.M.-B.; Supervision, J.M.-B.; Project Administration, D.S.M. and J.M.-B.; Funding Acquisition, J.M.-B. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conﬂicts of Interest: DSM reports institutional research grants from PharmaMar, Eisai, Immix BioPharma, and Novartis outside the submitted work; travel support from PharmaMar, Eisai, Celgene, Bayer, and Pﬁzer. PSB declares institutional research grants from PharmaMar, Eisai, Immix BioPharma, and Novartis outside the submitted work. MLA declares institutional research grants from PharmaMar, Eisai, Immix BioPharma, and Novartis outside the submitted work. JLMH declares institutional research grants from PharmaMar, Eisai, Immix BioPharma, and Novartis outside the submitted work. EBA declares institutional research grants from PharmaMar, Eisai, Immix BioPharma, and Novartis outside the submitted work. 4.7. Statistical Analysis All the categorical variables were reported as relative frequencies (%) and quantitative variables were expressed as median and ranges. OS and PFS were measured from the date of diagnosis (OS) or from the date of initial treatment within the clinical trial (PFS) to the ﬁnal event, and were estimated according to the Kaplan–Meier method. The associations between the variables of interest (i.e., gene expression and clinical outcomes) were performed by the log-rank test. Univariate and multivariate COX regression was carried out with continuous translational variables. All these associations were pre-planned to be performed in the whole series and in each treatment cohort. Correlations among gene expression levels and between IC50 values and In Vitro gene expression were performed using Spearman’s rank correlation coeﬃcient (ρ). All p-values reported were 2-sided, and the statistical 12 of 14 Cancers 2020, 12, 1128 signiﬁcance was deﬁned at p = 0.05. All the statistical procedures were performed with SPSS 22.0 software (IBM, Armonk, NY, USA). 5. Conclusions NH reports grants, personal fees and non-ﬁnancial support from PharmaMar, personal fees from Lilly, grants from Eisai, and grants from Novartis, outside the submitted work. MT is an employee of Synlab Diagnosticos Globales SAU. 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https://openalex.org/W2758824768	https://zenodo.org/records/1420570/files/5.%20Reduction%20of%20amine%20and%20biological%20antioxidants.pdf	English	null	Reduction of amine and biological antioxidants on NOx emissions powered by mango seed biodiesel	Revista Facultad de Ingeniería Universidad de Antioquia	2,017	cc-by-sa	4,808	Reduction of amine and biological antioxidants on NOx emissions powered by mango seed biodiesel s minas y antioxidantes biológicos en las emisiones de NOx en unidades impulsadas por biodiesel d Assistant professor, Department of Mechanical Engineering, MAM College of Engineering. 621105. Trichy, India ARTICLE INFO Received July 30, 2016 Accepted May 09, 2017 ARTICLE INFO Received July 30, 2016 Accepted May 09, 2017 ABSTRACT: This study scrutinizes the influence of amine and biological antioxidants on reduction of NOx emissions in a diesel engine fueled with B100 (100vol.% mango seed methyl ester) and B20 (20 vol.% mango seed methyl ester and 80 vol.% diesel fuel blend). Three amine antioxidants, p-phenylenediamine (PPD), Ethylendiamine (EDA) and N,N’-diphenyl- 1,4-phenylenediamine (DPPD) and three biological antioxidants, dichloromethane (DCM), alpha tocopherol acetate (α-T) and L-ascorbic acid (L-asc.acid) are tested in a kirloskar-make single cylinder four-stroke water cooled diesel engine of 5.9 KW rated power. There are five concentrations used in the antioxidant mixture of biodiesel blends. i.e., 0.005%-m, 0.010%-m, 0.025%-m, 0.05%-m and 0.1%-m. Where, %-m is the molar concentration employed in the antioxidant mixture. Results show that consequential reduction in NOx could be acquired by the accession of antioxidant additive DPPD with the 0.025% concentration from B20 fuel by 15.4% and B100 fuel by 39%. The DPPD additive increased the CO emissions over 7.42% for B100 fuel and 6.44% for B20 fuel. The biological antioxidant DCM exhibits 0.235 g/kWhr for B100 fuel and 0.297 g/kWhr for B20 fuel. Smoke emission is found to have increased with the addition of antioxidants. Slight increase in brake thermal efficiency (0.91%) is accomplished with the addition of antioxidants at full load. The experimental results are compared with analysis of variance and the result is merely the same as to that of experimentation. KEYWORDS Biodiesel, NOx reduction, DPPD, mango seed oil, prompt NO KEYWORDS Biodiesel, NOx reduction, DPPD, mango seed oil, prompt NO RESUMEN: Este estudio analiza la influencia de la amina y algunos antioxidantes biológicos en la reducción de las emisiones de NOx en un motor diesel alimentado con B100 (100% volumen de éster metílico de semillas de mango) y B20 (20% en volumen de semillas de mango y 80% en volumen de mezcla de combustible diesel), Se probaron tres antioxidantes de amina, p-fenilendiamina (PPD), etilendiamina (EDA) y N, N’-difenil-1,4-fenilendiamina (DPPD) y tres antioxidantes biológicos, diclorometano (DCM), acetato de alfa-tocoferol (α-T) y ácido L-ascórbico (L-asc.acid) en un motor diesel kirloskar de cuatro tiempos refrigerado por agua, 5,9 KW de potencia. Biodiesel, reducción de NOx, DPPD, aceite de semillas de mango, NO inmediatos Revista Facultad de Ingeniería, Universidad de Antioquia, No. 84, pp. 46-54, 2017 KEYWORDS Biodiesel, NOx reduction, DPPD, mango seed oil, prompt NO 2.1. Test fuels Mangifera induce is a comrade of the family of Anarcardiaceae and a sort of the genus Mangifera.It is customarily found in India, Pakistan and Philippines. All the antioxidants are weighed employing a high-precision electronic weighing balance and added to assessed quantity of mango seed biodiesel. To make 0.005%-m of antioxidant mixture, 50mg of antioxidant is appended to 1 kg of biodiesel. A 3000 rpm speed mixer is operated to devise a homogenous mixture of fuel and antioxidant. The antioxidants are mixed with B100 and B20 fuels at different concentrations such as 0.005%-m, 0.010%-m, 0.025%-m, 0.05%-m, 0.10%-m (%-m denotes molar concentration) with a constant engine speed of 1800 rpm. In this study, the reference fuel (B20) is produced by splash-blending method, which is the most perennial and cheapest process. The selected blending level is most incessantly used fraction in persistence and meticulous level biodiesel blend. Biodiesel is moderately compatible with diesel fuel and no phase separation is visually perceived in the blends [7]. The majority of the blending is splash blending each of two in the tanker of fuel or the tank of storage where the biodiesel and diesel fuel are pumped concomitantly into the tank [13]. McCormick et al. [6] explored various of tackles for reduction of NOx emissions from biodiesel. They detailed cetane improvers di-tert-butyl peroxide (DTBP) and Ethyl hexyl nitrate (EHN) are effectual for reducing NOx emissions (4%) in B20 blends. On the contrary, increasing fuel density, number of double bonds and quantified iodine number are equalized with increasing NOx emissions. They also accomplished that the properties of cetane number, density and iodine number are eminently correlated with one another. Gan and Ng [7] examined the eventual of antioxidants equallytert-butyl hydro quinone (TBHQ), butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are commingled in assorted concentrations (250,500,750 and 1000 ppm) in B10 and B20 blends of fuel. They examined that BHA is a covenanting antioxidant for reducing CO and NO emissions contemporaneously in the combustion of biodiesel blends. TBHQ is equally adroit of terminating NO formation more at the disbursement of higher level of incomplete combustion. Varatharajan et al. [8] explored the impact of antioxidant additives on NOx emission of jatropha biodiesel in diesel engine. The antioxidant additives α-tocopherol acetate, BHT, p-phenylenediamine, ethylenediamine and L-ascorbic acid are tested on single cylinder diesel engine. 2.1. Test fuels The experimental results exhibited that a 0.025%-m concentration of p-phenylenediamine additive is exquisite as NOx levels are decreased extremely comparable to sole jatropha biodiesel. While, the extension of antioxidants to neat biodiesel increased the CO and HC emissions. Conventionally, an antioxidant is a molecule which impedes the oxidation of other molecules. Free radicals are formed in oxidation and it is the main concern for the NOx generation in biodiesel combustion [9]. Antioxidants are computed to oxidizable organic materials to retard oxidation and to perpetuate the proficient entity of the substrates [10]. Reduction of amine and biological antioxidants on NOx emissions powered by mango seed biodiesel Hay cinco concentraciones usadas en la mezcla antioxidante de mezclas de biodiesel. Es decir, 0,005% -m, 0,010% -m, 0,025% -m, 0,05% -m y 0,1%, valores en los cuales %-m corresponde a la concentración molar empleada en la mezcla antioxidante. Los resultados muestran que la reducción consiguiente de NOx podría ser adquirida por la adhesión de aditivo antioxidante DPPD con la concentración de 0,025% de combustible B20 en un 15,4% y combustible B100 en un 39%. El aditivo DPPD aumentó las emisiones de CO más de 7,42% para el combustible B100 y 6,44% para el combustible B20. El DCM antioxidante biológico exhibe 0,235 g/kWh para combustible B100 y 0,297 g/kWh para combustible B20. Se ha comprobado que la emisión de humo ha aumentado con la adición de antioxidantes. Un ligero incremento en la eficiencia térmica del freno (0,91%) se logra con la adición de antioxidantes a plena carga. Los resultados experimentales se comparan con el análisis de varianza y el resultado es simplemente el mismo que el de la experimentación. * Corresponding author: Velmurugan Kolanjiappan e-mail: sksvel50@gmail.com ISSN 0120-6230 e-ISSN 2422-2844 DOI: 10.17533/udea.redin.n84a06 * Corresponding author: Velmurugan Kolanjiappan e-mail: sksvel50@gmail.com ISSN 0120-6230 DOI: 10.17533/udea.redin.n84a06 DOI: 10.17533/udea.redin.n84a06 46 V. Kolanjiappan; Revista Facultad de Ingeniería, No. 84, pp. 46-54, 2017 Introduction with aromatic amines. These peroxyl free radical generations are the focal source for the greater biodiesel NOx emissions. A free radical is the unpaired electron of a molecule and that monitors the rate of reaction oxidation [11]. Furthermore, antioxidants can reduce free radical disposition by four routes: chain breaking reactions, chelating the transition metal catalysts, scavenging the inaugurating radicals and reducing the combination of reactive radicals [12]. The empirical of this study is to peruse the NOx fuelled in blends of mango seed biodiesel. Antioxidant additives are chosen hinged on the effectiveness, cost and availability using the amended concentration proposed from the subsisting work. Utterly, the antioxidant additives are acquired from Lab chemicals at Chennai, India. The global cogitations for biodiesel fuel use are blooming with crude oil prices extending high levels. Biodiesel is produced from animal fats and vegetable oils by discrete esterification processes. The production of biodiesel is supplemented by 219.5% from 7828.4 millionliters in 2006 to about 17179.09 millionliters in 2009 [1]. Palash et al. [2] scrutinized the impacts of biodiesel fuels on NOx emissions and their reduction commences. They found that biodiesel and its blends have ardent impacts on HC, CO, and PM emissions owing to the NOx influencing factors of this fuel. NOx formation can be delineated by three mechanisms, i.e., thermal NOx, fuel NOx, and prompt NOx. Thermal NOx is the predominant correspondent to NOx emissions from a diesel engine [3]. Thermal NO formation is influenced by higher combustion and peak flame temperatures throughout complete combustion. High combustion temperature which may be above 1800°C, shatters the hefty triple bond of nitrogen molecules and initiates thermal NOx [4]. Mostly, generation of NOx rely on physical and chemical properties of fuel [5]. Table 1 Details of the dynamometer Make SAJ Test plant pvt. Ltd. Rating 11 kW Speed 9000 rpm Torque 50 Nm Type Eddy current Effective radius of arm 0.5 m Experiments are accomplished in a single-cylinder, water- cooled, naturally aspirated direct injection diesel engine of 5.9 KW rated power spanned with an eddy current dynamometer and it is represented in Figure 1. An eddy current dynamometer spanned to the engine is adopted as a loading device. The specification of the dynamometer is given in Table 1. The engine has hemispherical combustion chamber, with overhead valve arrangements completed by push rods and it retains the constant speed of 1800 rpm at all load conditions. Make SAJ Test plant pvt. Ltd. Rating 11 kW Speed 9000 rpm Torque 50 Nm Type Eddy current Effective radius of arm 0.5 m The speed of the engine is shown in the digital meter on the control panel. Engine cooling water inlet and outlet temperature are determined by a thermocouple. The water entailed for engine cooling is supplied from an overhead cooling water tank. The engine is loaded by proposing an electric current into the dynamometer. Fuel consumption is measured with the help of a data acquisition system and sensor. The Fuel consumption is divvied manually with the help of a digital stopwatch and burette. Exhaust emissions are measured with a NDIR (Non-Dispersive Infrared) based AVL Di Gas 444 gas analyser. The analyser afforded a CO computation range from 0 to 20% by volume with a resolution of 0.01%, NOx range of 0 to 5,000 ppm with an aspiration of 1 ppm and HC range of 0 to 20,000 ppm with a resolution of 1 ppm. The details of operating range, accuracy of the smoke meter and gas analyser are given in Table 2. Table 1 Details of the dynamometer Figure 1 Schematic diagram of the experimental set-up Figure 1 Schematic diagram of the experimental set-up Table 2 The operating range, accuracy of the smoke meter and gas analyser Table 2 The operating range, accuracy of the smoke meter and gas analyser Table 2 The operating range, accuracy of the smoke meter and gas analyser Sl.No Instrument Range Accuracy 1 AVL Di Gas analyser CO 0-10 % CO2 0-20 % HC 0-1000ppm NOx 0-500ppm ±0.02 ±0.03 ±20ppm ±10ppm 2 AVL Smoke meter 0-100 opacity in % ±2% 3 Dynamometer controller Load indicator 0-40 Nm ±0.2 Speed indicator 0-2000 rpm ±10 rpm 4 Thermocouple ±1°C 5 Burette for fuel measurement ±0.1 cc 6 Manometer ±1mm 7 Pressure transducer ±0.3 kg 8 Crank angle encoder 0-110 degrees ±1 The exhaust smoke emission is measured by AVL smoke meter. 2.2. Biodiesel production Transesterification of mango seed oil is executed under emanating conditions: 3.82 gram potassium hydroxide (KOH) catalyst per liter of mango seed oil, 5:1 molar ratio of methanol to mango seed oil, 55°C internal reaction temperature and 2 hour reaction time. After 8 hours of transesterification process, the glycerine is detrained in the bottom layer, and the upper layer composed the pure biodiesel. The glycerine is detached from the mixture, and the pure biodiesel is sent to a washing tank. The methyl ester is washed with pure water, which is heated at 55°C to decompose the water and methyl ester and rise the washing efficiency. The washing process is repeated three times as far as the pH value of methyl ester is decreased to 7. Finally, the methyl ester is heated at 100°C to remove any water and residue alcohol beneath the 200mmHg vaccum condition. Occasionally, the biodiesel and concoction of antioxidant fuel conquers the peroxyl free radical dispositions by reaction 47 V. Kolanjiappan; Revista Facultad de Ingeniería, No. 84, pp. 46-54, 2017 3. Results and discussions In this section, oxides of nitrogen, carbon monoxide, hydrocarbon, smoke emission and brake thermal efficiency of tested antioxidants are discussed. Among the five concentrations tested, the best concentration with best antioxidant additive is examined. The impacts of amine and biological antioxidants on NOx reducing exertion for B100 and B20 fuels are shown in the Figures 2 and 3. Temperature plays predominant role in NOx formation. High temperature and high oxygen concentrations ensued in higher formation of NOx [14]. 48 V. Kolanjiappan; Revista Facultad de Ingeniería, No. 84, pp. 46-54, 2017 From Figure 2, at full load, the B100 fuel shows the NOx reducing activity as DPPD (39%), PPD (35.5%), DCM (30.5%), EDA (28%), L-asc.acid (24.5%) and α-T (17.9%) is the lowest. From Figure 3, for B20 fuel, we initiated the higher NOx reduction at DPPD (15.4%) followed by PPD (13.5%), DCM (11.1%), L-asc.acid (8.2%), EDA (6.4%) and α-T (5.5%) is found to be the lowest. DPPD is the most efficacious of the antioxidant examined, relinquished more than 15% decrease in divvied NO emissions at all engine loads. All the antioxidant additives reduced the NO emissions equal to 0.025%-m concentrations and tackle to decrease afar. The same result is perceived by Varatharajan and Cheralathan [15] and he contemplated maximum NO reduction for B20 fuel with DPPD additive is 9.35 for neat biodiesel it is 28.36 with 0.025%-m concentrations. Figure 3 Effect of antioxidant additives on NO emission for B20 fuel Figure 3 Effect of antioxidant additives on NO emission for B20 fuel Figure 2 Effect of antioxidant additives on NO emission for B100 fuel Similar trends are discerned by Dunn [16] and he perceived increased antioxidant agitation at lower loadings (below 1000 ppm) and constant or lowered NO agitation at higher loadings. The possible cause for the antithetical relationship between rate of treatment and quantity of NO reduction is that all the amine based antioxidants comprise nitrogen in its chemical structure and at higher loadings, the profusion antioxidant reacts in oxygen and too provoke NO. The concord of specific NO emission of mango seed methyl ester with the optimum antioxidant additive to B100 fuel is shown in the Figure 4 and for B20 fuel is shown in the Figure 5. For B100 fuel, the NO produced by DPPD additive and neat biodiesel at full load is 1.92 and 3.1 g/kwhr respectively. 3. Results and discussions The corresponding NO emissions for B20 fuel are 1.73 and 2.12 g/kwhr consequently. Fenimore [17] presented the formation of NO through reactions of hydrocarbon radicals with molecular nitrogen and it is rendered in Eqs (1) to (4). Figure 2 Effect of antioxidant additives on NO emission for B100 fuel Figure 4 (a) Brake specific NOx emissions (B100) for experimental work and (b) ANOVA Figure 4 (a) Brake specific NOx emissions (B100) for experimental work and (b) ANOVA 49 V. Kolanjiappan; Revista Facultad de Ingeniería, No. 84, pp. 46-54, 2017 Figure 5 (a) Brake specific NOx emissions (B20) for experimental work and (b) ANOVA Fi 5 ( ) B k ifi NO i i (B2 gure 5 (a) Brake specific NOx emissions (B20) for experimental work and (b) ANOVA CH +N2àHCN + H (1) N + O2àNO + O (2) HCN + OHàCN + H2O (3) CN + O2àNO + O (4) There were five concentrations tested with B20 and B100 fuel. These fuels were mixed with 0.005%-m, 0.010%-m, 0.015%-m and 0.025%-m concentration of tested antioxidants and the best result was indicated in the figures with the best concentration. (4) 3.2. Effects on CO emissions Brezinsky et al. [1] distinguished increased formation of CH radicals in biodiesel combustion. This could be the pre-eminent cognition for increased formation of NOx. Peroxyl radical (HO2) processed by ester is nearly five times more active than the alkyl peroxyl radical commenced by hydrocarbons. The highly reactive ester peroxyl radicals are devastated by them [18]. From the results, it is apparent that more free radicals are formed in biodiesel combustion and prompt NO could play a formidable role in biodiesel NOx emissions. Lin and Lind [19] constituted increased production of peroxyl radicals and reduced array of OH radicals in biodiesel combustion. Figures 6 and 7 show the impact of amine and biological additives on brake specific CO emissions at various loads for B100 and B20 fuels. At full load, the DPPD additive has about 7.42% and 6.44% more CO emissions than the neat biodiesel and B20 fuel subsequently. Addition of antioxidant additives result in the increase of CO emissions in biodiesel, and that can be expounded by the additive reducing the oxidation capability of CO. But, peroxyl (HO2) and hydrogen peroxide (H2O2) radicals are formed during oxidation. Furthermore, these radicals are converted into hydroxyl (OH) radicals by consuming heat inside the combustion chamber. These OH radicals are mostly liable for the conversion of CO into CO2. This reduction in free radicals may have a remarkable influence on the formation of OH radicals and oxidation of CO and HC. Figures 6 and 7 represented the analysis of variance with respect to experimental work and the result was lower CO emissions than experimental work. DPPD additive reacts with peroxyl radical to form primary amine radical and that is very reactive. Moreover, the peroxyl radical reacts with amine radical and compare benzoquinodiamine and nitroxy radical. These reacted products efficiently decoy the free radicals. Figure 4 indicated almost same NOx emissions to experimental work. Figure 6 (a) Variation of CO emission (B100) for experimental work and (b) ANOVA Figure 6 (a) Variation of CO emission (B100) for experimental work and (b) ANOVA 50 V. Kolanjiappan; Revista Facultad de Ingeniería, No. 84, pp. 46-54, 2017 Figure 7 (a) Variation of CO emission (B20) for experimental work and (b) ANOVA Figure 7 (a) Variation of CO emission (B20) for experimental work and (b) ANOVA 3.3. Effects on HC emissions Figures 8 and 9 show the influence of amine and biological antioxidants on brake specific HC emissions at various loads for B100 and B20 fuels. Highest hydrocarbon emissions are recognized with Dichloromethane additive (0.235g/kwhr) for B100 fuel in Figure 8 and for B20 fuel it is 0.297g/kwhr in Figure 9. Lowest hydrocarbon emissions are inspected with ethylenediamine (0.194 g/kwhr) for B100 fuel in Figure 8 and for B20 it is 0.225g/kwhr at full load in Figure 9. It is found that addition of antioxidants led to increase in hydrocarbon emissions at full load. This increase in HC emissions could be owing to the reduction of oxidative free radical generation by antioxidants, and also the poor volatility of mixture and aromatic content which lowers the laity of combustion. Figures 8 and 9 indicated the individual representation of analysis of variance with respect to the experimental work. Figures 10 and 11 show the feature of the exhaust smoke emissions of B100 and B20 fuel containing the effective antioxidant additives. The results revealed that the smoke is almost persistent and imperceptible up to 2.36kw brake power and increased at high loads for all the blends. This is reasonable due to the fact that more fuel is combusted in the diffusion phase at high load than that of at low and intermediate load, leading to the increase of smoke emissions. The amine antioxidant DPPD additive produced lower smoke emissions out of the tested antioxidants and it was compared in analysis of variance in Figures 10 and 11. Figure 8 (a) Variation of HC emission (B100) for experimental work and (b) ANOVA Figure 8 (a) Variation of HC emission (B100) for experimental work and (b) ANOVA 51 V. Kolanjiappan; Revista Facultad de Ingeniería, No. 84, pp. 3.3. Effects on HC emissions 46-54, 2017 Figure 9 (a) Variation of HC emission (B20) for experimental work and (b) ANOVA Figure 9 (a) Variation of HC emission (B20) for experimental work and (b) ANOVA Figure 10 (a) Variation of Smoke emission (B100) for experimental work and (b) ANOVA Figure 11 (a) Variation of Smoke emission (B20) for experimental work and (b) ANOVA Figure 10 (a) Variation of Smoke emission (B100) for experimental work and (b) ANOVA Figure 10 (a) Variation of Smoke emission (B100) for experimental work and (b) ANOVA Figure 10 (a) Variation of Smoke emission (B100) for experimental work and (b) ANOVA Figure 11 (a) Variation of Smoke emission (B20) for experimental work and (b) ANOVA Figure 11 (a) Variation of Smoke emission (B20) for experimental work and (b) ANOVA Figure 11 (a) Variation of Smoke emission (B20) for experimental work and (b) ANO 52 52 V. Kolanjiappan; Revista Facultad de Ingeniería, No. 84, pp. 46-54, 2017 The DPPD additive increased the smoke density by 14.5% and 8.9% for B20 and B100 fuels appropriately at full load. There are many factors bestow to the increase of smoke density with antioxidant addition. The possible causes for the increase of smoke are increase of C-C bonds, reduction of oxygen squeak and increase of aromatic placate owing to the addition of antioxidants with fuels. The DPPD additive increased the smoke density by 14.5% and 8.9% for B20 and B100 fuels appropriately at full load. There are many factors bestow to the increase of smoke density with antioxidant addition. The possible causes for the increase of smoke are increase of C-C bonds, reduction of oxygen squeak and increase of aromatic placate owing to the addition of antioxidants with fuels. efficiencies are vaguely lower excluding the neat biodiesel in Figure 12. At full load, the brake thermal efficiency of DPPD with B20 fuel is 39.2%, but the base B20fuel brake thermal efficiency is 40.1%. The maximum brake thermal efficiency was obtained with DPPD additive with 0.025% molar concentration and the result was merely the same to the experimental with analysis of variance. 4. Conclusion 4. 5. 5. E. İleri and G. Koçar, “Experimental investigation of the effect of fuel injection advance on engine performance and exhaust emission parameters using canola oil methyl ester in a turbocharged direct-injection diesel engine,” Energy Fuels, vol. 23, no. 10, pp. 5191-5198, 2009. This paper has delineated the reputation of amine and biological antioxidants on NOx emissions powered by mango seed biodiesel in a direct injection diesel engine. The following denouements can be drawn hinged on the experimental results. g g pp 6. R. L. McCormick, J. R. Alvarez, and M. S. Graboski, “NOx solutions for biodiesel,” National Renewable Energy Laboratory, Golden, USA, Final Rep. NREL/SR- 510-31465, Feb. 2003. • The antioxidant is effective in controlling NOx emissions. But, they have notably increased the HC, CO and smoke emissions. 7. S. Gan and H. K. Ng, “Effects of antioxidant additives on pollutant formation from the combustion of palm oil methyl ester blends with diesel in a non-pressurised burner,” Energy Conversion and Management, vol. 51, no. 7, pp. 1536-1546, 2010. • Among the examined antioxidants DPPD observed to be efficacious for decreasing in NOx emissions than DEA, PPD, DCM, α-T and L-ascorbic acid. DPPD presented 39% reduction of NOxin B100 fuel and 15.4% in B20 fuel from 0.025%-m concentration. It is detected that NOx reduction is in the order of DPPD>PPD>DCM>L-ascorbic acid>EDA> α-T. pp 8. K. Varatharajan, M. Cheralathan, and R. Velraj, “Mitigation of NOx emissions from a jatropha biodiesel fuelled DI diesel engine using antioxidant additives,” Fuel, vol. 90, no. 8, pp. 2721-2725, 2011. pp 9. H. Sies, “Oxidative stress: oxidants and antioxidants,” Experimental Physiology, vol. 82, no. 2, pp. 291-295, 1997. • It is found that 0.025%-m concentration exhibits the best reduction in NOx among the five concentrations tested. Prompt NO could be the major cognition for biodiesel NOx effect. It is concluded that amine based antioxidants reduce the NOx formation than biological antioxidants for the reason that, the amine based antioxidant DPPD efficiently ambushes the free radicals. 10. D. Chaithongdee, J. Chutmanop, and P. Srinophakun, “Effect of antioxidants and additives on the oxidation stability of jatropha biodiesel,” Kasetsart J (Nat. Sci.), vol. 44, pp. 243-250, 2010. 11. M. Rios, S. N. Santiago, A. Sanders, and S. E. Mazzetto, “Antioxidative Activity of 5-n-Pentadecyl-2-tert- butylphenol Stabilizers in Mineral Lubricant Oil,” Energy Fuels, vol. 24, no. 5, pp. 3285-3291, 2010. 4. Conclusion İ̧ • The DPPD additive increased the CO emissions over 7.42% for B100 fuel and 6.44% for B20 fuel at full load. The use of antioxidant additive with biodiesel fuels lead to a remarkable increase in HC emissions by the biological antioxidant dichloromethane (DCM) and it is owing to the reduction of oxidative free radical generation by antioxidants, and also the poor volatility of mixture and aromatic content which lowers the laity of combustion. For B100 fuel, DCM exhibits 0.235 g/kWhr for B100 fuel and 0.297 g/kWhr for B20 fuel. This is owing to the element that the use of antioxidants in biodiesel hindered the conversion of CO. 12. E. İleri and G. Koçar, “Effects of antioxidant additives on engine performance and exhaust emissions of a diesel engine fueled with canola oil methyl ester-diesel blend,” Energy Conversion and Management, vol. 76, pp. 145-154, 2013. 13. H. Tang et al., “Quality survey of biodiesel blends sold at retail stations,” Fuel, vol. 87, no. 13-14, pp. 2951- 2955, 2008.̇ 14. E. İleri, A. D. Karaoglan, and A. Atmanli, “Response surface methodology based prediction of engine performance and exhaust emissions of a diesel engine fuelled with canola oil methyl ester,” J. Renew. Sust. Energy, vol. 5, no. 3, 2013. • Smoke density is slightly increased with the addition of antioxidants in biodiesel. Brake thermal efficiency is found to be significant. Though, slight increase in brake thermal efficiency (0.91%) is deduced with antioxidant fuel mixture at full load. • Smoke density is slightly increased with the addition of antioxidants in biodiesel. Brake thermal efficiency is found to be significant. Though, slight increase in brake thermal efficiency (0.91%) is deduced with antioxidant fuel mixture at full load. 15. K. Varatharajan and M. Cheralathan, “Effect of aromatic amine antioxidants on NOx emissions from a soybean biodiesel powered DI diesel engine,” Fuel Processing Technology, vol. 106, pp. 526-532, 2013. 3.5. Effects on brake thermal efficiency But, 0.91% increase in brake thermal efficiency is found with amine antioxidant DPPD. Addition of antioxidants to biodiesel fuels provocateur the reaction rate, and hence, engine brake thermal efficiency [20]. This increase in brake thermal efficiency is probably of the increased cylinder pressure with the addition of antioxidants. From the Figure 13, the experimental value is almost equal to the analysis of variance. Figures 12 and 13 indicate the variation of brake thermal efficiency with brake power for B100 and B20 fuel. At part loads, changes in brake thermal efficiencies owing to antioxidant additions are apparent. But, at full load Figure 12 (a) Brake thermal efficiency (B100) for experimental work and (b) ANOVA Figure 12 (a) Brake thermal efficiency (B100) for experimental work and (b) ANOVA Figure 13 (a) Brake thermal efficiency (B20) for experimental work and (b) ANOVA Figure 13 (a) Brake thermal efficiency (B20) for experimental work and (b) ANOVA 53 V. Kolanjiappan; Revista Facultad de Ingeniería, No. 84, pp. 46-54, 2017 Journal of Fuels and Lubricants, vol. 2, no. 1, pp. 789- 816, 2009.̧̇ 5. Refer 5. 16. R. Dunn, “Effect of antioxidants on the oxidative stability of methyl soyate (biodiesel),” Fuel Processing Technology, vol. 86, no. 10, pp. 1071-1085, 2005. 1. S. Garner, R. Sivaramakrishnan, and K. Brezinsky, “The high-pressure pyrolysis of saturated and unsaturated C7hydrocarbons,” Proc. Combust. Inst., vol. 32, no. 1, pp. 461-467, 2009. 17. C. Fenimore, “Formation of nitric oxide in premixed hydrocarbon flames,” Symposium (International) on Combustion, vol. 13, no. 1, pp. 373-380, 1971. pp 18. K. Ryu, “The characteristics of performance and exhaust emissions of a diesel engine using a biodiesel with antioxidants,” Bioresource Technology, vol. 101, no. 1, pp. 78-82, 2010. , 2. S. M. Palash et al., “Impacts of biodiesel combustion on NOx emissions and their reduction approaches,” Renewable and Sustainable Energy Reviews, vol. 23, pp. 473-490, 2013. pp 19. C. Y. Lin and H. A. Lin, “Effects of NOx –inhibitor agent on fuel properties of three-phase biodiesel emulsions,” Fuel Processing Technology, vol. 89, no. 11, pp. 1237- 1242, 2008. , 3. S. Fernando, C. Hall, and S. Jha, “NOx reduction from biodiesel fuels,” Energy Fuels, vol. 20, no. 1, pp. 376- 382, 2006. , 4. C. J. Mueller, A. L. Boehman, and G. C. 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https://openalex.org/W4292834097	https://link.springer.com/content/pdf/10.1007/s00521-022-07673-9.pdf	English	null	Hybrid deep learning diagonal recurrent neural network controller for nonlinear systems	Neural computing & applications	2,022	cc-by	12,641	Abstract In the present paper, a hybrid deep learning diagonal recurrent neural network controller (HDL-DRNNC) is proposed for nonlinear systems. The proposed HDL-DRNNC structure consists of a diagonal recurrent neural network (DRNN), whose initial values can be obtained through deep learning (DL). The DL algorithm, which is used in this study, is a hybrid algorithm that is based on a self-organizing map of the Kohonen procedure and restricted Boltzmann machine. The updating weights of the DRNN of the proposed algorithm are developed using the Lyapunov stability criterion. In this concern, simulation tasks such as disturbance signals and parameter variations are performed on mathematical and physical systems to improve the performance and the robustness of the proposed controller. It is clear from the results that the performance of the proposed controller is better than other existent controllers. Keywords Hybrid deep learning Diagonal recurrent neural network Nonlinear system Lyapunov stability Hybrid deep learning diagonal recurrent neural network controller for nonlinear systems Ahmad M. El-Nagar1 • Ahmad M. Zaki2 • F. A. S. Soliman2 • Mohammad El-Bardini1 Received: 23 June 2021 / Accepted: 25 July 2022 / Published online: 23 August 2022 The Author(s) 2022 Received: 23 June 2021 / Accepted: 25 July 2022 / Published online: 23 August 2022 The Author(s) 2022 Neural Computing and Applications (2022) 34:22367–22386 https://doi.org/10.1007/s00521-022-07673-9 (0123456789().,-volV)(0123456789(). ,- volV) Neural Computing and Applications (2022) 34:22367–22386 https://doi.org/10.1007/s00521-022-07673-9 (0123456789().,-volV)(0123456789(). ,- volV) ORIGINAL ARTICLE & Ahmad M. El-Nagar ahmed.elnagar@el-eng.menoﬁa.edu.eg Ahmad M. Zaki ahmed_zaki889@yahoo.com F. A. S. Soliman fouad.saad.soliman@gmail.com Mohammad El-Bardini dralbardini@el-eng.menoﬁa.edu.eg 1 Department of Industrial Electronics and Control Engineering, Faculty of Electronic Engineering, Menouﬁa University, Menof 32852, Egypt 2 Department of Electronics and Computers Engineering, Nuclear Materials Authority, El-Maadi, 530, Cairo, Egypt 1.2 Motivation It is evident from the literature review that DL applications are widely used for modeling systems and it does not cover the control research. Since nonlinear systems suffer from external disturbances and uncertainties, the main purpose of the present paper is to shed further light on the design of stable controllers for overcoming nonlinear system prob- lems. In this concern, self-organizing map (SOM) is an unsupervised learning algorithm trained using dimension- ality reduction (typically two-dimensional), discretized representation of input space of the training samples, and called a map. It differs from other ANN as it depends on the competitive learning and not the error-correction learning (like backpropagation with gradient descent). It uses a neighborhood function to preserve the topological properties of the input space to reduce data by creating a spatially organized representation, and also, it helps to discover the correlation between data [47–50]. On the other hand, the RBM is an unsupervised learning algorithm that makes inferences from input data without labeled respon- ses. The controllers and models based on NNs are always stuck with the initialized weights. If the initialized weights are not appropriate, the network gets stuck in local minima and leads the training process to a wrong ending and the network becomes infeasible to train therefore. Therefore, RBM is used to overcome this problem [46, 51]. Hence, a Machine learning (ML) is one of the applications of AI that can automatically learn from experience without explicit programming. ML focuses on the development of programs that can access data and use it to learn them [33]. In this signiﬁcance, the deep learning (DL) is a part of a wider family of ML based on ANN’s that learn represen- tations either supervised, unsupervised, or semi-supervised [34]. 1 Introduction control methods are not suitable for controlling nonlinear systems in practical applications [2, 3]. These problems can be overcome by optimal control techniques. Artiﬁcial intelligence (AI)-based controllers are one of these tech- niques, which have many advantages, such as [4, 5]: (1) it can lead to better performance when properly tuned. (2) It requires less tuning effort than non-optimal controllers. (3) It can be designed based on data from the real system or plant if an expert knowledge is not available. (4) It can be designed using a combination of linguistic and response- based information. [6–9]. The neural networks (NNs) are considered as one of AI, which are a series of algorithms to recognize underlying relationships in a set of data through a process, which like the operation of human brain [10]. Since many real-time implementations of nonlinear sys- tems involve nonlinearity, nonlinear systems play an important role in engineering research that are deﬁned as systems whose manner is not proportional to their inputs [1]. These systems suffer inherent uncertainty, time-vary- ing parameters and nonlinear dynamic behavioral. In this concern, non-optimal control suffers from some limitations due to the assumptions made for the control system such as linearity and time-invariance. Therefore, the non-optimal & Ahmad M. El-Nagar ahmed.elnagar@el-eng.menoﬁa.edu.eg Ahmad M. Zaki ahmed_zaki889@yahoo.com F. A. S. Soliman fouad.saad.soliman@gmail.com Mohammad El-Bardini dralbardini@el-eng.menoﬁa.edu.eg 1 Department of Industrial Electronics and Control Engineering, Faculty of Electronic Engineering, Menouﬁa University, Menof 32852, Egypt 2 Department of Electronics and Computers Engineering, Nuclear Materials Authority, El-Maadi, 530, Cairo, Egypt In this concern, there are various structures of NNs such as recurrent neural networks (RNNs) [11–14] and multi- layer feed-forward neural networks (MLFFNNs) [15, 16]. MLFFNN is called static network, where there are not tapped delay lines. Several MLFFNN models from obser- vational data were created for predicting the groundwater levels [17]. The NN controller was designed as a direct adaptive inverse control based on MLFFNN to control and estimate the model of nonlinear plants [18]. In [19], the researchers designed MLFFNN for classiﬁcation of non- linear mappings based on input and output samples. 1 Department of Industrial Electronics and Control Engineering, Faculty of Electronic Engineering, Menouﬁa University, Menof 32852, Egypt 2 Department of Electronics and Computers Engineering, 12 12 3 Neural Computing and Applications (2022) 34:22367–22386 22368 However, there are tapped delay lines in RNN, and this is called a dynamic network. 1.1 Literature review In [41], the parameters of the classical PID controller were tuned based on DL for controlling maglev train, which is a new type of the ground transports. The deep NNs (DNN) were introduced for dynamical systems modeling based on complex manner [42]. Three DNN structures are trained on successive data for studying validation of these networks in modeling of dynamical systems. In [43], DL was designed for analyzing the performance of a nonlinear continuous stirred tank reactor, which trained its weights tuned by hybrid algorithm. The DL was introduced as a hybrid algorithm with the fuzzy system for tuning the parameters of the PID controller [44], which was used for controlling the speed of brushless DC motor. In [45], DL controller was introduced, which is performed based on the MLFFNN and the RBM. It is used for initializing the weights values of a network for the nonlinear systems. In [46], DL was introduced for modeling the nonlinear systems based on Elman RNN and restricted Boltzmann machine (ERNN- RBM), which is considered as an unsupervised method for initializing only the ﬁrst layer. 1 Introduction The RNN is more robust than MLFFNN because it contains the MLFNN framework with tapped delay lines [20]. Various RNN structures exist in this regard, including Elman NNs; the feedback connec- tions from the output to the input of the hidden layer are performed via a context layer. Another RNN is Jordan NNs; the feedback connections from the output to the input layer are performed via a state layer. [21–23]. Recently, the fully connected RNN (FCRNN) was modiﬁed to provide a diagonal RNN (DRNN). In the hidden layer, the DRNN contains self-recurrent neurons that feed only their output back into themselves, not to other neurons in the same layer. [24–26]. In [27], in order to achieve high perfor- mance of the shunt active power ﬁlter, researchers designed a controller based on RNN. A self-organizing RNN for the nonlinear model predictive controller was designed to foresee the nonlinear systems behavior [28]. In [29], a ﬂexible manipulator was designed with a DRNN controller to limit backward vibration, which is performed based on a shaking control signal generator and an online identiﬁca- tion system. The DRNN was introduced as a controller and an observer for estimating the anonymous dynamics of the nonlinear system [30]. DRNN was developed to determine the optimal parameters of the PID controller for controlling induction motors [31]. In [32], based on the control inputs and current quadrotor states, researchers developed the PID controller using virtual sensing based on DRNNs and Kalman ﬁlters to predict the immeasurable cases of the quadrotor system. This is considered as the main advantage of DL because the initializing weights process is very critical issue. 1.2 Motivation In this concern, there are various applications of DL exist as follows: (1) In automation systems, an approach for detecting and assessing food waste trays based on hierar- chical DL algorithm was presented [35], (2) in medicine ﬁeld, a DL algorithm was used to classify and predict mutations from non-small cell lung cancer histopathology images [36], (3) in agriculture ﬁeld, a DL algorithm was introduced to locate paddy ﬁelds at the pixel level for a whole year long and for each temporal instance based on real imagery datasets of different landscapes from 2016 to 2018 [37], and (4) in recognition, a DL algorithm was used for real-time modeling of the human activity recognition with smartphones [38]. According to deﬁnitions [39, 40], the DL of NN’s includes two steps: ﬁrstly is the unsuper- vised training and secondly is using the weights from the unsupervised training for initializing the multilayer NNs. 123 123 Neural Computing and Applications (2022) 34:22367–22386 22369 <ð1Þ j ð Þ ¼ < ð1Þ 11 j ð Þ < ð1Þ 1n j ð Þ ... .. . ... < ð1Þ J1 j ð Þ < ð1Þ Jn j ð Þ 2 64 3 75 ð1Þ merge utilizing the features of SOM and RBM is proposed to improve the learning performance of the proposed net- work. To control the nonlinear systems, the hybrid deep learning DRNN controller (HDL-DRNNC) based on SOM and RBM is proposed. Initial weights for the DRNN are obtained using a hybrid deep learning (HDL) procedure, which is regarded as an unsupervised learning procedure. The HDL is performed based on RBM and self-organizing map (SOM) of the Kohonen procedure. ð1Þ Generally, < 1 ð Þ Jn j ð Þ denote the input weight between input neuron n and the hidden layer (1) neuron J. Hidden layer (1): the output of each node is denoted by vð1Þ j j ð Þ, which is speciﬁed as: Kð1Þ j j ð Þ ¼ vð1Þ j j 1 ð Þ<D1 j j ð Þ þ X n i¼1 < ð1Þ ji j ð Þexi j ð Þ þ Tj j ð Þ ; j ¼ 1; . . .; J ð2Þ vð1Þ j j ð Þ ¼ f Kð1Þ j j ð Þ ; j ¼ 1; . . • A new HDL for DRNN controller is proposed for nonlinear systems. mð m ¼ 1; ::; M ; ; ð5Þ vð2Þ m j ð Þ ¼ f Kð2Þ m j ð Þ ; m ¼ 1; ::; M ð6Þ ð5Þ ð Þ vð2Þ m j ð Þ ¼ f Kð2Þ m j ð Þ ; m ¼ 1; ::; M ð6Þ ð6Þ 4. The HDL-DRNNC pseudocode is explained in Sect. 5. The simulation results for the mathematical and physical nonlinear systems are introduced in Sect. 6. At ﬁnal, Sect. 7 exhibits the conclusion, which followed by the references. where <D2 j ð Þ ¼ <D2 1 j ð Þ <D2 M j ð ÞT denotes the where <D2 j ð Þ ¼ <D2 1 j ð Þ <D2 M j ð ÞT denotes the diagonal weight vector at the hidden layer (2), M denotes the nodes number, Tm j ð Þ denotes the threshold value for each node and < ð2Þ j ð Þ denotes the weights matrix between the hidden layer (1) and the hidden layer (2), which is deﬁned as: where <D2 j ð Þ ¼ <D2 1 j ð Þ <D2 M j ð Þ denotes the diagonal weight vector at the hidden layer (2), M denotes the nodes number, Tm j ð Þ denotes the threshold value for each node and < ð2Þ j ð Þ denotes the weights matrix between the hidden layer (1) and the hidden layer (2), which is deﬁned as: 1.2 Motivation .; J ð3Þ To ensure the stability of the adaptation parameters laws, the Lyapunov procedure is applied. The proposed HDL-DRNNC is trained quickly for keeping the trajectory and overcoming the system parameters variations and external disturbances. As shown in the simulation results, these features of the proposed HDL-DRNNC make it more robust than those of other controllers under the same conditions. ð2Þ ð3Þ where <D1 j ð Þ ¼ <D1 1 j ð Þ <D1 J j ð ÞT denotes the diag- onal weight vector at the hidden layer (1), J is the nodes number, Tj j ð Þ denotes the threshold value for every node, and fðÞ denotes hyperbolic tangent function, which is deﬁned as: 1.3 Novelties and contributions The main contributions of the paper are summarized as: • A new HDL for DRNN controller is proposed for nonlinear systems. • A new HDL for DRNN controller is proposed for nonlinear systems. f t ð Þ ¼ tanh t ð Þ ð4Þ • A new HDL for DRNN controller is proposed for nonlinear systems. f t ð Þ ¼ tanh t ð Þ ð4Þ and its derivative can be deﬁned by f 0 t ð Þ ¼ 1 f 2 t ð Þ. Hidden layer (2): the output of each node is denoted by vð2Þ m j ð Þ, which is speciﬁed as: and its derivative can be deﬁned by f 0 t ð Þ ¼ 1 f 2 t ð Þ. Hidden layer (2): the output of each node is denoted by vð2Þ m j ð Þ, which is speciﬁed as: • Developing the updating weights law for the DRNN of the proposed algorithm using Lyapunov theory to achieve stability. • Compared to other existing controllers, the HDL- DRNNC can handle problems of system uncertainties in both a mathematical system and a physical system. Kð2Þ m j ð Þ ¼ vð2Þ m j 1 ð Þ <D2 m j ð Þ þ X J j¼1 < ð2Þ mj j ð Þ vð1Þ j j ð Þ þ Tm j ð Þ ; m ¼ 1; ::; M Kð2Þ m j ð Þ ¼ vð2Þ m j 1 ð Þ <D2 m j ð Þ þ X J j¼1 < ð2Þ mj j ð Þ vð1Þ j j ð Þ þ Tm j ð Þ ; ¼ 1 M þ Tm j ð Þ ; The organization of the paper is as follows: the DRNN structure is exhibited in Sect. 2. The proposed HDL- DRNNC is introduced in Sect. 3. The weights updating based on Lyapunov stability criterion is introduced in Sect. 4. The HDL-DRNNC pseudocode is explained in Sect. 5. The simulation results for the mathematical and physical nonlinear systems are introduced in Sect. 6. At ﬁnal, Sect. 7 exhibits the conclusion, which followed by the references. The organization of the paper is as follows: the DRNN structure is exhibited in Sect. 2. The proposed HDL- DRNNC is introduced in Sect. 3. The weights updating based on Lyapunov stability criterion is introduced in Sect. 3.1 SOM of the Kohonen learning procedure u j ð Þ ¼ X M m¼1 < ð3Þ m j ð Þ vð2Þ m j ð Þ þ T j ð Þ u j ð Þ ¼ X M m¼1 < ð3Þ m j ð Þ vð2Þ m j ð Þ þ T j ð Þ ð3Þ ð8Þ u j ð Þ ¼ X m¼1 < ð3Þ m j ð Þ vð2Þ m j ð Þ þ T j ð Þ ð8Þ where T j ð Þ denotes the threshold value and < ð3Þ j ð Þ ¼ < ð3Þ 1 j ð Þ < ð3Þ M j ð Þ h iT denotes the weight vector between the hidden layer (2) and the output layer. The initializing weights values for the hidden layers of the DRNN, which is introduced in Sect. 2, are the main pur- pose of this section. The NN, which is used to initialize the weights of the hidden layers of the DRNN, is shown in Fig. 3. The weights of the NN are trained based on SOM of the Kohonen process [52]. The training is performed based on the hypothesis that one of the layer neurons responds most to the input, which is called the winner neuron. where T j ð Þ denotes the threshold value and < ð3Þ j ð Þ ¼ < ð3Þ 1 j ð Þ < ð3Þ M j ð Þ h iT denotes the weight vector between the hidden layer (2) and the output layer. 2 Diagonal recurrent neural network structure < ð2Þ j ð Þ ¼ < ð2Þ 11 j ð Þ < ð2Þ 1J j ð Þ ... .. . ... < ð2Þ M1 j ð Þ < ð2Þ MJ j ð Þ 2 64 3 75 ð7Þ As shown in Fig. 1, the structure of DRNN consists of four layers, namely two hidden layers, an input layer, and an output layer. ð7Þ Generally, < 2 ð Þ MJ j ð Þ denote the weight between the hid- den layer (1) neuron J and the hidden layer (2) neuron M. Input layer: the external input vector is represented by E j ð Þ ¼ ex1 ½ j ð Þ exn j ð ÞT and < 1 ð Þ j ð Þ is the input weight matrix, which links the input vector to the hidden layer (1) neurons and it is represented as: Output layer: its output is denoted by u j ð Þ, which is calculated as: 123 123 123 u j ð Þ ¼ X M m¼1 < ð3Þ m j ð Þ vð2Þ m j ð Þ þ T j ð Þ ð8Þ where T j ð Þ denotes the threshold value and < ð3Þ j ð Þ ¼ < ð3Þ 1 j ð Þ < ð3Þ M j ð Þ h iT denotes the weight vector between the hidden layer (2) and the output layer. 22370 22370 Neural Computing and Applications (2022) 34:22367–22386 3 Hybrid deep learning diagonal recurrent neural network controller 1 Structure of DRNN 123 22371 Neural Computing and Applications (2022) 34:22367–22386 Fig. 2 Structure of HDL- DRNNC Fig. 2 Structure of HDL- DRNNC Fig. 2 Structure of HDL- DRNNC Fig. 3 NN based on SOMK unsupervised learning previously. The number of neurons in the output layer of the NN (Fig. 3) is equal to the number of output neurons of the hidden layer (2) of the DRNN and the number of weights for the hidden layer (2) of the DRNN are equal to the number of weights for the NN. After the training of NN is completed, the values of the hidden layer (2) weights of the DRNN < ð2Þ mj j ð Þ will be equal to the values of NN weights, -ð2Þ lf j ð Þ; l ¼ m ¼ 1; :::; M ð Þ. f The SOM of the Kohonen (SOMK) procedure is sum- marized as: Step 1: All the weights -ðQÞ lf ; Q ¼ 1 ; 2 of the NN, which is shown in Fig. 3, are initialized at zero values. Step 2: Enter the values of E j ð Þ to the NN. Step 3: The winner neuron x is selected using the Euclidean distance between the input and the neuron weights -ðQÞ x as: Step 1: All the weights -ðQÞ lf ; Q ¼ 1 ; 2 of the NN, which is shown in Fig. 3, are initialized at zero values. Step 1: All the weights -ðQÞ lf ; Q ¼ 1 ; 2 of the NN, which is shown in Fig. 3, are initialized at zero values. Step 2: Enter the values of E j ð Þ to the NN. Step 3: The winner neuron x is selected using the Euclidean distance between the input and the neuron weights -ðQÞ x as: Fig. 3 NN based on SOMK unsupervised learning Step 2: Enter the values of E j ð Þ to the NN. Step 3: The winner neuron x is selected using the Euclidean distance between the input and the neuron weights -ðQÞ x as: equal to the number of input neurons of the hidden layer (2) of the DRNN where their values are If j ð Þ ¼ vð1Þ j j ð Þ; f ¼ j ¼ 1; :::; J ð Þ. 3 Hybrid deep learning diagonal recurrent neural network controller For training the hidden layer (1) of the DRNN, the number of neurons in the input layer of the NN (Fig. 3), which is used to initialize the weights of the hidden layers, is equal to the number of input neurons of the hidden layer (1) of the DRNN where their values are If j ð Þ ¼ exi j ð Þ; f ¼ i ¼ 1; ::; n ð Þ. f denotes the number of neurons in the input layer of the NN (Fig. 3) and i denotes the number of the input neurons of the hidden layer (1) of the DRNN. The number of neurons in the output layer of the NN (Fig. 3) is equal to the number of output neurons of the hidden layer (1) of the DRNN and the weights number for the hidden layer (1) of the DRNN are equal to the weights number for the NN. After the training of NN is completed, the values of the hidden layer (1) weights of the DRNN < ð1Þ ji j ð Þ will be equal to the values of NN weights, -ð1Þ lf j ð Þ; l ¼ j ¼ 1; :::; J ð Þ. The initializing weights process for DRNN controllers is very critical issue. Where, if this process is zero or not suitable, then the DRNN will stumble in local minimum and it will lead to wrong network termination as learning due to the initial layers learning of a network will become impossible [46]. This issue may be leading the controller to become unstable. For this reason, DL is proposed. In this regard, any NN with more than one hidden layer is referred to as a deep network, which can be learned by DL. The proposed HDL-DRNNC consists of DRNN that can be trained by DL. The DL algorithm based on SOM and RBM is considered as an unsupervised learning for initializing the weights values of the DRNN. DRNN’s two hidden layers, which are described in the previous section, are trained using SOM algorithm. On the other hand, the RBM algorithm is used for training the DRNN output layer. The proposed HDL-DRNNC structure is shown in Fig. 2. For training the hidden layer (2) of the DRNN, the number of neurons in the input layer of NN (Fig. 3) are NN Fig. 3.2 Restricted Boltzmann machine Fig. 4 Structure of RBM Initializing the weights values for the output layer of the DRNN is performed based on RBM [45, 53]. The RBM that is used in this section is illustrated in Fig. 4. Where all the weights of the output layer are equal to zero, RBM contains two main layers: ﬁrstly, the visible layer, which contains a visible nodes group S and secondly, the hidden layers, which contains a hidden nodes group D [46, 54]. Initializing the weights values for the output layer of the DRNN is performed based on RBM [45, 53]. The RBM that is used in this section is illustrated in Fig. 4. Where all the weights of the output layer are equal to zero, RBM contains two main layers: ﬁrstly, the visible layer, which contains a visible nodes group S and secondly, the hidden layers, which contains a hidden nodes group D [46, 54]. For training the DRNN output layer, the number of neurons in the input layer of the RBM is equal to the number of input neurons of the output layer of the DRNN where their values are Si j ð Þ ¼ vð2Þ m j ð Þ; i ¼ m ¼ 1; ::; M ð Þ. The number of neurons in the output layer of the RBM is equal to the number of neurons in the output layer of the DRNN and the weights number of the output layer of the DRNN equals to the weights number of the RBM. After the training of RBM is completed, the values of the output layer weights of the DRNN < 3 ð Þ m j ð Þ will be equal to the RBM weights values, Xji j ð Þ; j ¼ 1; :::; P ð Þ and i ¼ m ¼ 1; ::; M ð Þ. In this paper, P ¼ 1. Fig. 4 Structure of RBM Fig. 4 Structure of RBM ex -ðQÞ x ¼ min l ex -ðQÞ x ð9Þ ð9Þ where Q, l and x are the number of the NN layer, the index of any neuron and the index of the winner neuron, respectively. Based on the approach in [53, 55], Hinton introduced contrastive divergence (CD) for training RBM. The RBM input is Sðr 1Þ, which shifts to the visible layer at time ðr 1Þ. 3 Hybrid deep learning diagonal recurrent neural network controller The number of the input neurons of the hidden layer (2) are denoted by j, where f is deﬁned 12 123 Neural Computing and Applications (2022) 34:22367–22386 22372 3.2 Restricted Boltzmann machine Then, the hidden layer output is obtained as: Step 4: Calculate the Gaussian neighborhood function as: q Q ð Þ xl j ð Þ ¼ ‘ exp ðx lÞ2 2f ! ; 0 \q Q ð Þ xl j ð Þ 1 ð10Þ ð10Þ Dj r 1 ð Þ ¼ F X N i XjiSi r 1 ð Þ þ Bj ! ; r ¼ 1; :::::; <; j ¼ 1; ::; P and i ¼ 1; ::; N where ‘ and f are constants. where ‘ and f are constants. Step 5: The updating law of the weights of the NN Step 5: The updating law of the weights of the NN ðQÞ ð14Þ ð14Þ -ðQÞ lf ðjÞ, where Q ¼ 1, is obtained as: -ðQÞ lf ðjÞ, where Q ¼ 1, is obtained as: where Xji represents the weight between a visible node i and a hidden node j and Si represents the binary state of the visible node. P and N are the hidden nodes number and the visible nodes number, respectively. B ¼ B1 BP ½ T represents the hidden nodes biases and F denotes sigmoid activation function F zð Þ ¼ 1= 1 þ exp z ð Þ ð Þ. f D-ð1Þ lf ðjÞ ¼ q ð1Þ xl j ð ÞðexðjÞ -ð1Þ lf ðjÞÞ ð11Þ -ð1Þ lf ðj þ 1Þ ¼ -ð1Þ lf ðjÞ þ D-ð1Þ lf ðjÞ; l ¼ j ¼ 1; :::; J ð Þ and f ¼ i ¼ 1; :::; n ð Þ ð12Þ ð11Þ ð12Þ The updating law of the weights of the NN -ðQÞ lg ðjÞ, where Q ¼ 2, is obtained as: The inverse layer reconstructs the data from the hidden layer. As a result, S rð Þ is obtained at r as follows: -ð2Þ lf ðj þ 1Þ ¼ -ð2Þ lf ðjÞ þ q ð2Þ xl j ð ÞðexðjÞ -ð2Þ lf ðjÞÞ; l ¼ m ¼ 1; :::; M ð Þ and f ¼ j ¼ 1; :::; J ð Þ Si rð Þ ¼ F Qi rð Þ ð Þ ¼ F X P j¼1 XijDj r 1 ð Þ þ Ai ! 3.2 Restricted Boltzmann machine ð15Þ ð15Þ ð13Þ where Xij represents the weight between a hidden node j and a visible node i, Dj represents the binary state of a hidden node and A ¼ A1 AN ½ T represents the Fig. 5 HDL-DRNNC block diagram with nonlinear system 123 Neural Computing and Applications (2022) 34:22367–22386 22373 Fig. 6 Output response for the mathematical system (Task 1) Fig. 6 Output response for the mathematical system (Task 1) Fig. 6 Output response for the mathematical system (Task 1) 4 Weights updating based on Lyapunov stability visible nodes biases. Subsequently, S rð Þ transfers to the visible layer and the hidden layer output is obtained as: Dj rð Þ ¼ F Qj rð Þ ¼ F X N i¼1 XjiSi rð Þ þ Bj ! ð16Þ The performance function is denoted by El j ð Þ, which is deﬁned as: The performance function is denoted by El j ð Þ, which is deﬁned as: ð16Þ El j ð Þ ¼ 1 2 !d j ð Þ !a j ð Þ ð Þ2¼ 1 2 e2 x j ð Þ ð20Þ CD < case: The parameters learning rules for the weights and biases of nonlinear RBM are clariﬁed as [55]: CD < case: The parameters learning rules for the weights and biases of nonlinear RBM are clariﬁed as [55]: ð20Þ where !dðjÞ and !aðjÞ denote the reference input and the actual output, respectively. The DRNN is trained to mini- mize the error signal [56]. X < r¼1 Dj rð Þ Dj r 1 ð Þ Si rð Þ F0 Qj rð Þ þ Si rð Þ Si r 1 ð Þ ð Þ Djðr 1Þ F0 Qi rð Þ ð Þ ! ð17Þ ð17Þ To achieve stability, the updating weights of DRNN of the proposed HDL-DRNNC are developed using Lyapunov stability criteria. Two conditions must be met in order to the system to be asymptotically stable, as outlined in Eqs. (21 and 22) Bj j þ 1 ð Þ ¼ Bj j ð Þ þ e X < r¼1 Dj rð Þ Dj r 1 ð Þ F0 Qj rð Þ ! ð18Þ Ai j þ 1 ð Þ ¼ Ai j ð Þ þ e X < r¼1 Si rð Þ Si r 1 ð Þ ð Þ F0 Qi rð Þ ð Þ ! Bj j þ 1 ð Þ ¼ Bj j ð Þ < Bj j þ 1 ð Þ ¼ Bj j ð Þ þ e X < r¼1 Dj rð Þ Dj r 1 ð Þ F0 Qj rð Þ ! 4 Weights updating based on Lyapunov stability Rx j ð Þ [ 0 for all j except j ¼ 0 ð21Þ DRx j ð Þ ¼ Rx j þ 1 ð Þ RxðjÞ 0 ð22Þ ð21Þ ð22Þ ð21Þ ð Þ ð22Þ ð22Þ ð18Þ where Rx j ð Þ is a positive deﬁnite function. The updating weight equation can be expressed as a common form: Ul j þ 1 ð Þ ¼ UlðjÞ gDUlðjÞ ð23Þ ð23Þ where Ul j ð Þ and DUl j ð Þ denote a generalized weight vector and its desired modiﬁcation and the learning rate is denoted by g. ð19Þ ð19Þ where e is the RBM learning rate and j is the iteration number. When the parameters of RBM are learned, hence the output layer of the DRNN can be initialized based on the weights of RBM Xji j þ 1 ð Þ. Theorem 1 To achieve the stability of the controlled process, the updating equation for the DRNN weights of the proposed scheme is obtained as the following: Ul j þ 1 ð Þ ¼ Ul j ð Þ þ g b Ul j ð Þ 1 þ r b e2 x j ð Þ þ r b ex j ð Þ U2 l j ð Þ oex j ð Þ oUl j ð Þ 2k ð24Þ where b ; r and k are positive constants. Ul j þ 1 ð Þ Ul j þ 1 ð Þ ¼ Ul j ð Þ þ g b Ul j ð Þ 1 þ r b e2 x j ð Þ þ r b ex j ð Þ U2 l j ð Þ oex j ð Þ oUl j ð Þ 2k ð24Þ ¼ Ul j ð Þ þ g b Ul j ð Þ 1 þ r b e2 x j ð Þ þ r b ex j ð Þ U2 l j ð Þ oex j ð Þ oUl j ð Þ 2k ð24Þ ð24Þ where b ; r and k are positive constants. where b ; r and k are positive constants. 123 3 Fig. 8 Output response for the mathematical system (Task 2) Fig. 7 Control signal (Task 1) Fig. 9 Control signal (Task 2) 22374 Neural Computing and Applications (2022) 34:22367– Neural Computing and Applications (2022) 34:22367–22386 22374 Fig. 4 Weights updating based on Lyapunov stability 8 Output response for the mathematical system (Task 2) Fig. 7 Control signal (Task 1) 22374 Neural Computing and Applications (2022) 34:22367 Fig. 7 Control signal (Task 1) Fig. 7 Control signal (Task 1) Fig. 8 Output response for the mathematical system (Task 2) Fig. 7 Control signal (Task 1) Fig. 8 Output response for the mathematical system (Task 2) Fig. 8 Output response for the mathematical system (Task 2) i l i l k Fig. 9 Control signal (Task 2) 123 Fig. 9 Control signal (Task 2) Neural Computing and Applications (2022) 34:22367–22386 22375 Fig. 10 Output response for the mathematical system (Task 3) Fig 11 Control signal (Task 3) Fig. 12 Output response for the mathematical system (Task 4) Fig. 10 Output response for the mathematical system (Task 3) Fig. 11 Control signal (Task 3) Fig. 10 Output response for the mathematical system (Task 3) Fig. 10 Output response for the mathematical system (Task 3) Fig. 10 Output response for the mathematical system (Task 3) Fig. 11 Control signal (Task 3) Fig 11 Control signal (Task 3) Fig. 11 Control signal (Task 3) Fig. 11 Control signal (Task 3) Fig 12 Output response for the mathematical system (Task 4) Fig. 12 Output response for the mathematical system (Task 4) 12 23 Neural Computing and Applications (2022) 34:22367–22386 22376 Proof Suppose the next Lyapunov function: Rx j ð Þ ¼ Ra j ð Þ þ RbðjÞ þ Rc j ð Þ ð25Þ where Ra j ð Þ ¼ r 2 ex j ð ÞUl j ð Þ ð Þ2,Rb j ð Þ ¼ b 2 Ul j ð Þ ð Þ2, Rc j ð Þ ¼ k 2 DUl j ð Þ ð Þ2, DRa j ð Þ, DRb j ð Þ and DRc j ð Þ are deﬁned as: DRa j ð Þ ¼ Ra j þ 1 ð Þ RaðjÞ ¼ r 2 ex j þ 1 ð ÞUl j þ 1 ð Þ ð Þ2 r 2 ex j ð ÞUl j ð Þ ð Þ2 ð26Þ Fig. 13 Control signal (Task 4) Fig. 14 EVS schematic diagram Fig. 13 Control signal (Task 4) Fig. 13 Control signal (Task 4) Fig. 14 EVS schematic diagram Fig. 4 Weights updating based on Lyapunov stability 14 EVS schematic diagram Proof Suppose the next Lyapunov function: Rx j ð Þ ¼ Ra j ð Þ þ RbðjÞ þ Rc j ð Þ ð25Þ ð25Þ where Ra j ð Þ ¼ r 2 ex j ð ÞUl j ð Þ ð Þ2,Rb j ð Þ ¼ b 2 Ul j ð Þ ð Þ2, Rc j ð Þ ¼ k 2 DUl j ð Þ ð Þ2, DRa j ð Þ, DRb j ð Þ and DRc j ð Þ are deﬁned as: where Ra j ð Þ ¼ r 2 ex j ð ÞUl j ð Þ ð Þ2,Rb j ð Þ ¼ b 2 Ul j ð Þ ð Þ2, Rc j ð Þ ¼ k 2 DUl j ð Þ ð Þ2, DRa j ð Þ, DRb j ð Þ and DRc j ð Þ are deﬁned as: b 2 Ul j ð Þ ð Þ2, DRa j ð Þ ¼ Ra j þ 1 ð Þ RaðjÞ ¼ r 2 ex j þ 1 ð ÞUl j þ 1 ð Þ ð Þ2 r 2 ex j ð ÞUl j ð Þ ð Þ2 ð26Þ Fig. 14 EVS schematic diagram Fig. 14 EVS schematic diagram Fig. 15 Output response for the EVS (Task 1) Fig. 15 Output response for the EVS (Task 1) Fig. 15 Output response for the EVS (Task 1) Neural Computing and Applications (2022) 34:22367–22386 22377 Fig. 17 Output response for the EVS (Task 2) Fig. 18 The EVS control signal (Task 2) 0 150 300 450 600 750 900 1050 0 5 10 15 20 25 30 35 Time (Sec.) )tl o V ( la n g is l o rt n o C DRNNC HDL-DRNNC Fig. 16 The EVS control signal (Task 1) Neural Computing and Applications (2022) 34:22367–22386 2 Fig. 17 Output response for the EVS (Task 2) 0 150 300 450 600 750 900 1050 0 5 10 15 20 25 30 35 Time (Sec.) )tl o V ( la n g is l o rt n o C DRNNC HDL-DRNNC Fig. 16 The EVS control signal (Task 1) 0 150 300 450 600 750 900 1050 0 5 10 15 20 25 30 35 Time (Sec.) )tl o V ( la n g is l o rt n o C DRNNC HDL-DRNNC Fig. 16 The EVS control signal (Task 1) Fig. 4 Weights updating based on Lyapunov stability 19 Output response for the EVS (Task 3) DRb j ð Þ ¼Rb j þ 1 ð Þ RbðjÞ ¼ b 2 Ul j þ 1 ð Þ ð Þ2 b 2 Ul j ð Þ ð Þ2 ð27Þ DRc j ð Þ ¼ Rc j þ 1 ð Þ RcðjÞ ¼ k 2 DUl j þ 1 ð Þ ð Þ2 k 2 DUl j ð Þ ð Þ2 ð28Þ DRb j ð Þ ¼Rb j þ 1 ð Þ RbðjÞ ¼ b 2 Ul j þ 1 ð Þ ð Þ2 b 2 Ul j ð Þ ð Þ2 DRc j ð Þ ¼ Rc j þ 1 ð Þ RcðjÞ ¼ k 2 DUl j þ 1 ð Þ ð Þ2 k 2 DUl j ð Þ ð Þ2 DRb j ð Þ ¼Rb j þ 1 ð Þ RbðjÞ ¼ b 2 Ul j þ 1 ð Þ ð Þ2 b 2 Ul j ð Þ ð Þ2 ð27Þ DRc j ð Þ ¼ Rc j þ 1 ð Þ RcðjÞ ¼ k 2 DUl j þ 1 ð Þ ð Þ2 k 2 DUl j ð Þ ð Þ2 ð28Þ The term r 2 ex j þ 1 ð ÞUl j þ 1 ð Þ ð Þ2 can be deﬁned based on Taylor series in the linear form as [24]: r 2 ex j þ 1 ð ÞUl j þ 1 ð Þ ð Þ2 ¼ r 2 ex j ð ÞUl j ð Þ ð Þ2 þ o r 2 ex j ð ÞUl j ð Þ ð Þ2 oUl j ð Þ DUl j ð Þ Table 1 The mathematical system MAE values Task 1 Task 2 Task 3 Task 4 DRNNC 0.5802 0.6603 0.6732 0.6385 FCRNNC [62] 0.5891 0.6686 0.6800 0.6471 ERNN-RBM [46] 0.2513 0.2869 0.2915 0.2813 DRNNC-SOM 0.0306 0.0518 0.0386 0.0386 FFNNHLC [61] 0.1057 0.1455 0.1537 0.1133 AIT2-TSK-FLC-RL [63] 0.07203 0.1038 0.1241 0.0728 FFNN-RBM [45] 0.04478 0.0655 0.0770 0.0475 HDL-DRNNC 0.0107 0.0164 0.0191 0.0154 Table 1 The mathematical system MAE values ð28Þ The term r 2 ex j þ 1 ð ÞUl j þ 1 ð Þ ð Þ2 can be deﬁned based on Taylor series in the linear form as [24]: r 2 ex j þ 1 ð ÞUl j þ 1 ð Þ ð Þ2 ¼ r 2 ex j ð ÞUl j ð Þ ð Þ2 þ o r 2 ex j ð ÞUl j ð Þ ð Þ2 oUl j ð Þ DUl j ð Þ þ HOT ð29Þ ex j þ 1 ð Þ ¼ ex j ð Þ þ oex j ð Þ oUl j ð Þ DUl j ð Þ ð32Þ ð32Þ ð29Þ where HOT denotes to the higher order term, which can be ignored. 4 Weights updating based on Lyapunov stability 17 Output response for the EVS (Task 2) Fig. 18 The EVS control signal (Task 2) Fig. 18 The EVS control signal (Task 2) 12 3 Neural Computing and Applications (2022) 34:22367–22386 22378 DRb j ð Þ ¼Rb j þ 1 ð Þ RbðjÞ ¼ b 2 Ul j þ 1 ð Þ ð Þ2 b 2 Ul j ð Þ ð Þ2 ð27Þ DRc j ð Þ ¼ Rc j þ 1 ð Þ RcðjÞ ¼ k 2 DUl j þ 1 ð Þ ð Þ2 k 2 DUl j ð Þ ð Þ2 ð28Þ The term r 2 ex j þ 1 ð ÞUl j þ 1 ð Þ ð Þ2 can be deﬁned based on Taylor series in the linear form as [24]: r 2 ex j þ 1 ð ÞUl j þ 1 ð Þ ð Þ2 ¼ r 2 ex j ð ÞUl j ð Þ ð Þ2 þ o r 2 ex j ð ÞUl j ð Þ ð Þ2 oUl j ð Þ DUl j ð Þ Fig. 19 Output response for the EVS (Task 3) Table 1 The mathematical system MAE values Task 1 Task 2 Task 3 Task 4 DRNNC 0.5802 0.6603 0.6732 0.6385 FCRNNC [62] 0.5891 0.6686 0.6800 0.6471 ERNN-RBM [46] 0.2513 0.2869 0.2915 0.2813 DRNNC-SOM 0.0306 0.0518 0.0386 0.0386 FFNNHLC [61] 0.1057 0.1455 0.1537 0.1133 AIT2-TSK-FLC-RL [63] 0.07203 0.1038 0.1241 0.0728 FFNN-RBM [45] 0.04478 0.0655 0.0770 0.0475 HDL-DRNNC 0.0107 0.0164 0.0191 0.0154 Fig. 19 Output response for the EVS (Task 3) Fig. 19 Output response for the EVS (Task 3) Fig. 4 Weights updating based on Lyapunov stability Therefore, Eq. (29) can be rewritten as: Equation (32) can be rewritten as: ex j þ 1 ð Þ ex j ð Þ ¼ Dex j ð Þ ¼ oex j ð Þ oUl j ð Þ DUl j ð Þ ð33Þ ð33Þ r 2 ex j þ 1 ð Þ Ul j þ 1 ð Þ ð Þ2 r 2 ex j ð Þ Ul j ð Þ ð Þ2 ¼ r 2 o ex j ð Þ Ul j ð Þ ð Þ2 oUl j ð Þ DUl j ð Þ ð30Þ Then, by replacing the term oex j ð Þ oUl j ð Þ DU j ð Þ in Eq. (31), we obtain the following: ð30Þ DRa j ð Þ ¼ r 2 ex j þ 1 ð ÞUl j þ 1 ð Þ ð Þ2 r 2 ex j ð ÞUl j ð Þ ð Þ2 ¼r ex j ð ÞU2 l j ð ÞDex j ð Þ þ r e2 x j ð ÞUl j ð ÞDUl j ð Þ ð34 The right side of the previous equation is rewritten as follows: ð34Þ r 2 o ex j ð Þ Ul j ð Þ ð Þ2 oUl j ð Þ DUl j ð Þ ¼r ex j ð ÞU2 l j ð Þ oex j ð Þ oUl j ð Þ DUl j ð Þ þ r e2 x j ð ÞUl j ð ÞDUl j ð Þ Similarity, Similarity, DRb j ð Þ ¼ b Ul j ð ÞDUl j ð Þ and DRc ¼ k DUl j ð Þ ð Þ2: ð31Þ The second condition for stability is determined as: Similarity, Similarity, 123 3 Neural Computing and Applications (2022) 34:22367–22386 22379 Fig. 20 The EVS control signal (Task 3) Fig. 20 The EVS control signal (Task 3) Fig. 4 Weights updating based on Lyapunov stability 20 The EVS control signal (Task 3) k DUl j ð Þ ð Þ2þDUl j ð Þ r ex j ð Þ U2 l j ð Þ Dex j ð Þ DUl j ð Þ þr e2 x j ð Þ Ul j ð Þ þ b Ul j ð Þ þ n ¼ 0 ð37Þ DRx j ð Þ ¼ r ex j ð ÞU2 l j ð ÞDex j ð Þ þ r e2 x j ð ÞUl j ð ÞDUl j ð Þ þ b Ul j ð ÞDUl j ð Þ þ k DUl j ð Þ ð Þ2 0 ð35Þ Table 2 The mathematical system RMSE values Task 1 Task 2 Task 3 Task 4 DRNNC 1.94485 2.0513 2.0525 2.0500 FCRNNC [62] 1.94556 2.0521 2.0533 2.0506 ERNN-RBM [46] 0.90912 0.9589 0.9590 0.9584 DRNNC-SOM 0.17832 0.2117 0.1893 0.1885 FFNNHLC [61] 0.41366 0.4446 0.4480 0.4361 AIT2-TSK-FLC-RL [63] 0.12039 0.1595 0.1847 0.12489 FFNN-RBM [45] 0.13242 0.1528 0.1634 0.1397 HDL-DRNNC 0.03797 0.0461 0.0495 0.0427 Table 2 The mathematical system RMSE values ð37Þ The general quadratic equation is determined as: c v2 þ b v þ a ¼ 0 c v2 þ b v þ a ¼ 0 ð38Þ ð38Þ The roots of Eq. (38) are calculated as: v1;2 ¼ b ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ b2 4ca p 2c ð39Þ ð39Þ From Eqs. (37) and (38), obviously, DUl k ð Þ acts as v in Eq. (38) and the values of c; b and a in Eq. 6 Simulation results ð45Þ So, by replacing DUl j ð Þ in Eq. (23), the updating equation for the parameters of the DRNN of the HDL- DRNNC can be given as in Eq. (24). A comparison of the proposed HDL-DRNNC and DRNNC is performed under the same conditions with zero initial weights to show the performance of the hybrid learning algorithm. In the present paper, assign J ¼ M ¼ l ¼ 10; n ¼ 3, R ¼ 10, N ¼ 10, < ¼ 1 and P ¼ 1. In order to evaluate the performance and demon- strate the robustness of the proposed controller, the mean absolute error (MAE) and the root-mean-square error (RMSE) are used. MAE and RMSE are clariﬁed as [57, 58]: 4 Weights updating based on Lyapunov stability Therefore, b r b ex j ð Þ U2 l j ð Þ Dex j ð Þ DUl j ð Þ þ 1 þ r b e2 x j ð Þ Ul j ð Þ 2 4k n ¼ 0 ð41Þ ð41Þ Table 3 EVS parameters Symbol and abbreviation Value Symbol and abbreviation Value m (kg) 800 A (m2) 1.8 Caf (mH) 1.776 q (kg/m3) 1.25 J (kgm2) 0.05 Cd 0.3 re (m) 0.25 lrr 0.015 Ca þ Cf (mH) 6.008 G 11 <a þ <f (X) 0.2 B (NMs) 0.0002 Symbol and abbreviation Value Symbol and abbreviation Value Neural Computing and Applications (2022) 34:22367–22386 22380 Table 5 MAE values for the EVS Task 1 Task 2 Task 3 DRNNC 0.56730 1.17533 0.97630 FCRNNC [62] 0.50638 1.51069 1.31162 ERNN-RBM [46] 0.45990 0.95267 0.74690 DRNNC-SOM 0.43245 0.40114 0.16700 FFNNHLC [61] 0.47226 1.04951 0.86402 AIT2-TSK-FLC-RL [63] 0.28022 0.39834 0.29394 FFNN-RBM [45] 0.22735 0.21747 0.14997 HDL-DRNNC 0.07896 0.11124 0.06934 Table 4 EVS parameters variation values Symbol and abbreviation Value m 900 Cd 0.2 J 0.04 re 0.3 Ca þ Cf 4.008 Ra þ Rf 0.25 lrr 0.025 Table 4 EVS parameters variation values Symbol and abbreviation Value m 900 Cd 0.2 J 0.04 re 0.3 Ca þ Cf 4.008 Ra þ Rf 0.25 lrr 0.025 and, n can be calculated as: T b Table 6 RMSE values for the EVS Task 1 Task 2 Task 3 DRNNC 1.23253 2.61144 2.50004 FCRNNC [61] 1.16820 2.51704 2.40280 ERNN-RBM [46] 1.13236 2.05436 1.96370 DRNNC-SOM 1.08478 0.85328 0.6678 FFNNHLC [62] 1.14823 2.44867 2.33094 AIT2-TSK-FLC-RL [63] 0.73576 1.24810 1.12558 FFNN-RBM [45] 0.66215 1.03518 0.89828 HDL-DRNNC 0.39100 0.63858 0.53724 ð42Þ ð43Þ ex3 j ð Þ ¼ ex j ð Þ 2ex j 1 ð Þ þ ex j 2 ð Þ. The output layer contains one output u j ð Þ. Algorithm 1 summarizes the proposed HDL-DRNNC pseudocode for reader’s convenience. ð44Þ 4 Weights updating based on Lyapunov stability (37) are obtained as: DRx j ð Þ ¼ r ex j ð ÞU2 l j ð ÞDex j ð Þ þ r e2 x j ð ÞUl j ð ÞDUl j ð Þ þ b Ul j ð ÞDUl j ð Þ þ k DUl j ð Þ ð Þ2 0 c ¼ k; b ¼ b r b ex j ð Þ U2 l j ð Þ Dex j ð Þ DUl j ð Þ þ 1 þ r b e2 x j ð Þ Ul j ð Þ and a ¼ n ð40Þ ð35Þ ð35Þ ð35Þ ð40Þ Equation (35) can be rewritten as: DRx j ð Þ ¼ r ex j ð ÞU2 l j ð ÞDex j ð Þ þ r e2 x j ð ÞUl j ð ÞDUl j ð Þ þ b Ul j ð ÞDUl j ð Þ þ k DUl j ð Þ ð Þ2¼ n Equation (35) can be rewritten as: DRx j ð Þ ¼ r ex j ð ÞU2 l j ð ÞDex j ð Þ þ r e2 x j ð ÞUl j ð ÞDUl j ð Þ þ b Ul j ð ÞDUl j ð Þ þ k DUl j ð Þ ð Þ2¼ n There is a single unique solution for Eq. (38), if ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ b2 4ca p ¼ 0. Therefore, ð36Þ ð36Þ ð36Þ b 4ca p ¼ 0. 5 Steps of the proposed HDL-DRNNC The system block diagram with the proposed HDL- DRNNC is shown in Fig. 5. The error signal ex j ð Þ is the difference between the reference input !dðjÞ and the actual output of the nonlinear system !aðjÞ. The proposed controller input is ex j ð Þ and its output is the control signal u j ð Þ, which forward to the nonlinear system. MAE ¼ 1 KL X KL j¼1 ex j ð Þ j j ð46Þ RMSE ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 KL XKL j¼1 ex j ð Þ ð Þ2 s ð47Þ MAE ¼ 1 KL X KL j¼1 ex j ð Þ j j ð46Þ ð46Þ As shown in Figs. 1 and 2, the ﬁrst layer of the HDL- DRNNC contains three inputs, which are the error signal ex1 j ð Þ ¼ ex j ð Þ, the change of error signal ex2 j ð Þ ¼ ex j ð Þ ex j 1 ð Þ and the change of the change of error signal RMSE ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 KL XKL j¼1 ex j ð Þ ð Þ2 s ð47Þ ð47Þ 123 22381 Neural Computing and Applications (2022) 34:22367–22386 where KL denotes iterations number. where KL denotes iterations number. where KL denotes iterations number. 6.2 Case 2: physical system In this section, the proposed controller is used for con- trolling a physical system, which is the electrical vehicle system (EVS). Nowadays, EVSs are increasingly advanc- ing because of the importance of environmental protection and lack of energy sources [64]. The control of EVSs is important role in order to determine a high-performance EVS with an optimal balance of travelling range per charge, maximum speed and acceleration performance [64]. EVSs are basically time-variant (e.g. the EVS parameters and the road condition are consistently varying) nonlinear system, which making the control of an EVS quite cumbersome [64]. Therefore, the control of EVS should be designed robustly and adaptively to improve the system in both dynamic and steady performance states. 12 123 Neural Computing and Applications (2022) 34:22367–22386 22382 controllers. It is evident that the proposed HDL-DRNNC is capable of responding to the uncertainty effects (the parameters variation and disturbance) as compared to the DRNNC. where T denotes the sampling period. where T denotes the sampling period. where T denotes the sampling period. where T denotes the sampling period. Figures 6 and 7 exhibit the system response and the control signal for the proposed HDL-DRNNC and DRNNC. It is clear that there is an error between the set- point and the system output under using the DRNNC at the beginning of simulation task. However, the proposed controller using hybrid learning algorithm based on SOM and RBM is able to track the set-point without a steady- state error. The MAE and RMSE values for the proposed HDL- DRNNC scheme are clearly smaller than those obtained for other schemes. Due to use of DL to initialize the weight values of the proposed HDL-DRNNC, it can reduce the impact of the system uncertainties caused by external dis- turbance, parameter variations, and random noise com- pared with other schemes. The hybrid algorithm is used because it gave better results than the DRNNC-based SOM algorithm as shown in Tables 1 and 2. ð49Þ ð50Þ ð49Þ ð50Þ !a j þ 1 ð Þ ¼ G1 j þ 1 ð Þ Tables 1 and 2 illustrate the values of MAE and RMSE for the proposed HDL-DRNNC, the DRNNC and other schemes, that are published previously such as feed-for- ward neural network based on RBM (FFNN-RBM) [45], ERNN-RBM [46], feed-forward neural network with hybrid learning controller (FFNNHLC) [61], FCRNNC [62] and adaptive interval type-2 Takagi–Sugeno–Kang fuzzy logic controller based on reinforcement learning (AIT2-TSK-FLC-RL) [63]. On the other hand, the pro- posed HDL-DRNNC is compared with DRNNC based on SOM (DRNNC-SOM) to show the beneﬁts of hybrid learning. 6.1.2 Task 2: uncertainty due to disturbance To evaluate the robustness of the proposed HDL-DRNNC, a disturbance value of 50% of its desired output is added to the system output at j ¼ 2950. Figure 8 illustrates that the system output tracks the set-point without a steady-state error after adding a disturbance value to the measured output for the proposed HDL-DRNNC. After adding a disturbance value for the DRNNC, there is still a steady- state error. Figure 9 exhibits the control signal of the sys- tem for both controllers. In contrast with the DRNNC, the proposed HDL-DRNNC clearly responds to disturbance effects. 6.1 Case1: mathematical system A non-afﬁne nonlinear system is used to test the perfor- mance of the proposed controller, which is given as [59, 60]: 6.1.4 Task 4: uncertainty due to noise G1 j þ 1 ð Þ ¼ a1 j ð Þ G2 j ð Þ þ a2 j ð Þ sin G1 j ð Þ ð Þ ð48Þ G2 j þ 1 ð Þ ¼ a3 j ð Þ cos G2 j ð Þ ð Þ sin G1 j ð Þ ð Þ þ a4 j ð Þ u j ð Þ þ a5 j ð Þ tanh u j ð Þ ð Þ ð48Þ A random noise signal is added at j ¼ 2950 instant. The output response and the control signal of the system are shown in Figs. 12 and 13, respectively. In contrast to the output response based on DRNNC, the proposed HDL- DRNNC has the ability to recover from the impact of random noise more quickly. The proposed HDL-DRNNC is more robust than the DRNNC. þ a5 j ð Þ tanh u j ð Þ ð Þ ð49Þ !a j þ 1 ð Þ ¼ G1 j þ 1 ð Þ ð50Þ where a1 j ð Þ ¼ 0:5; a2 j ð Þ ¼ 0:3; a3 j ð Þ ¼ 1; a4 j ð Þ ¼ 2 and a5 j ð Þ ¼ 2. ð49Þ ð50Þ The reference signal in this task is deﬁned as: ð51Þ !d j ð Þ ¼ 1 þ 0:5 sin 0:05jT ð Þ ð51Þ !d j ð Þ ¼ 1 þ 0:5 sin 0:05jT ð Þ ð51Þ 6.1.1 Task 1: tracking the reference signal trajectory The reference signal in this task is deﬁned as: 6.2.3 Task 3: uncertainty due to random noise A random noise signal is added at t ¼ 240 sec. The EVS response and its control signal are shown in Figs. 19 and 20. It is clear that the EVS response for the proposed HDL- DRNNC is quickly recovering from the impact of random noise as compared to the output response based on DRNNC. The robustness of the proposed HDL-DRNNC is better than that compared with DRNNC. ð53Þ q h ð Þ ¼ 1 Ca þ Cf 0 2 4 3 5 and Z h ð Þ ¼ h2 ð54Þ C1 ¼ <a þ <f h1 þ Caf h1 h2 ð55Þ C2 ¼ 0:5 q A Cd re G 2 h2 2 þ lrr m g tanh h2 ð Þ ð56Þ ð55Þ The analyses of the MAE and RMSE values for the proposed HDL-DRNNC, the DRNNC and other schemes are presented in Tables 5 and 6. It is clear that the MAE and RMSE values for the proposed HDL-DRNNC are smaller than those obtained for other schemes. Compared with other schemes, HDL-DRNNC has the ability to reduce the impact of system uncertainties. ð56Þ where x and i denote to angular speed and angular speed of the motor. Cf ; Ca; <f and <a denote the ﬁeld inductance, armature inductance, the ﬁeld resistance and armature resistance, respectively.J denotes the inertia of the motor, re denotes the tire radius of the EVS, which includes the tires with gearing system, Caf is the mutual inductance between the armature and the ﬁeld windings, and q, A and m denote the air density, the frontal area of the vehicle and the mass of the EVS, respectively. lrr,B,G and Cd denote the rolling resistance coefﬁcient, the viscous coefﬁcient, the gearing ratio and the drag coefﬁcient, respectively. The values of EVS parameters are listed in Table 3. The main features of the proposed HDL-DRNNC are gathered as follows: (1) It has a swift learning control due to its use of hybrid DL, which uses SOM and RBM to initialize the weights values, (2) the controller is stable as it uses the Lyapunov stability method to update the weight values and it guarantees the stability, and (3) it is suc- cessful for reducing the system uncertainties and tracking the performance output for both mathematical system and physical system. 6.2.1 Task 1: tracking the reference signal trajectory Figures 15 and 16 exhibit the EVS response and its control signal when the desired input is given as: 6.1.3 Task 3: Uncertainty due to disturbance with parameters variation At j ¼ 2950 instant and after the system output reaches the reference input, the system parameters are varied as fol- lows: a1 j ð Þ ¼ 0:35; a2 j ð Þ ¼ 0:35; a3 j ð Þ ¼ At j ¼ 2950 instant and after the system output reaches the reference input, the system parameters are varied as fol- lows: a1 j ð Þ ¼ 0:35; a2 j ð Þ ¼ 0:35; a3 j ð Þ ¼ 1:5; a4 j ð Þ ¼ 2 :5 and a5 j ð Þ ¼ 1 with an effect 40% dis- turbance. The output response and the control signal of the system are shown in Figs. 10 and 11, respectively, for both Neural Computing and Applications (2022) 34:22367 22386 22383 Neural Computing and Applications (2022) 34:22367–22386 6.2.2 Task 2: uncertainty due to parameters variation with disturbance Figure 14 shows the schematic diagram of an EVS and the mathematical model is given as [63–66]: _h ¼V h ð Þ þ q h ð Þu; !a ¼Z h ð Þ ð52Þ ð52Þ In this task, the EVS parameters are varied as in Table 4 with an effect 40% disturbance after the system output reaches the reference input at t ¼ 240 sec. The system response and its control signal for both controllers are exhibited as in Figs. 17 and 18. It is clear that the robust- ness of the proposed HDL-DRNNC is better than the DRNNC due to its ability of reducing the effect of system uncertainties. where h ¼ i x ¼ h1 h2 , V h ð Þ; q h ð Þ and Z h ð Þ are deﬁned as: as: V h ð Þ ¼ 1 Ca þ Cf C1 1 J þ m re G 2 0 B @ 1 C A Caf h2 1 B h2 re G C2 2 666664 3 777775 ð53Þ q h ð Þ ¼ 1 Ca þ Cf 0 2 4 3 5 and Z h ð Þ ¼ h2 ð54Þ C1 ¼ <a þ <f h1 þ Caf h1 h2 ð55Þ C2 ¼ 0:5 q A Cd re G 2 h2 2 þ lrr m g tanh h2 ð Þ ð56Þ V h ð Þ ¼ 1 Ca þ Cf C1 1 J þ m re G 2 0 B @ 1 C A Caf h2 1 B h2 re G C2 2 666664 3 777775 ð 7 Conclusion !d ¼ 0:02t 0\t 210 10 210\t 420 15 420\t 630 0:0237t þ 30 630\t 840 2 840\t 1050 8 > > > > < > > > > : ð57Þ !d ¼ 0:02t 0\t 210 10 210\t 420 15 420\t 630 0:0237t þ 30 630\t 840 2 840\t 1050 8 > > > > < > > > > : ð57Þ In the present paper, the HDL-DRNNC is proposed for nonlinear systems. The HDL-DRNNC uses the DRNN, which can be learned from HDL. In order to guarantee the stability of the proposed controller, the updating weights of the DRNN are derived using the Lyapunov stability crite- rion. Two nonlinear systems, namely mathematical and physical, are used to estimate the performance of the pro- posed controller. According to the obtained results, the proposed HDL-DRNNC can overcome uncertainty and track the performance of the controlled systems. By com- paring MAE and RMSE indicators, it is evident that the response of mathematical and physical systems based on ð57Þ In this task, the set-point changing is carried for testing the proposed HDL-DRNNC, which is compared with the DRNNC. It is clear that the EVS response using the pro- posed HDL-DRNNC reaches the set-point faster than the DRNNC. 123 12 3 3 Neural Computing and Applications (2022) 34:22367–22386 22384 Neurocomputing 321:28–35. https://doi.org/10.1016/j.neucom. 2018.08.034 HDL-DRNNC is able to recover fast from the effects of uncertainties as compared with the response of mathe- matical and physical systems based on DRNNC and other existing controllers. As conclusion, HDL-DRNNC robust- ness has superior performance and a faster ability to recover from uncertainty as compared to DRNNC and other controllers. In the future work, the authors will try to implement practically the proposed algorithm using microcontrollers for controlling a real system. HDL-DRNNC is able to recover fast from the effects of uncertainties as compared with the response of mathe- matical and physical systems based on DRNNC and other existing controllers. As conclusion, HDL-DRNNC robust- ness has superior performance and a faster ability to recover from uncertainty as compared to DRNNC and other controllers. In the future work, the authors will try to implement practically the proposed algorithm using microcontrollers for controlling a real system. 8. Fourati F, Chtourou M (2007) A greenhouse control with feed- forward and recurrent neural networks. Simul Model Pract The- ory 15:1016–1028. https://doi.org/10.1016/j.simpat.2007.06.001 9. 7 Conclusion Kouziokas GN, Chatzigeorgiou A, Perakis K (2018) Multilayer feed forward models in groundwater level forecasting using meteorological data in public management. Water Resour Manag 32:5041–5052. https://doi.org/10.1007/s11269-018-2126-y 10. Shaﬁq MA (2016) Direct adaptive inverse control of nonlinear plants using neural networks. In: Future technologies conference (FTC). IEEE, pp 827–830 11. 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IEEE Trans Industr Inf 8:746–756. https://doi.org/10.1109/TII.2012.2205582 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/. 14. 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https://openalex.org/W3000538307	https://europepmc.org/articles/pmc7151176?pdf=render	English	null	Design and Development of a Wearable Exoskeleton System for Stroke Rehabilitation	Healthcare	2,020	cc-by	12,464	Received: 12 November 2019; Accepted: 9 January 2020; Published: 15 January 2020 Abstract: For more than a decade, many countries have been actively developing robotic assistive devices to assist in the rehabilitation of individuals with limb disability to regain function in the extremities. The exoskeleton assistive device in this study has been designed primarily for hemiplegic stroke patients to aid in the extension of ﬁngers to open up the palm to simulate the eﬀects of rehabilitation. This exoskeleton was designed as an anterior-support type to achieve palmar extension and acts as a robotic assistive device for rehabilitation in bilateral upper limb task training. Testing results show that this wearable exoskeleton assistive device with human factor consideration using percentile dimensions can provide comfortable wear on patients as well as adequate torque to pull individual ﬁngers into ﬂexion towards the palm for rehabilitation. We hope this exoskeleton device can help stroke patients with loss of function in the upper extremities to resume motor activities in order to maintain activities of daily living. Keywords: stroke rehabilitation; wearable assistive device; exoskeleton; 3D printing Healthcare 2020, 8, 18; doi:10.3390/healthcare8010018 healthcare healthcare healthcare www.mdpi.com/journal/healthcare Design and Development of a Wearable Exoskeleton System for Stroke Rehabilitation Yang-Kun Ou 1 , Yu-Lin Wang 2,3,4, Hua-Cheng Chang 5 and Chun-Chih Chen 6,* 6 Research and Development, AirTAC International Group, Tainan 74148, Taiwan * Correspondence: scottfefe@gmail.com Received: 12 November 2019; Accepted: 9 January 2020; Published: 15 January 2020 1. Introduction There are two hundred million people all over the world suﬀering from loss of limb function [1], and most of these functions could be recovered with rehabilitation. Rehabilitation is mainly the use of other objects to force the aﬀected limb to resume activity and has been shown in studies to aid in the paretic limb to recover [2]. Passive and consecutive activities can achieve the eﬀects of physical therapy, can reduce muscle spasticity [3], and can stimulate activity in the cerebral cortex [4]. For more than a decade, many countries around the world are actively developing assistive devices using robotic technologies to help patients with loss of limb function due to various causes to undergo repetitive rehabilitation [5,6]: Jansen et al. designed a particular type of hybrid assistive limb exoskeleton for patients with spinal cord injury undergoing rehabilitation and underwent clinical trial with 21 patients; after training of 90 days, all patients showed signiﬁcant improvement in their functional and ambulatory mobility without the exoskeleton [7]. Many researchers made lower extremity exoskeleton for gait rehabilitation [8–11] with various types of actuators such as regenerative magnetorheological actuator, series elastic actuator, electric motor actuator, etc. Some devices even enhance lower extremity performance [12–15] to provide better mobility to patients with knee injuries Healthcare 2020, 8, 18; doi:10.3390/healthcare8010018 www.mdpi.com/journal/healthcare 2 of 14 Healthcare 2020, 8, 18 or other kinds of loss of function in the lower extremities. For the arms, many types of assistive exoskeleton device have been described [16–21], and most devices can be combined with other adapted equipment. However, only a few assistive exoskeleton rehabilitation devices for the hand have been described, mostly due to the complexity in the structure of the hand and the large range of motion that the ﬁngers, making design for a hand assistive device very diﬃcult. Bataller et al. [22] presented a design for a ﬁnger exoskeleton device with servomotors made from 3D printing that is low in cost and can be mass-produced for sports or rehabilitation for individual ﬁngers. Iqbal et al. [23] described a hand exoskeleton rehabilitation device to facilitate tendon therapy exercises: this device covered only the proximal interphalangeal joint and utilizes the upward- and downward-movements of the said joint to bring about ﬂexion and extension movements. Hence, the use of an exoskeleton assistive device for therapy of the individual with loss of limb function is a method that is both practical and convenient. 1. Introduction Among the many causes of death, cerebrovascular disease places second in the world; colloquially known as “stroke,” it is the rupture of blood vessel in various parts of the brain and is one of the major causes of loss of limb function [24]. Stroke is deﬁned by World Health Organization (WHO) as “rapidly developing clinical signs of focal (or global) disturbance of cerebral function, lasting more than 24 h or leading to death, with no apparent cause other than of vascular origin”. Common symptoms include weakness or numbness in one side of the face or of limbs, diﬃculty in swallowing or speech, vertigo, severe headache, hemiparesis, and loss of intellectual abilities. With recent advances in medicine, most stroke patients survive, but there is often damage to the motor neuron after the acute phase of the disease. It is found that 73–88% stroke survivors suﬀer the sequela of hemiparesis, accompanied by long-term loss of function [25,26]. Recovery after stroke depends on the diﬀerent methods of rehabilitation as well as other treatments [27]. According to the American Heart Association (AHA), 55–75% of stroke patients suﬀer from upper limb dysfunction but persistent rehabilitation can usually recover partial function and only a few could attain complete recovery. The main reason only a few can recover is because most patients after stroke only rely on the unaﬀected side to perform normal daily activities. For example, before stroke occurred, an individual pours water from a pitcher with his right hand and drinks from the cup using the left. But after stroke occurred, his right arm became paretic so he switched to performing both the tasks of pouring and drinking with the left hand. As a result, what started as mere weakness in the right limb, after the transfer of all activities to the unaﬀected left limb, may eventually lose its function completely [28]. Past studies found that only 5–20% patients regain their upper limb functions; by one year after stroke, there are still 33% patients with no function in the upper limb—this shows the diﬃculty in upper limb rehabilitation. Normal upper limb function is a very important key in maintaining independent living; when the upper limb loses its function, activities of daily living are aﬀected, thereby aﬀecting the capability to live independently. This is also the reason behind the lack of patients’ participation in activities. 2. The Human Hand Structure 2. The Human Hand Structure The exoskeleton assistive device is to be worn directly over the hand; therefore, it must take into consideration the range of motion (ROM) and degrees of freedom (DOF) for each and every joint in the hand. With the exception of the thumb, every ﬁnger is made up of 3 joints and 4 bones—the joints of the ﬁngers are metacarpophalangeal (MCP), proximal interphalangeal (PIP), and distal interphalangeal (DIP); the bones of the ﬁngers are the metacarpals, proximal phalanx, middle phalanx, and distal phalanx. The thumb has no middle phalanx and is made up of two joints, metacarpophalangeal (MCP) and interphalangeal (IP). As shown in Figure 1, every MCP has two DOFs, while every PIP, DIP, and IP have one DOF, making up a total of 19 DOFs in each hand. The large number of DOFs makes any assistive device design for the hand quite challenging [36] and is made even more diﬃcult by the complex structure of the bones of the hand: there is a great anatomical variation in the shape and dimensions of individual bones [37], the location on the device where the ﬁnger joint aligns is hard to accommodate to everyone’s hand size; for example, for the PIP, because of the variation in ﬁnger bone length, some may fall near the proximal phalanx while others fall near the distal, and the same scenario also applies to the DIP; while the MCP may not have this problem, because of the variation in palm width, the thumb is often either compressed or too far out and therefore often excluded from exoskeleton designs, making it hard to develop an exoskeleton for rehabilitation that can accommodate a large number of people. The exoskeleton assistive device is to be worn directly over the hand; therefore, it must take into consideration the range of motion (ROM) and degrees of freedom (DOF) for each and every joint in the hand. With the exception of the thumb, every finger is made up of 3 joints and 4 bones—the joints of the fingers are metacarpophalangeal (MCP), proximal interphalangeal (PIP), and distal interphalangeal (DIP); the bones of the fingers are the metacarpals, proximal phalanx, middle phalanx, and distal phalanx. The thumb has no middle phalanx and is made up of two joints, metacarpophalangeal (MCP) and interphalangeal (IP). 1. Introduction , ,  for the design of the exoskeleton assistive device to accommodate approximately 80–90% users;  for the device to be light-weight, low-cost, and easy to fit onto the forearm. 2. The Human Hand Structure 2. The Human Hand Structure As shown in Figure 1, every MCP has two DOFs, while every PIP, DIP, and IP have one DOF, making up a total of 19 DOFs in each hand. The large number of DOFs makes any assistive device design for the hand quite challenging [36] and is made even more difficult by the complex structure of the bones of the hand: there is a great anatomical variation in the shape and dimensions of individual bones [37], the location on the device where the finger joint aligns is hard to accommodate to everyone’s hand size; for example, for the PIP, because of the variation in finger bone length, some may fall near the proximal phalanx while others fall near the distal, and the same scenario also applies to the DIP; while the MCP may not have this problem, because of the variation in palm width, the thumb is often either compressed or too far out and therefore often excluded from exoskeleton designs, making it hard to develop an exoskeleton for rehabilitation that can accommodate a large number of people. Figure 1. The human phalanx and finger joint. Figure 1. The human phalanx and ﬁnger joint. Figure 1 The human phalanx and finger joint Figure 1. The human phalanx and ﬁnger joint. 3 E k l t St t l D i 3. Exoskeleton Structural Design 1. Introduction Therefore, the recovery of upper limb function to restore normal activities is a very important issue [29,30]. In addition to exoskeleton assistive devices, recent years have also seen the development of methods speciﬁcally for limb rehabilitation, such as mirror therapy published in 1999 by Altschuler et al. to train upper limb function in stroke patients [31]: in this method, the paretic hand is kept inside a mirror-box while the mirror reﬂects the image of the normal, non-paretic hand, giving the illusion of it being the paretic hand. The visual eﬀect from the mirrored reﬂection stimulates the premotor are of the brain as well as the posterior prefrontal cortex to engage the patient to perform activities in both hands simultaneously, which will in turn improve the rehabilitation of the paretic hand. This method has been demonstrated to be eﬀective by many studies [32,33]. Furthermore, bilateral training of the upper limbs has also been shown to have a signiﬁcant rehabilitative eﬀect; studies have shown that, when compared with unilateral training, bilateral training can increase the frequency of training and that the eﬀect is signiﬁcantly better than unilateral training [34,35]. Therefore, this study hypothesizes the design of this particular exoskeleton rehabilitation device to achieve the following: 3 of 14 Healthcare 2020, 8, 18 ■ for the healthy (non-paretic) hand to assist the paretic hand to undergo bilateral extension-ﬂexion training simultaneously; ■ for the exoskeleton rehabilitative device to allow also for the rehabilitation of the ﬁngers; ■ for the design of the exoskeleton assistive device to accommodate approximately 80–90% users; ■ for the device to be light-weight, low-cost, and easy to ﬁt onto the forearm. Healthcare 2020, 8, x FOR PEER REVIEW 3 of 13  for the design of the exoskeleton assistive device to accommodate approximately 80–90% users;  for the device to be light-weight, low-cost, and easy to fit onto the forearm. ■ for the healthy (non-paretic) hand to assist the paretic hand to undergo bilateral extension-ﬂexion training simultaneously; Healthcare 2020, 8, x FOR PEER REVIEW 3 of 13 ■ for the exoskeleton rehabilitative device to allow also for the rehabilitation of the ﬁngers; ■ for the design of the exoskeleton assistive device to accommodate approximately 80–90% users; ■ for the device to be light-weight, low-cost, and easy to ﬁt onto the forearm. 3 Exoskeleton Structural Design 3. Exoskeleton Structural Design Schematic of the exoskeleton. The exoskeleton interphalangeal joint is shown in Figure 3. The breadth of the ﬁve-ﬁnger joints was referenced using the largest male ring size. At MCP, the bending angle is set to be 0–70◦, and at PIP, it is set at 0–90◦. Because the mechanical pulling force is exerted only against the palmar surface, the patient’s ﬁnger ﬂexion is unaﬀected, thus allowing for greater room for activity during therapy. Joints at 0◦are equipped with safety baﬄe plates to ensure that the exoskeleton does not cause overextension of the ﬁngers during therapy. The exoskeleton forearm was made with Poly Lactic Acid (PLA) material via 3D printing, with a total length of 290.10 mm and width of 121.87 mm, mainly to assist in ﬁnger movements of the hemiplegic arm. Every ﬁnger joint is equipped with a mechanical connecting rod, and there are 5 sets of servomotors to drive the connecting rod to control movement of every ﬁnger. The mechanical drive is on the middle phalanx where it approximates the PIP, but the Figure 2. Schematic of the exoskeleton. Figure 2. Schematic of the exoskeleton. Figure 2. Schematic of the exoskeleton. Figure 2. Schematic of the exoskeleton. Figure 2. Schematic of the exoskeleton. Figure 2. Schematic of the exoskeleton. The exoskeleton interphalangeal joint is shown in Figure 3. The breadth of the five-finger joints was referenced using the largest male ring size. At MCP, the bending angle is set to be 0–70°, and at PIP, it is set at 0–90°. Because the mechanical pulling force is exerted only against the palmar surface, the patient’s finger flexion is unaffected, thus allowing for greater room for activity during therapy. Joints at 0° are equipped with safety baffle plates to ensure that the exoskeleton does not cause overextension of the fingers during therapy. The exoskeleton forearm was made with Poly Lactic Acid (PLA) material via 3D printing, with a total length of 290.10 mm and width of 121.87 mm, mainly to assist in finger movements of the hemiplegic arm. Every finger joint is equipped with a mechanical connecting rod, and there are 5 sets of servomotors to drive the connecting rod to control movement of every finger. 3 Exoskeleton Structural Design 3. Exoskeleton Structural Design 3. Exoskeleton Structural Design Current exoskeleton assistive devices on the market are fashioned as full skin coverage on the dorsal surface of the hand with a retractive design in which the palmar portion of the fingers are restrained with Velcro fasteners. When making a fist, the exoskeleton usually exerts force from the dorsal portion of the hand and, when extending the fingers, it uses external tension of the exoskeleton to pull on the Velcro fasteners to open up the palm, but this design is complicated by the aforementioned difficulty of varying lengths of finger segments, making it difficult to produce a single device that can fit all sizes. Furthermore, because of hypertonia (spasticity), the paretic hand of hemiplegic patients is clasped into a fist at resting state and it is easy for the hand to form a fist but Current exoskeleton assistive devices on the market are fashioned as full skin coverage on the dorsal surface of the hand with a retractive design in which the palmar portion of the ﬁngers are restrained with Velcro fasteners. When making a ﬁst, the exoskeleton usually exerts force from the dorsal portion of the hand and, when extending the ﬁngers, it uses external tension of the exoskeleton to pull on the Velcro fasteners to open up the palm, but this design is complicated by the aforementioned diﬃculty of varying lengths of ﬁnger segments, making it diﬃcult to produce a single device that can ﬁt all sizes. Furthermore, because of hypertonia (spasticity), the paretic hand of hemiplegic patients is clasped into a ﬁst at resting state and it is easy for the hand to form a ﬁst but extremely diﬃcult 4 of 14 Healthcare 2020, 8, 18 to extend the ﬁngers to open up the palm from a closed ﬁst. It should be kept in mind that the main task in rehabilitation is to assist in allowing the ﬁngers to perform extension and ﬂexion at will. The exoskeleton assistive device presented in this study is designed mainly for hemiplegic stroke patients to simulate a rehabilitation therapy session to achieve ﬁnger extension. The exoskeleton is designed to exert force against the palmar surface of the hand to assist the patient in achieving ﬁnger extension. Figure 2 is a schematic diagram of the phalanx and ﬁnger joints of the exoskeleton. 3 Exoskeleton Structural Design 3. Exoskeleton Structural Design In this design, the phalanx part is made up of only the proximal and middle phalanx, and the PIP on the exoskeleton is where the proximal phalanx approximates the metacarpal bone. The exoskeleton PIP will align directly with the patient’s proximal phalanx; this ensures that, when the patient is wearing the device during therapy, the ﬁnger joint will align with the PIP. The exoskeleton DIP is where the middle phalanx approximates the distal phalanx and, for patients with shorter ﬁngers, may end up aligning with the patient’s distal phalanx (rather than the DIP) but can nevertheless still achieve complete ﬁnger extension. Two sizes—M and L—are set to accommodate users with all glove sizes. The dimensions of our device were based on the Humanscale Manual [38], which contains over 60,000 bits of ergonomic and human engineering statistics for the human head, hands, and feet; is divided into ages 0.5–13 years and adults; and contains length, width, and angle dimensions from the 1st to the 99th percentiles. Dimensions for the M size of our device is based on the female 90th percentile data, whereas the L size is based on the male 90th percentile for individual angles, lengths, and ﬁnger joint widths of the ﬁve ﬁngers of the hand. The exoskeleton thumb is designed as a detachable segment in order to accommodate diﬀerent palm widths; as detailed in Figure 2, the detachable thumb is made with a movable joint that allows for thumb abduction and adduction and has various attachment sites to connect to the main body of the exoskeleton to adapt to diﬀerent palm widths. Healthcare 2020, 8, x FOR PEER REVIEW 4 of 13 The exoskeleton is designed to exert force against the palmar surface of the hand to assist the patient in achieving finger extension. Figure 2 is a schematic diagram of the phalanx and finger joints of the exoskeleton. In this design, the phalanx part is made up of only the proximal and middle phalanx, and the PIP on the exoskeleton is where the proximal phalanx approximates the metacarpal bone. The exoskeleton PIP will align directly with the patient’s proximal phalanx; this ensures that, when the patient is wearing the device during therapy, the finger joint will align with the PIP. 3 Exoskeleton Structural Design 3. Exoskeleton Structural Design The exoskeleton DIP is where the middle phalanx approximates the distal phalanx and, for patients with shorter fingers, may end up aligning with the patient’s distal phalanx (rather than the DIP) but can nevertheless still achieve complete finger extension. Two sizes—M and L—are set to accommodate users with all glove sizes. The dimensions of our device were based on the Humanscale Manual [38], which contains over 60,000 bits of ergonomic and human engineering statistics for the human head, hands, and feet; is divided into ages 0.5–13 years and adults; and contains length, width, and angle dimensions from the 1st to the 99th percentiles. Dimensions for the M size of our device is based on the female 90th percentile data, whereas the L size is based on the male 90th percentile for individual angles, lengths, and finger joint widths of the five fingers of the hand. The exoskeleton thumb is designed as a detachable segment in order to accommodate different palm widths; as detailed in Figure 2, the detachable thumb is made with a movable joint that allows for thumb abduction and adduction and has various attachment sites to connect to the main body of the exoskeleton to adapt to different palm widths. Figure 2. Schematic of the exoskeleton. The exoskeleton interphalangeal joint is shown in Figure 3. The breadth of the five-finger joints was referenced using the largest male ring size. At MCP, the bending angle is set to be 0–70°, and at PIP, it is set at 0–90°. Because the mechanical pulling force is exerted only against the palmar surface, the patient’s finger flexion is unaffected, thus allowing for greater room for activity during therapy. Joints at 0° are equipped with safety baffle plates to ensure that the exoskeleton does not cause overextension of the fingers during therapy. The exoskeleton forearm was made with Poly Lactic Acid (PLA) material via 3D printing, with a total length of 290.10 mm and width of 121.87 mm, mainly to assist in finger movements of the hemiplegic arm. Every finger joint is equipped with a mechanical connecting rod, and there are 5 sets of servomotors to drive the connecting rod to control movement of every finger. The mechanical drive is on the middle phalanx where it approximates the PIP, but the main source of mechanical drive is still located at the PIP and the DIP is linked to the PIP via Figure 2. 3 Exoskeleton Structural Design 3. Exoskeleton Structural Design The mechanical drive is on the middle phalanx where it approximates the PIP, but h f h l d ll l d h I d h I l k d h I The exoskeleton interphalangeal joint is shown in Figure 3. The breadth of the ﬁve-ﬁnger joints was referenced using the largest male ring size. At MCP, the bending angle is set to be 0–70◦, and at PIP, it is set at 0–90◦. Because the mechanical pulling force is exerted only against the palmar surface, the patient’s ﬁnger ﬂexion is unaﬀected, thus allowing for greater room for activity during therapy. Joints at 0◦are equipped with safety baﬄe plates to ensure that the exoskeleton does not cause overextension of the ﬁngers during therapy. The exoskeleton forearm was made with Poly Lactic Acid (PLA) material via 3D printing, with a total length of 290.10 mm and width of 121.87 mm, mainly to assist in ﬁnger movements of the hemiplegic arm. Every ﬁnger joint is equipped with a mechanical connecting rod, and there are 5 sets of servomotors to drive the connecting rod to control movement of every ﬁnger. The mechanical drive is on the middle phalanx where it approximates the PIP, but the The exoskeleton interphalangeal joint is shown in Figure 3. The breadth of the five-finger joints was referenced using the largest male ring size. At MCP, the bending angle is set to be 0–70°, and at PIP, it is set at 0–90°. Because the mechanical pulling force is exerted only against the palmar surface, the patient’s finger flexion is unaffected, thus allowing for greater room for activity during therapy. Joints at 0° are equipped with safety baffle plates to ensure that the exoskeleton does not cause overextension of the fingers during therapy. The exoskeleton forearm was made with Poly Lactic Acid (PLA) material via 3D printing, with a total length of 290.10 mm and width of 121.87 mm, mainly to assist in finger movements of the hemiplegic arm. Every finger joint is equipped with a mechanical connecting rod, and there are 5 sets of servomotors to drive the connecting rod to control movement of every finger. The mechanical drive is on the middle phalanx where it approximates the PIP, but The exoskeleton interphalangeal joint is shown in Figure 3. The breadth of the ﬁve-ﬁnger joints was referenced using the largest male ring size. 3 Exoskeleton Structural Design 3. Exoskeleton Structural Design At MCP, the bending angle is set to be 0–70◦, and at PIP, it is set at 0–90◦. Because the mechanical pulling force is exerted only against the palmar surface, the patient’s ﬁnger ﬂexion is unaﬀected, thus allowing for greater room for activity during therapy. Joints at 0◦are equipped with safety baﬄe plates to ensure that the exoskeleton does not cause overextension of the ﬁngers during therapy. The exoskeleton forearm was made with Poly Lactic Acid (PLA) material via 3D printing, with a total length of 290.10 mm and width of 121.87 mm, mainly to assist in ﬁnger movements of the hemiplegic arm. Every ﬁnger joint is equipped with a mechanical connecting rod, and there are 5 sets of servomotors to drive the connecting rod to control movement of every ﬁnger. The mechanical drive is on the middle phalanx where it approximates the PIP, but the 5 of 14 Healthcare 2020, 8, 18 main source of mechanical drive is still located at the PIP and the DIP is linked to the PIP via connected rods. Every ﬁnger uses one servomotor to achieve extension; when on the highest voltage of 7.4v, the drive is up to 37kg/cm. According to ﬁeld testing, the process of movement is transmitted to the exoskeleton PIP and can provide a pulling force as high as 5 kg. Because, in the hemiplegic patient, the hand muscles have become rigid (spastic) and there may be varying degrees of hemiplegia as well as changes in the grip strength, the paretic hand is often clasped into a ﬁst during therapy. Therefore a microcontroller module is necessary to control the servomotor with a larger torque, of which the internal control is programmed to 0◦at the ﬁnger joint to serve as a limit control so that the motor will automatically stop when the angle of 0◦has been achieved at the ﬁnger joint to avoid injury from overextension of the ﬁngers. Also, there is an external emergency stop button for patients to press when they encounter any discomfort while using the device during a therapy session, which shuts oﬀ the power to the exoskeleton arm. The entire exoskeleton with the motors and electrical wiring weighs a total of 800 g. 3 1 Static Analy Static Analysis y Static analysis was performed using Solidworks on two components of the device: the middle open-up exoskeleton and the control movement point (please refer back to Figure 3) with the following configuration: Static analysis was performed using Solidworks on two components of the device: the middle open-up exoskeleton and the control movement point (please refer back to Figure 3) with the following conﬁguration: 3.1. 3 1 Static Analy Static Analysis Static Analysis Static analysis was performed using Solidworks on two components of the device: the middle k l t d th t l t i t ( l f b k t Fi 3) ith th Material: Acrylonitrile Butadiene Styrene (ABS plastic) Material: Acrylonitrile Butadiene Styrene (ABS plastic) n-up exoskeleton and the control movement point (please refer back to Figure 3) with the owing configuration: Material: Acrylonitrile Butadiene Styrene (ABS plastic) Material: Acrylonitrile Butadiene Styrene (ABS plastic) n-up exoskeleton and the control movement point (please owing configuration: Material: Acrylonitrile Butadiene Styrene (ABS plastic) Material: Acrylonitrile Butadiene Styrene (ABS plastic) n-up exoskeleton and the control movement point (please refer owing configuration: Weight: middle open-up exoskeleton at 2.6 g; control movement point at 1.98 g B d diti fi d d i bl ( l Fi 4) Weight: middle open-up exoskeleton at 2.6 g; control movement point at 1.98 g g g Material: Acrylonitrile Butadiene Styrene (ABS plastic) Boundary conditions: fixed end in blue (please see Figure 4) F t d t bl i d d f f 5 k f d Boundary conditions: ﬁxed end in blue (please see Figure 4) y y ( p ) Weight: middle open-up exoskeleton at 2.6 g; control movem Boundary conditions: fixed end in blue (please see Figure 4) F t d t bl i d d f f 5 k f d Boundary conditions: ﬁxed end in blue (please see Figure 4) y y ( p ) Weight: middle open-up exoskeleton at 2.6 g; control moveme Force exerted at blue: maximum downward force of 5 kgf, designated force of 2 kgf, and safety index 2.5 G id fi it l t l i Force exerted at blue: maximum downward force of 5 kgf, designated force of 2 kgf, and safety index 2.5 Boundary conditions: fixed end in blue (please see Figure 4) Force exerted at blue: maximum downward force of 5 kgf, designated force of 2 kgf, and safety Grid: finite element analysis Grid: ﬁnite element analysis index 2.5 (a) (b) Figure 4. Fixed end in blue for (a) middle open-up exoskeleton and (b) control movement point. Results of stress distribution analysis (Figure 5) with stress concentrator at corner was 1.08 M2 approximating 1 103 kgf/mm2 for the middle open up exoskeleton and 3 54123e + 007 N (a) (b) Figure 4. 3 Exoskeleton Structural Design 3. Exoskeleton Structural Design Healthcare 2020, 8, x FOR PEER REVIEW 5 of 13 patient, the hand muscles have become rigid (spastic) and there may be varying degrees of hemiplegia as well as changes in the grip strength, the paretic hand is often clasped into a fist during therapy. Therefore a microcontroller module is necessary to control the servomotor with a larger torque, of which the internal control is programmed to 0° at the finger joint to serve as a limit control so that the motor will automatically stop when the angle of 0° has been achieved at the finger joint to avoid injury from overextension of the fingers. Also, there is an external emergency stop button for patients to press when they encounter any discomfort while using the device during a therapy session, which shuts off the power to the exoskeleton arm. The entire exoskeleton with the motors and electrical wiring weighs a total of 800 g. Healthcare 2020, 8, x FOR PEER REVIEW 5 of 13 patient, the hand muscles have become rigid (spastic) and there may be varying degrees of hemiplegia as well as changes in the grip strength, the paretic hand is often clasped into a fist during therapy. Therefore a microcontroller module is necessary to control the servomotor with a larger torque, of which the internal control is programmed to 0° at the finger joint to serve as a limit control so that the motor will automatically stop when the angle of 0° has been achieved at the finger joint to avoid injury from overextension of the fingers. Also, there is an external emergency stop button for patients to press when they encounter any discomfort while using the device during a therapy session, which shuts off the power to the exoskeleton arm. The entire exoskeleton with the motors Figure 3. Schematic of the exoskeleton. Figure 3. Schematic of the exoskeleton. a total of 800 g. Figure 3. Schematic of the exoskeleton. Figure 3. Schematic of the exoskeleton. a total of 800 g. Figure 3. Schematic of the exoskeleton. Figure 3. Schematic of the exoskeleton. total of 800 g. Figure 3. Schematic of the exoskeleton. Figure 3. Schematic of the exoskeleton. The exoskeleton assistive device in glove to be worn on the non-paretic han 4. Electromechanical Integration Design The exoskeleton assistive device in 4. Electromechanical Integration Design 6 gauge module (BF350-3AA), which functions mainly to extract the bending angle data during finger extension-flexion of the non-paretic hand and to relay the signal back to the microcontroller module (TI-MSP430). Through an algorithm, the bending angle of each finger in the non-paretic hand is sent via Wi-Fi to the exoskeleton to set the servomotor in motion to transmit the corresponding degree of electrical power to pull on the connecting rods on the device in order to bring about movements in the paretic hand to mimic those of the non-paretic hand while simultaneously collecting signals from the sensors to allow the mimicking movements to occur simultaneously with the non-paretic hand, achieving the effect of mirror therapy in the upper limbs. Figure 7 illustrates the signal transmission. Movements of the upper limb is reconstructed using an algorithm through signal filtering sequence to exclude noise from background and unintentional movements. Feature extraction is used to draw out the feature of each movement in mirror therapy, and feature reduction is used to scale down the computational complexity and to augment movement discrimination. For signal filtering, in order to lower the high-frequency noise error of the signal of the acceleration and the angular velocity, the 6 y p glove to be worn on the non-paretic hand. Each fingertip on the glove is fitted with a set of strain gauge module (BF350-3AA), which functions mainly to extract the bending angle data during finger extension-flexion of the non-paretic hand and to relay the signal back to the microcontroller module (TI-MSP430). Through an algorithm, the bending angle of each finger in the non-paretic hand is sent via Wi-Fi to the exoskeleton to set the servomotor in motion to transmit the corresponding degree of electrical power to pull on the connecting rods on the device in order to bring about movements in the paretic hand to mimic those of the non-paretic hand while simultaneously collecting signals from the sensors to allow the mimicking movements to occur simultaneously with the non-paretic hand, achieving the effect of mirror therapy in the upper limbs. Figure 7 illustrates the signal transmission. Movements of the upper limb is reconstructed using an algorithm through signal filtering sequence to exclude noise from background and unintentional movements. 3 1 Static Analy Static Analysis Fixed end in blue for (a) middle open-up exoskeleton and (b) control movement point. Figure 4. Fixed end in blue for (a) middle open-up exoskeleton and (b) control movement point. (b) oskeleton and (b) control movement point. (b) (a) Figure 4. Fixed end in blue for (a) middle open-u (a) ) contro (b) in blue (a) Results of stress distribution analysis (Figure 5) with stress concentrator at corner was 1. Figure 4. Fixed end in blue for (a) middle open-up exoskeleton and (b) control movement point. Figure 4. Fixed end in blue for (a) middle open-up exoskeleton and (b) control movement point. 6 of 14 Healthcare 2020, 8, 18 Results of stress distribution analysis (Figure 5) with stress concentrator at corner was 1.081 + 07 N/M2 approximating 1.103 kgf/mm2 for the middle open-up exoskeleton and 3.54123e + 007 N/m2 approximating 3.613 kgf/mm2 for the control movement point, inadequate to cause structural collapse for either component. Healthcare 2020, 8, x FOR PEER REVIEW 6 of 14 (a) (b) Figure 5. Stress distribution analysis for (a) middle open-up exoskeleton and (b) control movement point. Comme l Figure 5. Stress distribution analysis for (a) middle open-up exoskeleton and (b) control movement point. (a) (b) Figure 5. Stress distribution analysis for (a) middle open-up exoskeleton and (b) control movement C (a) (b) (a) (b) Figure 5. Stress distribution analysis for (a) middle open-up exoskeleton and (b) control movement point. Comm Figure 5. Stress distribution analysis for (a) middle open-up exoskeleton and (b) control movement point. (a) (b) Figure 5 St e di t ibutio a aly i fo (a) iddle o e u e o keleto a d (b) o t ol o e e t Strain analysis (Figure 6) showed maximum deformation to be 0.16 mm for the middle open-up exoskeleton and 0.11 mm for the control movement point, inadequate to cause structural collapse for either component. please remo Strain analysis (Figure 6) showed maximum deformation to be 0.16 mm for the middle open-up exoskeleton and 0.11 mm for the control movement point, inadequate to cause structural collapse for either component. point. Strain analysis (Figure 6) showed maximum deformation to be 0.16 mm for the middle open-up exoskeleton and 0.11 mm for the control movement point, inadequate to cause structural collapse for either component. Commented please remo (a) (b) Figure 6. 3 1 Static Analy Static Analysis Strain analysis for (a) middle open-up exoskeleton and (b) control movement point 4. Electromechanical Integration Design Th k l t i ti d i i thi t d l i l d h bilit ti i ti C p (a) (b) Figure 6. Strain analysis for (a) middle open-up exoskeleton and (b) control movement point 4 Electromechanical Integration Design C p Figure 6. Strain analysis for (a) middle open-up exoskeleton and (b) control movement point. (b) exoskeleton and (b) control movement point (b) (a) Figure 6. Strain analysis for (a) middle open-up (a) nalysis (a) contro (b) 4. Electromechanical Integration Design Figure 6. Strain analysis for (a) middle open-up exoskeleton and (b) control movement point Figure 6. Strain analysis for (a) middle open-up exoskeleton and (b) control movement point. 4.1. Acceleration and Velocity 4.1. Acceleration and Velocity 4.1. Acceleration and Velocity 4.1. Acceleration and Velocity 4.1. Acceleration and Velocity 4.1. Acceleration and Velocity If the motor angular velocity remains constant (for example, 2 deg/s) and consistent with the speed transmitted to the part of the device in contact with a user’s hand, it would take 10 s for the proximal end of the index finger to achieve full extension from a flexed position, before the velocity increases steadily (Figure 8a), while the thumb would take 9.5 s (Figure 8b), before the velocity also increases steadily. If the motor angular velocity remains constant (for example, 2 deg/s) and consistent with the speed transmitted to the part of the device in contact with a user’s hand, it would take 10 s for the proximal end of the index ﬁnger to achieve full extension from a ﬂexed position, before the velocity increases steadily (Figure 8a), while the thumb would take 9.5 s (Figure 8b), before the velocity also increases steadily. If the motor angular velocity remains constant (for example, 2 deg/s) and consistent with the speed transmitted to the part of the device in contact with a user’s hand, it would take 10 s for the proximal end of the index finger to achieve full extension from a flexed position, before the velocity increases steadily (Figure 8a), while the thumb would take 9.5 s (Figure 8b), before the velocity also increases steadily. (a) (b) Figure 8. (a) Acceleration of exoskeleton index finger. (b) Acceleration of exoskeleton thumb. (a) (b) Figure 8. (a) Acceleration of exoskeleton index finger. (b) Acceleration of exoskeleton thumb. Figure 8. (a) Acceleration of exoskeleton index ﬁnger. (b) Acceleration of exoskeleton thumb. (b) (b) (a) (a) (b) (b) (a) (a) Figure 8. (a) Acceleration of exoskeleton index finger. (b) Acceleration of exoskeleton thumb. Figure 8. (a) Acceleration of exoskeleton index finger. (b) Acceleration of exoskeleton thumb. Figure 8. (a) Acceleration of exoskeleton index ﬁnger. (b) Acceleration of exoskeleton thumb. The exoskeleton assistive device in glove to be worn on the non-paretic han 4. Electromechanical Integration Design The exoskeleton assistive device in 4. Electromechanical Integration Design Feature extraction is used to draw out the feature of each movement in mirror therapy, and feature reduction is used to scale down the computational complexity and to augment movement discrimination. For signal filtering, in order to lower the high-frequency noise error of the signal of the acceleration and the angular velocity, the The exoskeleton assistive device in this study also includes a neoprene rehabilitation assistive glove to be worn on the non-paretic hand. Each ﬁngertip on the glove is ﬁtted with a set of strain gauge module (BF350-3AA), which functions mainly to extract the bending angle data during ﬁnger extension-ﬂexion of the non-paretic hand and to relay the signal back to the microcontroller module (TI-MSP430). Through an algorithm, the bending angle of each ﬁnger in the non-paretic hand is sent via Wi-Fi to the exoskeleton to set the servomotor in motion to transmit the corresponding degree of electrical power to pull on the connecting rods on the device in order to bring about movements in the paretic hand to mimic those of the non-paretic hand while simultaneously collecting signals from the sensors to allow the mimicking movements to occur simultaneously with the non-paretic hand, achieving the eﬀect of mirror therapy in the upper limbs. Figure 7 illustrates the signal transmission. Movements of the upper limb is reconstructed using an algorithm through signal ﬁltering sequence to exclude noise from background and unintentional movements. Feature extraction is used to draw out the feature of each movement in mirror therapy, and feature reduction is used to scale down the computational complexity and to augment movement discrimination. For signal ﬁltering, in order to lower the high-frequency noise error of the signal of the acceleration and the angular velocity, the calibrated signal needs to go through a low-pass ﬁlter (for example, moving average ﬁlter, Butterworth ﬁlter, or Chebyshev ﬁlter) to ﬁlter high-frequency noises. 7 of 14 7 of 13 Healthcare 2020, 8, 18 Healthcare 2020 8 x FO Figure 7. Schematic of signal transmission. Figure 7. Schematic of signal transmission. Figure 7. Schematic of signal transmission. Figure 7. Schematic of signal transmission. Figure 7. Schematic of signal transmission. Figure 7. Schematic of signal transmission. p g y 5 1 P D fi i i 5.1. Parameter Deﬁnitions The exoskeleton in th 5.1. Parameter Definitions The exoskeleton in this study includes only the proximal and middle phalanges; the parameters of the finger joint are defined as shown in Figure 10. Point O is the point where the lower-support joint attaches to the core of the exoskeleton, akin to the MCP joint of the hand, and therefore serves as the origin of the coordinates where the X and Y axes are both zero. Point A is the fixing point for the connecting rod where the relationship between the MCP and PCP is controlled; point B is the position of the PIP at rest, while B’ is the shifted PIP position after movement. Similarly, C is the position of DIP at rest, while C’ is the shifted DIP position after movement. Point C is the terminal end of the exoskeleton when it moves the connecting rod moves with it and affects the rest of the finger joints. OB തതതത is the proximal phalanx, BC തതതത is the middle phalanx, θଵ is the DOF of the MCP as well as change in MCP angle, whereas θଶ is that of PIP. The relationships between every point, line, and angle are known, and every phalangeal joint has only one DOF, with a total of 10 DOFs. Therefore the actuation of the device is movement on a level plane, where finger joint movement is brought about using mechanical connecting rods; therefore, θଶ changes in accordance with θଵ and the l d iti f h fi t f th ti t b i di t d i th t ti The exoskeleton in this study includes only the proximal and middle phalanges; the parameters of the ﬁnger joint are deﬁned as shown in Figure 10. Point O is the point where the lower-support joint attaches to the core of the exoskeleton, akin to the MCP joint of the hand, and therefore serves as the origin of the coordinates where the X and Y axes are both zero. Point A is the ﬁxing point for the connecting rod where the relationship between the MCP and PCP is controlled; point B is the position of the PIP at rest, while B’ is the shifted PIP position after movement. Similarly, C is the position of DIP at rest, while C’ is the shifted DIP position after movement. p g y 5 1 P D fi i i 5.1. Parameter Deﬁnitions The exoskeleton in th Point C is the terminal end of the exoskeleton when it moves the connecting rod moves with it and aﬀects the rest of the ﬁnger joints. OB is the proximal phalanx, BC is the middle phalanx, θ1 is the DOF of the MCP as well as change in MCP angle, whereas θ2 is that of PIP. The relationships between every point, line, and angle are known, and every phalangeal joint has only one DOF, with a total of 10 DOFs. Therefore the actuation of the device is movement on a level plane, where ﬁnger joint movement is brought about using mechanical connecting rods; therefore, θ2 changes in accordance with θ1 and the angles and positions of each ﬁnger movement of the patient can be indicated using the actuation tracks of points O, B, and C. of the finger joint are defined as shown in Figure 10. Point O is the point where the lower-support joint attaches to the core of the exoskeleton, akin to the MCP joint of the hand, and therefore serves as the origin of the coordinates where the X and Y axes are both zero. Point A is the fixing point for the connecting rod where the relationship between the MCP and PCP is controlled; point B is the position of the PIP at rest, while B’ is the shifted PIP position after movement. Similarly, C is the position of DIP at rest, while C’ is the shifted DIP position after movement. Point C is the terminal end of the exoskeleton when it moves the connecting rod moves with it and affects the rest of the finger joints. OB തതതത is the proximal phalanx, BC തതതത is the middle phalanx, θଵ is the DOF of the MCP as well as change in MCP angle, whereas θଶ is that of PIP. The relationships between every point, line, and angle are known, and every phalangeal joint has only one DOF, with a total of 10 DOFs. Therefore the actuation of the device is movement on a level plane, where finger joint movement is brought about using mechanical connecting rods; therefore, θଶ changes in accordance with θଵ and the angles and positions of each finger movement of the patient can be indicated using the actuation tracks of points O, B, and C. ach finger movement of the patient can be ind C. Figure 10. Schematic of finger joint parameters. 4 2 Force Sensitive Resistor Sensor 4.2. Force Sensitive Resistor Sensor 4.2. Force Sensitive Resistor Sensor 4.2. Force Sensitive Resistor Sensor Five sensors were installed to measure force sensitivity of the hands against the exoskeleton device and found that resistance and force have an inverse and linear relationship with the R2 for the five sensors ranging between 0.9213 and 0.9588, as shown in Figure 9, where the y-axis is the force sensing resistance in Ohms and the x-axis is the force in kgf Five sensors were installed to measure force sensitivity of the hands against the exoskeleton device and found that resistance and force have an inverse and linear relationship with the R2 for the five sensors ranging between 0.9213 and 0.9588, as shown in Figure 9, where the y-axis is the force sensing resistance in Ohms and the x-axis is the force in kgf. Five sensors were installed to measure force sensitivity of the hands against the exoskeleton device and found that resistance and force have an inverse and linear relationship with the R2 for the ﬁve sensors ranging between 0.9213 and 0.9588, as shown in Figure 9, where the y-axis is the force sensing resistance in Ohms and the x-axis is the force in kgf. 8 of 14 8 of 13 Healthcare 2020, 8, 18 H l h 2020 8 FO 18 Healthcare 2020, 8, x FOR PEER REVIEW Figure 9. Force sensitivity analysis. 10 11 12 13 14 15 16 17 18 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 Sensor1 Sensor2 Sensor3 Sensor4 Sensor5 Figure 9. Force sensitivity analysis. Figure 9. Force sensitivity analysis. 5. Operating Analysis 10 11 12 13 14 15 16 17 18 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 Sensor1 Sensor2 Sensor3 Sensor4 Sensor5 Healthcare 2020, 8, x FOR PEER REVIEW Figure 9. Force sensitivity analysis. 10 11 12 13 14 15 16 17 18 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 Sensor1 Sensor2 Sensor3 Sensor4 Sensor5 Figure 9. Force sensitivity analysis. Figure 9. Force sensitivity analysis. 5. Operating Analysis 10 11 12 13 14 15 16 17 18 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 Sensor1 Sensor2 Sensor3 Sensor4 Sensor5 5.2. Analysis of Movement 5.2. Analysis of Movement The relationship between points O and B is a linear one and between points B and C; OB and BC are indicated with the distance formulae below: The relationship between points O and B is a linear one and between points B and C; OB തതതത and BC തതതത are indicated with the distance formulae below: The relationship between points O and B is a linear one and between points B and C; OB and BC are indicated with the distance formulae below: The relationship between points O and B is a linear one and between points B and C; OB തതതത and BC തതതത are indicated with the distance formulae below: OB = q (XB −XO)2 + (YB −YO)2 (1) BC = q (XC −XB)2 + (YC −YB)2 (2) OB തതതത= ඥ(𝑋஻−𝑋ை)ଶ+ (𝑌஻−𝑌ை)ଶ (1) BC തതതത= ඥ(𝑋஼−𝑋஻)ଶ+ (𝑌஼−𝑌஻)ଶ (2) Bᇱ dictates movement changes of OB തതതത, Bᇱ is B multiplied by the the formula is denoted by Rot(θ) as follows: (1) BC = q (XC −XB)2 + (YC −YB)2 (2) Bᇱ dictates movement changes of OB തതതത, Bᇱ is B multiplied by the the formula is denoted by Rot(θ) as follows: (2) the Whereas the position of B′ dictates movement changes of OB, B′ is B multiplied by the rotation matrix of θ1, of which the formula is denoted by Rot(θ) as follows: Bᇱ= B(x, y) × Rot(θଵ) (3) Whereas the position of B′ dictates movement changes of OB, B′ is B multiplied by the rotation matrix of θ1, of which the formula is denoted by Rot(θ) as follows: Bᇱ= B(x, y) × Rot(θଵ) (3) B′ = B(x, y) × Rot(θ1) (3) is more complex: as shown in Figure 5, movement of C s of θଵ , θଶ, and point B, and its position is in turn altered when (3) of C when The movement pattern by C′ is more complex: as shown in Figure 5, movement of C simultaneously aﬀects the parameters of θ1, θ2, and point B, and its position is in turn altered when B transforms into B′. The relationship between points C′ and O is formulated below: B transforms into Bᇱ. 5.2. Analysis of Movement 5.2. Analysis of Movement The relationship between points Cᇱ and O is formulated below: Cᇱ ୓ (x, y)= Bᇱ ୓ (x, y)+Rotை(θଵ)× Cᇱ(x, y) ୆ᇲ (4) OC′(x, y)= OB′(x, y)+RotO(θ1)×B′C′(x, y) (4) nal end of the exoskeleton assistive device, its movement affects other s post-movement position can be used to back-trace positions and angles (4) ther l Since C′ is the terminal end of the exoskeleton assistive device, its movement aﬀects other parameters the most and its post-movement position can be used to back-trace positions and angles of other points. p p p p g of other points. 6. Test Results p g y 5 1 P D fi i i 5.1. Parameter Deﬁnitions The exoskeleton in th Figure 10. Schematic of ﬁnger joint parameters. Figure 10. Schematic of finger joint parameters. Figure 10. Schematic of ﬁnger joint parameters. Healthcare 2020, 8, 18 9 of 14 5.2. Analysis of Movement 5.2. Analysis of Movement Simulated Results The relations various parts of th However, while individual ﬁngers are moving, other ﬁngers also bend slightly—this phenomenon is most apparent in the ring ﬁnger and is caused by the connectedness among tendons in the human hand and is part of a normal ﬁnger reaction. It also shows that the bending angle algorithm can gauge in a precise manner the changes in the hand when bending and can respond appropriately. Healthcare 2020, 8, x FOR PEER REVIEW 10 of 13 precise manner during simulation. However, while individual fingers are moving, other fingers also bend slightly—this phenomenon is most apparent in the ring finger and is caused by the connectedness among tendons in the human hand and is part of a normal finger reaction. It also shows that the bending angle algorithm can gauge in a precise manner the changes in the hand when bending and can respond appropriately. Healthcare 2020, 8, x FOR PEER REVIEW 10 of 13 precise manner during simulation. However, while individual fingers are moving, other fingers also bend slightly—this phenomenon is most apparent in the ring finger and is caused by the connectedness among tendons in the human hand and is part of a normal finger reaction. It also shows that the bending angle algorithm can gauge in a precise manner the changes in the hand when Figure 12. Actual movements of each finger joint. Figure 12. Actual movements of each ﬁnger joint. bending and can respond appropriately. Figure 12. Actual movements of each finger joint. Figure 12. Actual movements of each finger joint. Figure 12. Actual movements of each ﬁnger joint. Figure 12. Actual movements of each finger joint. When comparing the tracks from movements of the hand with formulae-derived position points of each phalangeal joint, as an example illustrated in Figure 13, it can be seen that the movement arcs for points Bᇱ and Cᇱ are identical to those from operating the assistive glove. Since the use of different methods to validate the movement patterns result in the same movement arc, it therefore confirms the appropriateness and the practicality of the constructs in this study. What should also not be overlooked is that it provides greater DOFs to the finger joints and its cost is cheaper compared to other exoskeleton devices on the market. Simulated Results The relations various parts of th The range of motion in other existing exoskeleton models fall between 0~55° for MCP and 0~65° for PIP [39]; in contrast, the exoskeleton in this study offers 0~70° for MCP and 0~90° for PIP and is contrary to other designs that exclude the thumb or utilize a fixed thumb [39,40]. The exoskeleton in this study offers a range of motion up to 35° for the thumb— a greater angle means more room for motion and can provide better rehabilitation results for stroke patients with upper limb hemiplegia, as detailed in Table 1. When comparing the tracks from movements of the hand with formulae-derived position points of each phalangeal joint, as an example illustrated in Figure 13, it can be seen that the movement arcs for points B′ and C′ are identical to those from operating the assistive glove. Since the use of diﬀerent methods to validate the movement patterns result in the same movement arc, it therefore conﬁrms the appropriateness and the practicality of the constructs in this study. What should also not be overlooked is that it provides greater DOFs to the ﬁnger joints and its cost is cheaper compared to other exoskeleton devices on the market. The range of motion in other existing exoskeleton models fall between 0∼55◦for MCP and 0∼65◦for PIP [39]; in contrast, the exoskeleton in this study oﬀers 0∼70◦ for MCP and 0∼90◦for PIP and is contrary to other designs that exclude the thumb or utilize a ﬁxed thumb [39,40]. The exoskeleton in this study oﬀers a range of motion up to 35◦for the thumb—a greater angle means more room for motion and can provide better rehabilitation results for stroke patients with upper limb hemiplegia, as detailed in Table 1. When comparing the tracks from movements of the hand with formulae-derived position points of each phalangeal joint, as an example illustrated in Figure 13, it can be seen that the movement arcs for points Bᇱ and Cᇱ are identical to those from operating the assistive glove. Since the use of different methods to validate the movement patterns result in the same movement arc, it therefore confirms the appropriateness and the practicality of the constructs in this study. What should also not be overlooked is that it provides greater DOFs to the finger joints and its cost is cheaper compared to other exoskeleton devices on the market. Simulated Results The relations various parts of th Simulated Results The relations various parts of th The relationship formulae between the points can be used to obtain the dimensions of the various parts of the exoskeleton assistive device; and with the aforementioned range of bending angle in MCP and PIP, the position and angle of individual points during movement could be simulated and compared to actual measurements. various parts of the exoskeleton assistive device; and with the aforementioned range of bending angle in MCP and PIP, the position and angle of individual points during movement could be simulated and compared to actual measurements. Figure 11 is the scatterplot based on alternating between the minimum and maximum angles of θଵfrom 0~70° Post-movement points Bᇱand Cᇱcomputed from Equations (3) and (4) using the Figure 11 is the scatterplot based on alternating between the minimum and maximum angles of θ1 from 0∼70◦. Post-movement points B′ and C′ computed from Equations (3) and (4) using the positions of B and C and the varying angles of θ1 are compared to those from the outer appearance based on Solidworks design layout. The positions of B′ and C′ simulated from the formulae as a result of movement matched completely with the movement arc on the design layout. θଵ from 0 70 . Post movement points B and C computed from Equations (3) and (4) using the positions of B and C and the varying angles of θଵ are compared to those from the outer appearance based on Solidworks design layout. The positions of Bᇱ and Cᇱ simulated from the formulae as a result of movement matched completely with the movement arc on the design layout. Figure 11. Schematic of simulated finger joint movement arc. Figure 11. Schematic of simulated ﬁnger joint movement arc. Figure 11. Schematic of simulated finger joint movement arc. Figure 11. Schematic of simulated ﬁnger joint movement arc. 10 of 14 10 of 14 Healthcare 2020, 8, 18 Figure 12 demonstrates the actual movement of individual ﬁngers. In this demonstration, the assistive glove is worn on the left hand while the exoskeleton device is on the right. The bending angle of ﬁngers in the left hand drives varying degrees of bending, and the ﬁngers can move in a precise manner during simulation. 7. Discussion and Conclusions The design concept of the exoskeleton assistive device in this study stems from multiple medical studies for the rehabilitation of hemiplegic stroke patients; therefore, it can provide a better therapeutic eﬀect in the rehabilitation process. The assistive device weighs only around 800 g in its entirety; is portable; provides a more powerful torque to pull on the ﬁngers; and can accommodate hemiplegic stroke patients with varying degrees of disease severity, diﬀering palm sizes, diﬀering ﬁnger segment lengths, and diﬀering ﬁnger breadth to cater to most patients. As a whole, this device is more than capable of achieving therapeutic goals in addition to being safe and convenient to use and can easily be adapted for general use. Currently, the prooﬁng of all parts for testing purposes brings the cost to within $650 USD, which is lower than the market price; furthermore, other current exoskeleton rehabilitation devices are mostly used in rehabilitation institutions, while the device in this study oﬀers hemiplegic stroke patients the option to undergo rehabilitation in the comfort of his or her own home and anticipates to improve further on the portability, safety, and cost to allow patients to use the device at home for self-rehabilitation. This paper describes the design of an exoskeleton assistive device for the hand based on principles of mirror therapy with an innovative design, in which ﬁnger movements are powered from the palmar side (hence, the term “lower-support type”) and was made from 3D printing while able to retain structural integrity as demonstrated by static analysis and force sensitivity analysis. Three-dimensional printing is low in cost and therefore could easily be made widely accessible; therefore, this device can oﬀer the most beneﬁt at a reduced cost for upper extremity rehabilitation and hereafter can improve the function and the quality of life of patients. Many current studies of exoskeleton rehabilitation devices remain at the testing level in institutions and cannot capture problems and diﬃculties encountered in real-life use, but the assistive device in this study has already worked with several hospitals for on-site testing and is in the process of improving the structural design using feedback from real-life testing. Stroke rehabilitation is a rather dull process that is ongoing and repetitive, making it diﬃcult for patients to go through the entire process with patience. Simulated Results The relations various parts of th The range of motion in other existing exoskeleton models fall between 0~55° for MCP and 0~65° for PIP [39]; in contrast, the exoskeleton in this study offers 0~70° for MCP and 0~90° for PIP and is contrary to other designs that exclude the thumb or utilize a fixed thumb [39,40]. The exoskeleton in this study offers a range of motion up to 35° for the thumb— a greater angle means more room for motion and can provide better rehabilitation results for stroke patients with upper limb hemiplegia, as detailed in Table 1. Figure 13. Actual movement of the finger joint arc. bl 1 C i f k l t h bilit ti d i Figure 13. Actual movement of the finger joint arc. Figure 13. Actual movement of the ﬁnger joint arc. Figure 13. Actual movement of the finger joint arc. Figure 13. Actual movement of the finger joint arc. Figure 13. Actual movement of the ﬁnger joint arc. Figure 13. Actual movement of the finger joint arc. Figure 13. Actual movement of the finger joint arc. Figure 13. Actual movement of the ﬁnger joint arc. 11 of 14 Healthcare 2020, 8, 18 Table 1. Comparison of exoskeleton rehabilitation devices. Table 1. Comparison of exoskeleton rehabilitation devices. Table 1. Comparison of exoskeleton rehabilitation devices. Labels MCP PIP DIP Transverse DOF—Thumb Weight Clinical Testing The design in this study 70◦ 90◦ N.A. 35◦ 800g No Susanto et al., 2015 [39] 55◦ 65◦ N.A. N.A. >1kg Yes Pu et al., 2014 [40] 90◦ 80◦ 100◦ N.A. 700g No Note: MCP = metacarpophalangeal joint; PIP = proximal interphalangeal joint; DIP = distal interphalangeal joint; DOF = degree of freedom. References 1. Braithwaite, J.; Mont, D. Disability and Poverty: A Survey of World Bank Poverty Assessment and Implications; World Bank: Washington, DC, USA, 2008. 1. Braithwaite, J.; Mont, D. Disability and Poverty: A Survey of World Bank Poverty Assessment and Implications; World Bank: Washington, DC, USA, 2008. 1. Braithwaite, J.; Mont, D. 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Discussion and Conclusions In order to motivate stroke patients to actively participate in the rehabilitation process, further developments may see the addition of VR (virtual reality) elements to enrich the rehabilitation experience to speed up patient recovery. Because VR can incorporate entertaining game themes, can increase the level of attention in stroke patients during therapy, can reduce the sense of loss from the loss of function due to the disease, and is signiﬁcantly more eﬀective compared to conventional therapy [41,42], it is set to be the next direction of this study. Author Contributions: Conceptualization, Y.-K.O.; methodology, Y.-K.O.; hardware design, C.-C.C.; software design, H.-C.C.; collected the data, Y.-L.W.; writing—original draft preparation, Y.-K.O.; writing—review and editing, Y.-K.O. and C.-C.C. All authors have read and agreed to the published version of the manuscript. Funding: This study was partially funded by the Ministry of Science and Technology of Taiwan (MOST) (grant number 106-2221-E-218-020-MY3) and by the Allied Advanced Intelligent Biomedical Research Center (A2IBRC) under the Higher Education Sprout Project of the Ministry of Education. Acknowledgments: We would like to thank three anonymous reviewers and the editors for their comments. Acknowledgments: We would like to thank three anonymous reviewers and the editors for their comments. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 12 of 14 Healthcare 2020, 8, 18 References Nef, T.; Guidali, M.; Riener, R. ARMin III—Arm therapy exoskeleton with an ergonomic shoulder actuation. Appl. Bionics Biomech. 2009, 6, 127–142. [CrossRef] 19. Nef, T.; Guidali, M.; Riener, R. ARMin III—Arm therapy exoskeleton with an ergonomic shoulder actuation. Appl. Bionics Biomech. 2009, 6, 127–142. [CrossRef] 20. 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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2419163642	http://publikationen.ub.uni-frankfurt.de/files/44529/elife-14618.pdf	English	null	A mitofusin-dependent docking ring complex triggers mitochondrial fusion in vitro	eLife	2,016	cc-by	13,510	Competing interest: See page 21 Mitochondria constitute a remarkably dynamic network with an organization and ultra-structure that is regulated by fusion and fission of mitochondrial outer and inner membranes (Labbe´ et al., 2014; Westermann, 2010). Fusion and fission are crucial for all mitochondrial functions including oxidative phosphorylation, calcium signalling, apoptosis and lipid metabolism. Defects in mitochon- drial fusion and fission are associated with numerous pathologies and severe neurodegenerative dis- eases (Dorn, 2013; Liesa et al., 2009). Received: 21 January 2016 Accepted: 01 June 2016 Published: 02 June 2016 Received: 21 January 2016 Accepted: 01 June 2016 Published: 02 June 2016 Mitochondrial fusion and fission both depend on large GTPases of the Dynamin-Related Protein (DRP) family (Labbe´ et al., 2014; Low and Lo¨we, 2010). To promote fission, soluble DRPs assemble into spirals around membrane compartments. GTP hydrolysis causes the spirals to constrict, reduc- ing the diameter of the compartments, ultimately followed by their separation (Bui and Shaw, 2013). In contrast to fission, the role of DRPs in lipid bilayer fusion remains poorly understood. Among the three families of transmembrane DRPs implicated in fusion, Mitofusins and OPA1 medi- ate fusion of the mitochondrial outer and inner membranes, respectively, whereas atlastins promote homotypic fusion of ER tubules (McNew et al., 2013). GTP binding and hydrolysis participate in trans auto-oligomerization of atlastins and OPA1 through their respective GTPase domains (Rujiviphat et al., 2012; Klemm et al., 2011; Byrnes et al., 2013; DeVay et al., 2009; Meglei and McQuibban, 2009; Moss et al., 2011; Saini et al., 2014). The resulting homotypic tethering of ER RESEARCH ARTICLE A mitofusin-dependent docking ring complex triggers mitochondrial fusion in vitro Tobias Brandt1†, Laetitia Cavellini2†, Werner Ku¨ hlbrandt1, Mickae¨ l M Cohen2 1Max Planck Institute of Biophysics, Frankfurt, Germany; 2Laboratoire de Biologie Mole´ culaire et Cellulaire des Eucaryotes, Institut de Biologie Physico-Chimique, Sorbonne Universite´ s, Paris, France Abstract Fusion of mitochondrial outer membranes is crucial for proper organelle function and involves large GTPases called mitofusins. The discrete steps that allow mitochondria to attach to one another and merge their outer membranes are unknown. By combining an in vitro mitochondrial fusion assay with electron cryo-tomography (cryo-ET), we visualize the junction between attached mitochondria isolated from Saccharomyces cerevisiae and observe complexes that mediate this attachment. We find that cycles of GTP hydrolysis induce progressive formation of a docking ring structure around extended areas of contact. Further GTP hydrolysis triggers local outer membrane fusion at the periphery of the contact region. These findings unravel key features of mitofusin-dependent fusion of outer membranes and constitute an important advance in our understanding of how mitochondria connect and merge. DOI: 10.7554/eLife.14618.001 For correspondence: werner. kuehlbrandt@biophys.mpg.de (WK); cohen@ibpc.fr (MMC) †These authors contributed equally to this work Competing interest: See page 21 Funding: See page 21 Received: 21 January 2016 Accepted: 01 June 2016 Published: 02 June 2016 Introduction Membrane fusion underlies fundamental biological processes such as fertilization, virus entry into host cells or intracellular protein trafficking. Protein and lipid trafficking mainly involves SNAREs (Sol- uble N-ethyl maleimide sensitive factor Attachment protein Receptors) that are expressed on all intracellular compartments undergoing fusion except peroxisomes and mitochondria (Cai et al., 2007; Escobar-Henriques and Anton, 2013). †These authors contributed equally to this work Reviewing editor: Nikolaus Pfanner, University of Freiburg, Germany Copyright Brandt 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. Brandt et al. eLife 2016;5:e14618. DOI: 10.7554/eLife.14618 1 of 23 Research article Research article Biophysics and structural biology Cell biology eLife digest Yeast and other eukaryotic cells contain distinct compartments that have specific roles. For example, compartments called mitochondria – which are surrounded by two layers of membrane – provide the energy needed for many cell processes. The organization of the network of mitochondria in a cell has a large effect on their capacity to provide energy. Mitochondria can fuse together to make larger compartments or divide to make smaller ones. Defects in fusion or division of mitochondria can reduce the amount of energy that is provided, which, in humans and animals can lead to diseases that affect various organs, especially those in the nervous system. When two mitochondria fuse they must first attach to each other and then merge their outer membranes. Proteins called mitofusins are known to be involved in these processes, but the molecular details of how they take place were not clear. Brandt, Cavellini et al. investigated how mitochondria isolated from budding yeast cells attach to each other. The experiments found that two mitochondria first become loosely attached by mitofusins. These proteins then promote a tighter attachment in which the outer membranes of the two mitochondria come into contact over a larger area. This contact area is determined by a linear arrangement of proteins referred to as the docking ring. Brandt, Cavellini et al. further observed that local fusion between the outer membranes takes place at the edge of the contact area in the path of the docking ring. Future research will need to address how mitochondria attach to each other in living cells a how the process is regulated. and mitochondrial inner membranes is accompanied by conformational rearrangements of the DRPs that are thought to trigger subsequent fusion of lipid bilayers. Based on structural insights gained from BDLP (Bacterial Dynamin-Like Protein) (Low and Lo¨we, 2006; Low et al., 2009), a close rela- tive of the yeast mitofusin Fzo1, mitofusins may promote outer membrane tethering and fusion through similar processes of oligomerization and conformational rearrangement (Cohen et al., 2011; Escobar-Henriques and Anton, 2013). Reviewing editor: Nikolaus Pfanner, University of Freiburg, Germany As seen with spirals formed during membrane scission, DRPs are characterized by their ability to assemble into higher-order macromolecular structures (Ingerman et al., 2005; Low et al., 2009; Mears and Hinshaw, 2008). Whether DRPs participate in the formation of such structures during membrane attachment and fusion is unknown. In particular, the precise orchestration of events from the initial attachment of membranes to their ultimate fusion and the requirement for GTP binding and hydrolysis during these steps is elusive. By combining the power of an in vitro mitochondrial fusion assay with cryo-electron tomography (cryo-ET), we undertook to visualize the junction between mitochondria, resulting in an unprecedented dissection of the outer membrane fusion process. Results Cryo-ET reveals distinct populations of attached mitochondria The in vitro mitochondrial fusion assay (Figure 1A) allows us to distinguish between discrete steps of the mitochondrial fusion process (i.e. attachment, fusion of outer membranes and fusion of inner membranes) (Hoppins et al., 2009; Meeusen et al., 2006, 2004). Purified mitochondria from wild- type yeast cells were brought into contact by centrifugation and were incubated at 4˚C for 10 min to promote mitofusin-dependent attachment (Meeusen et al., 2004). Subsequent incubation for 45 min at 25˚C allows fusion of outer membranes but not inner membranes (Figure 1A; top) unless energy is regenerated (Meeusen et al., 2004). Consistent with this, fusion reactions recurrently yielded 6 to 8% intermediates with fused outer membranes (Figure 1B). Prolonged incubation of centrifuged mitochondria at 4˚C (Figure 1A; bottom) decreases fusion of outer membranes but sta- bilizes attached intermediates (Cohen et al., 2011) with approximately 25% of mitochondria in close contact (Figure 1C). We reasoned that with an imaging technique of sufficient resolution, it should Brandt et al. eLife 2016;5:e14618. DOI: 10.7554/eLife.14618 2 of 23 Research article Biophysics and structural biology Cell biology Biophysics and structural biology Cell biology Figure 1. In vitro outer membrane fusion and attachment assays. (A) Purified mitochondria are brought into contact by incubation on ice promotes mitofusin-dependent attachment, which is essential for subsequent fusion of outer membra Prolonged incubation on ice prevents fusion of outer membranes but stabilizes attached intermediates (bottom). Upon inner membranes does not occur unless energy is regenerated. (B) Top: Fusion reactions were performed by mixing mit expressing either the outer membrane protein OM45 tagged with GFP (OM45-GFP) or the mitochondrial matrix targete Bottom: Fluorescence microscopy of a fusion reaction. Co-localization of GFP and mCherry indicate intermediates with f arrows), scale bars 1 mm. Top right: Fusion efficiency. Error bar represents the s.d. from three independent experiments. Figure 1. In vitro outer membrane fusion and attachment assays. (A) Purified mitochondria are brought into contact by centrifugation. A 10 min incubation on ice promotes mitofusin-dependent attachment, which is essential for subsequent fusion of outer membranes at room temperature (top). Prolonged incubation on ice prevents fusion of outer membranes but stabilizes attached intermediates (bottom). Upon incubation at 25˚C, fusion of inner membranes does not occur unless energy is regenerated. Figure 1. In vitro outer membrane fusion and attachment assays. (A) Purified mitochondria are brought into contact by centrifugation. A 10 min incubation on ice promotes mitofusin-dependent attachment, which is essential for subsequent fusion of outer membranes at room temperature (top). Prolonged incubation on ice prevents fusion of outer membranes but stabilizes attached intermediates (bottom). Upon incubation at 25˚C, fusion of inner membranes does not occur unless energy is regenerated. (B) Top: Fusion reactions were performed by mixing mitochondria isolated from cells expressing either the outer membrane protein OM45 tagged with GFP (OM45-GFP) or the mitochondrial matrix targeted mCherry (Mito-mCherry). Bottom: Fluorescence microscopy of a fusion reaction. Co-localization of GFP and mCherry indicate intermediates with fused outer membranes (white arrows), scale bars 1 mm. Top right: Fusion efficiency. Error bar represents the s.d. from three independent experiments. (C) Representative transmission electron micrograph of in vitro attachment reactions with mitochondria isolated from wild-type cells. DOI: 10 7554/eLife 14618 003 Results (B) Top: Fusion reactions were performed by mixing mitochondria isolated from cells expressing either the outer membrane protein OM45 tagged with GFP (OM45-GFP) or the mitochondrial matrix targeted mCherry (Mito-mCherry). Bottom: Fluorescence microscopy of a fusion reaction. Co-localization of GFP and mCherry indicate intermediates with fused outer membranes (white arrows), scale bars 1 mm. Top right: Fusion efficiency. Error bar represents the s.d. from three independent experiments. (C) Representative transmission electron micrograph of in vitro attachment reactions with mitochondria isolated from wild-type cells. DOI 10 7554/ Lif 14618 003 Figure 1. In vitro outer membrane fusion and attachment assays. (A) Purified mitochondria are brought into contact by centrifugation. A 10 min incubation on ice promotes mitofusin-dependent attachment, which is essential for subsequent fusion of outer membranes at room temperature (top). Prolonged incubation on ice prevents fusion of outer membranes but stabilizes attached intermediates (bottom). Upon incubation at 25˚C, fusion of inner membranes does not occur unless energy is regenerated. (B) Top: Fusion reactions were performed by mixing mitochondria isolated from cells expressing either the outer membrane protein OM45 tagged with GFP (OM45-GFP) or the mitochondrial matrix targeted mCherry (Mito-mCherry). Bottom: Fluorescence microscopy of a fusion reaction. Co-localization of GFP and mCherry indicate intermediates with fused outer membranes (white arrows), scale bars 1 mm. Top right: Fusion efficiency. Error bar represents the s.d. from three independent experiments. (C) Representative transmission electron micrograph of in vitro attachment reactions with mitochondria isolated from wild-type cells. DOI: 10.7554/eLife.14618.003 Brandt et al. eLife 2016;5:e14618. DOI: 10.7554/eLife.14618 3 of 23 Brandt et al. eLife 2016;5:e14618. DOI: 10.7554/eLife.14618 3 of 23 Brandt et al. eLife 2016;5:e14618. DOI: 10.7554/eLife.14618 Research article Biophysics and structural biology Cell biology Figure 2. Cryo-ET of docked intermediates (79% of sampled wild-type mitochondria, see Table 1). (A at different z-heights (section planes indicated in B) through the center (left) or edge (right) of a cont and the perpendicular minor axis lb (see B); scale bars 100 nm. Dense protein complexes (yellow arro outer membrane (dl, blue bracket) but not in the center of the contact area (1–3 nm, green bracket, d (right) reveal interstitial densities between the outer membranes. (B) 3D rendering of outer membran Figure 2. Cryo-ET of docked intermediates (79% of sampled wild-type mitochondria, see Table 1). (A) Slices and zooms through tomographic volumes at different z-heights (section planes indicated in B) through the center (left) or edge (right) of a contact area defined by its major axis, la (red bracket), and the perpendicular minor axis lb (see B); scale bars 100 nm. Dense protein complexes (yellow arrows) are visible at a distance of 6–9 nm from the outer membrane (dl, blue bracket) but not in the center of the contact area (1–3 nm, green bracket, ds). Sections through the edge of a contact area (right) reveal interstitial densities between the outer membranes. (B) 3D rendering of outer membranes (red and orange) of two closely apposed mitochondria and protein densities around the contacts area (blue; the same color scheme is used in all figures). DOI: 10 7554/eLife 14618 004 Figure 2. Cryo-ET of docked intermediates (79% of sampled wild-type mitochondria, see Table 1). (A) Slices and zooms through tomographic volumes at different z-heights (section planes indicated in B) through the center (left) or edge (right) of a contact area defined by its major axis, la (red bracket), and the perpendicular minor axis lb (see B); scale bars 100 nm. Dense protein complexes (yellow arrows) are visible at a distance of 6–9 nm from the outer membrane (dl, blue bracket) but not in the center of the contact area (1–3 nm, green bracket, ds). Sections through the edge of a contact area (right) reveal interstitial densities between the outer membranes. (B) 3D rendering of outer membranes (red and orange) of two closely apposed mitochondria and protein densities around the contacts area (blue; the same color scheme is used in all figures). Brandt et al. eLife 2016;5:e14618. DOI: 10.7554/eLife.14618 DOI: 10 7554/eLife 14618 004 Figure 2 continued on next page 4 of 23 4 of 23 Brandt et al. eLife 2016;5:e14618. DOI: 10.7554/eLife.14618 Brandt et al. eLife 2016;5:e14618. DOI: 10.7554/eLife.14618 Brandt et al. eLife 2016;5:e14618. DOI: 10.7554/eLife.14618 Figure 2 continued The following figure supplement is available for figure 2: Figure supplement 1. Cryo-ET of major mitochondrial population of wild-type attachment intermediates (Docked, 79%, see Table 1). DOI: 10.7554/eLife.14618.005 Research article Biophysics and structural biology Cell biology Biophysics and structural biology Cell biology Biophysics and structural biology Cell biology plement 1. Cryo-ET of major mitochondrial population of wild-type attachment intermediates (Docked, 79%, see Table 1). be possible to visualize the contact sites and detect complexes that mediate mitochondrial attachment. Cryo-ET revealed two main populations of attached mitochondria, characterized by morphologi- cally distinct contacts between outer membranes. 79% of the contact areas were organized as regions where opposing outer membranes approached one another to a distance ds of 1–3 nm (Figure 2A, green bracket; Figure 2—figure supplement 1A), without observable density between the membranes. The contact areas displayed an average surface of 22,600 ± 3100 nm2 with flattened outer membranes, prompting us to term this state ‘docked’ intermediates (Figure 2A). At the edges of the contact areas, where the distance dl between outer membranes reached 6–9 nm (Figure 2A, blue brackets; Figure 2—figure supplement 1B), we detected defined protein densities between the two outer membranes (Figure 2A; yellow arrows). Tomographic reconstruction (Figure 2—fig- ure supplement 1C) and 3D rendered volumes (Figure 2B) of mitochondria revealed that contact areas are delimited by a dense ring-like structure, termed the docking ring (Video 1). Contact areas of the remaining 21% of attached mitochondria were smaller (6900 ± 1700 nm2) with an average distance of 6.7 ± 0.5 nm between outer membranes (Figure 3). In contrast to docked intermediates, there were clear densities between the apposed outer membranes over the complete contact area (Figure 3A; Figure 3—figure supplement 1). Frequently, this density appeared to consist of more or less regular repeats of globular protein units (Figure 3B; red arrows). These units, with an average spacing of 4.6 ± 0.7 nm (n = 13), occasionally extended two protrusions toward each membrane (Figure 3B; inserted zoom). We refer to these attached mitochondria as tethered intermediates, as they appeared to be tethered by proteins. GTP hydrolysis is required for formation of docking rings y y q g g The ring of densities (docked intermediates, Figure 2B) and the regular repeat of globular protein densities (tethered intermediates, Figure 3) indicate that protein complexes may be responsible for promoting mitochondrial attachment. As the homotypic attachment of outer membranes and their subsequent fusion depends on the GTPase activity of mitofusins (Cohen et al., 2011; Koshiba et al., 2004), we assessed the impact of GTP hydrolysis on the formation of the tethered and docked inter- mediates. Cryo-ET of in vitro attachment reactions treated with a non-hydrolysable GTP analog (GMP-PNP) revealed two distinct populations of attached mitochondria with different distances between their outer membranes (Figure 4). g In contrast to non-treated samples, only 14% of mitochondria formed contacts reminiscent of docked intermediates and with significantly smaller contact areas (Table 1). In the remaining 86% of attached mitochondria, the outer membranes were 6.3 ± 0.2 nm apart (Figure 4A, blue bracket). Densities were observed between apposed membranes with frequent repeats of 3–4 nm globular structures with, occasionally, two pro- trusions extending towards each membrane (Figure 4A, inserted zoom and Figure 4B, red arrows). These densities and their regular spac- ing (4.3 ± 0.6 nm; n = 24) were strikingly similar to those seen in wild-type tethered intermedi- ates (Figure 3B). Moreover, among the attached intermediates we identified (Table 1), this sub- g In contrast to non-treated samples, only 14% of mitochondria formed contacts reminiscent of docked intermediates and with significantly smaller contact areas (Table 1). In the remaining 86% of attached mitochondria, the outer membranes were 6.3 ± 0.2 nm apart (Figure 4A, blue bracket). Densities were observed between apposed membranes with frequent repeats of 3–4 nm globular structures with, occasionally, two pro- trusions extending towards each membrane (Figure 4A, inserted zoom and Figure 4B, red arrows) These densities and their regular spac Video 1. 3D rendering of docked mitochondria as shown in Figure 2. Outer membranes in red and orange, distinct density in blue. DOI: 10.7554/eLife.14618.006 5 of 23 Brandt et al. eLife 2016;5:e14618. DOI: 10.7554/eLife.14618 Research article Research article Biophysics and structural biology Ce ure 3. Cryo-ET analysis of tethered intermediates (21% of sampled wild-type mitochondria, see Table 1). (A) 3D rendering of two closely app ochondria shown in B, top row and Figure 3—figure supplement 1. (B) Slices and zooms (indicated by black and red dashed boxes) through mographic volumes; scale bars 100 nm. Figure 3. Cryo-ET analysis of tethered intermediates (21% of sampled wild-type mitochondria, see Table 1). (A) 3D rendering of two closely apposed mitochondria shown in B, top row and Figure 3—figure supplement 1. (B) Slices and zooms (indicated by black and red dashed boxes) through tomographic volumes; scale bars 100 nm. Blue bracket: outer membrane distance; red arrows, interstitial density. DOI: 10.7554/eLife.14618.007 Figure supplement 1. Cryo-ET analysis of the minor mitochondrial population of wild-type attachment intermediates (Tethered, 21%, see Table 1). DOI: 10.7554/eLife.14618.008 GTP hydrolysis is required for formation of docking rings Blue bracket: outer membrane distance; red arrows, interstitial density. OI: 10.7554/eLife.14618.007 e following figure supplement is available for figure 3: ure supplement 1. Cryo-ET analysis of the minor mitochondrial population of wild-type attachment intermediates (Tethered, 21%, see Table OI: 10.7554/eLife.14618.008 Research article Biophysics and structural biology Cell Biophysics and structural biology Cell biology Biophysics and structural biology Cell biology Figure 3. Cryo-ET analysis of tethered intermediates (21% of sampled wild-type mitochondria, see Table 1). (A) 3D rendering of two closely apposed mitochondria shown in B, top row and Figure 3—figure supplement 1. (B) Slices and zooms (indicated by black and red dashed boxes) through tomographic volumes; scale bars 100 nm. Blue bracket: outer membrane distance; red arrows, interstitial density. DOI 10 7554/ Lif 14618 007 Figure 3. Cryo-ET analysis of tethered intermediates (21% of sampled wild-type mitochondria, see Table 1). (A) 3D rendering of two closely apposed mitochondria shown in B, top row and Figure 3—figure supplement 1. (B) Slices and zooms (indicated by black and red dashed boxes) through tomographic volumes; scale bars 100 nm. Blue bracket: outer membrane distance; red arrows, interstitial density. DOI: 10.7554/eLife.14618.007 6 of 23 6 of 23 Figure 3. Cryo-ET analysis of tethered intermediates (21% of sampled wild-type mitochondria, see Table 1). (A) 3D rendering of two closely apposed mitochondria shown in B, top row and Figure 3—figure supplement 1. (B) Slices and zooms (indicated by black and red dashed boxes) through tomographic volumes; scale bars 100 nm. Blue bracket: outer membrane distance; red arrows, interstitial density. DOI: 10.7554/eLife.14618.007 The following figure supplement is available for figure 3: Figure supplement 1. Cryo-ET analysis of the minor mitochondrial population of wild-type attachment intermediates (Tethered, 21%, see Table 1). Brandt et al. eLife 2016;5:e14618. DOI: 10.7554/eLife.14618 Brandt et al. eLife 2016;5:e14618. DOI: 10.7554/eLife.14618 Research article Research article Biophysics and structural biology Cell biology Figure 4. Cryo-ET of mitochondrial attachment intermediates upon GMP-PNP treatment (Tethered, 86%, see Table 1). (A-B) Example slices an (black and red dashed boxes) through tomographic volumes; scale bars 100 nm; blue bracket: distance between outer membranes; red arrows regularly spaced interstitial protein densities. Note the repeat distance of 4.3 nm. (C) 3D rendering of two closely apposed mitochondria shown The zoomed densities correspond to the regularly spaced interstitial protein densities zoomed in A. (D) Histogram of distances between outer membranes for all major populations of attached intermediates Figure 4. Cryo-ET of mitochondrial attachment intermediates upon GMP-PNP treatment (Tethered, 86%, see Table 1). (A-B) Example slices and zooms (black and red dashed boxes) through tomographic volumes; scale bars 100 nm; blue bracket: distance between outer membranes; red arrows: regularly spaced interstitial protein densities. Note the repeat distance of 4.3 nm. (C) 3D rendering of two closely apposed mitochondria shown in A. The zoomed densities correspond to the regularly spaced interstitial protein densities zoomed in A. (D) Histogram of distances between outer membranes for all major populations of attached intermediates. DOI: 10.7554/eLife.14618.009 The following figure supplement is available for figure 4: Figure 4 continued on next page Figure 4. Cryo-ET of mitochondrial attachment intermediates upon GMP-PNP treatment (Tethered, 86%, see Table 1). (A-B) Example slices and zooms (black and red dashed boxes) through tomographic volumes; scale bars 100 nm; blue bracket: distance between outer membranes; red arrows: regularly spaced interstitial protein densities. Note the repeat distance of 4.3 nm. (C) 3D rendering of two closely apposed mitochondria shown in A. The zoomed densities correspond to the regularly spaced interstitial protein densities zoomed in A. (D) Histogram of distances between outer membranes for all major populations of attached intermediates. DOI: 10 7554/eLife 14618 009 The following figure supplement is available for figure 4: Figure 4 continued on next page 7 of 23 Brandt et al. eLife 2016;5:e14618. DOI: 10.7554/eLife.14618 Research article Biophysics and structural biology Cell biology Figure 4 continued Figure supplement 1. Cryo-ET of a tethered mitochondrial intermediate upon GMP-PNP treatment (Tethered, 86%, see Table 1). DOI: 10.7554/eLife.14618.010 Figure supplement 1. Cryo-ET of a tethered mitochondrial intermediate upon GMP-PNP treatment (Tethered, 86%, see Table 1). DOI: 10.7554/eLife.14618.010 1A; Video 2) and statistics (Figure 4D; Figure 4—figure supplement 1B–D). Brandt et al. eLife 2016;5:e14618. DOI: 10.7554/eLife.14618 These observations suggest that inhibition of GTP hydrolysis induces the accumulation of teth- ered mitochondria at the expense of docked intermediates (21% tethered intermediates in wild-type conditions against 86% upon GMP-PNP treatment). GTP hydrolysis mediates the transition from globular protein repeats to docking rings To find out if inhibition of GTP hydrolysis blocks formation of the docking ring at an early stage of the mitochondrial attachment process, we introduced GMP-PNP at distinct time points of the in vitro fusion reaction and analyzed the ratios of tethered and docked intermediates by cryo-ET (Figure 5A). Docked intermediates were predominantly found when GMP-PNP was added 10, 20 or 40 min after centrifugation (Figure 5B, blue squares), indicating that the ring had been already formed and that it was not dissolved by GMP-PNP. In contrast, addition of GMP-PNP prior to or immediately after centrifugation resulted in strong accumulation of tethered intermediates (Figure 5B, red circles), while docked intermediates were suppressed. Strikingly, addition of GMP- PNP two minutes after centrifugation resulted in equal proportions of tethered and docked inter- mediates (Figure 5B). These observations demonstrate not only that tethering precedes docking, but also that transi- tion from one state to the other is controlled by GTP hydrolysis. Consequently, the tethered interme- diate with its repeating globular protein densities represents an early stage of the mitochondrial outer membrane fusion process. GTP hydrolysis then allows this stage to evolve towards the docked state with the docking ring. Consistent with this, a discrete category of intermediates was detected among the mixed popula- tion of tethered and docked intermediates when GMP-PNP was added two minutes after centrifuga- tion. This category was characterized by apposed membrane regions sandwiching protein densities identical to those in tethered intermediates and regions reminiscent of docked contact areas where outer membranes approached to within less than 3 nm (Figure 5—figure supplement 1 and Video 3). This hybrid category of attached mitochondria probably represents a transition state from mitochondrial tethering towards mitochondrial docking. Brandt et al. eLife 2016;5:e14618. DOI: 10.7554/eLife.14618 Biophysics and structural biology Cell biology Biophysics and structural biology Cell biology The question of how docking promotes the formation of intermediates with fused outer membranes (Figure 6—figure supplement 1) was nonetheless puzzling. Fusion may occur any- where within the contact area devoid of visible densities because this area corresponds to regions where membrane contact is closest. Alternatively, the docking ring might trigger fusion over the whole rim of the contact area, similar to the ’vertex ring’ that assembles at the edge of docked vacuoles (Wang et al., 2002; Wickner, 2010). This ring, composed of SNAREs, small GTPases, tethering factors and lipid microdomains, promotes membrane fusion at the periphery of contact regions between vacuoles, generating a lumenal vesicle that degrades in the fused organelle. In the context of mitochondrial fusion, such an intralumenal outer membrane vesicle would block the subsequent attachment of inner membranes. Indeed, we did not find fused outer membrane intermediates of this kind. Video 2. 3D rendering of tethered mitochondria upon addition of GMP-PNP as shown in Figure 4. Outer membranes in red and orange, distinct density in blue. DOI: 10.7554/eLife.14618.012 Biophysics and structural biology Cell biology Video 2. 3D rendering of tethered mitochondria upon addition of GMP-PNP as shown in Figure 4. Outer membranes in red and orange, distinct density in blue. DOI: 10.7554/eLife.14618.012 Video 2. 3D rendering of tethered mitochondria upon addition of GMP-PNP as shown in Figure 4. Outer membranes in red and orange, distinct density in blue. DOI: 10.7554/eLife.14618.012 vacuoles, generating a lumenal vesicle that degrades in the fused organelle. In the context of mitochondrial fusion, such an intralumenal outer membrane vesicle would block the subsequent attachment of inner membranes. Indeed, we did not find fused outer membrane intermediates of this kind. However, close inspection of large populations of attached intermediates (from 87 high magnifi- cation micrographs) by Transmission Electron Microscopy (TEM) of stained plastic sections revealed a sub-category of mitochondria in close contact, presumably in the docked configuration, in which the outer membranes were fused near the rim of the contact region (Figure 6—figure supplement 2A and B). While the switch from docking to fusion is bound to be a rapid, transient process and cryo-ET can only sample specimen volumes of the order of 10–7 nanoliters, we succeeded in captur- ing such a docked intermediate. final GTP hydrolysis step triggers the transition from docking to sion Of all attached mitochondria, docked intermediates would be the most suitable for outer membrane fusion. The large contact surface and close approach of outer membranes would poise them for fusion. The proportion of docked intermediates increases when outer membrane fusion can proceed but decreases when fusion is inhibited, as we showed by inhibiting GTP hydrolysis. Table 1. Characteristics of mitochondrial attachment intermediates identified in this study. Abbreviations: o.e. overexpression, i.a. inter alia. Table 1. Characteristics of mitochondrial attachment intermediates identified in this study. Abbreviations: o.e. overexpression, i.a. inter alia. Condition (n) Contact type (n) % Densities organization Distance ± SE / nm Contact area ± SE / nm2 Wild-Type (52) Docked (41) 79 Docking ring 2.1 ± 0.1 22600 ± 3100 Tethered (11) 21 Interstitial/Few 6.7 ± 0.5 6900 ± 1700 Wild-Type + GMP-PNP (22) Tethered (19) 86 Interstitial/Few 6.3 ± 0.2 10100 ± 1300 Other (3) 14 i.a. Docking ring 2.6 ± 0.3 11600 ± 3900 Fzo1 O.E. (19) Abortive (14) 74 Interstitial/ Numerous 8.8 ± 0.4 5000 ± 900 Other (5) 26 Docking ring 2.5 ± 0.4 4200 ± 1200 DOI: 10.7554/eLife.14618.011 DOI: 10.7554/eLife.14618 8 of 23 Brandt et al. eLife 2016;5:e14618. DOI: 10.7554/eLife.14618 8 of 23 Research article Biophysics and structural biology Cell biology The tomographic volume shows that the outer membranes of two mitochondria were locally fused to form a toroid, 40 nm pore on one side of the contact area (Figure 6A). The toroid pore formed in the path of the docking ring (Figure 6B and Video 4). Densi- ties in the vicinity of the fusion pore were sparse, suggesting that in this region the docking ring structure was in the process of disassembly (Figure 6B). These observations provide a proof of prin- ciple that the docked intermediates are competent for effective outer membrane fusion, raising the question of which molecular events trigger the transition from docking to outer membrane fusion. q gg g To this end, we compared the effect of GMP-PNP on in vitro fusion efficiency either before teth- ering (i.e. after centrifugation; see Figure 5B) or after docking (i.e. after 10 min incubation on ice; see Figure 5B) (Figure 6C; see Figure 1B). Strikingly, the extent of outer membrane fusion impairment was similar (40 to 50%), irrespective of whether GMP-PNP was added at the beginning or at the end of the 10 min incubation period (Figure 6D). This indicates that it is not important whether the mitochondria were tethered or docked. Hence, GTP hydrolysis is not only required for the transition from tethering to docking but also for the transition from docking to fusion. Fzo1 overexpression inhibits mitochondrial docking To further evaluate the functional relationship between mitofusins, mitochondrial docking and mito- chondrial fusion, we took advantage of the fact that both the absence or the accumulation of mitofu- sins inhibits mitochondrial fusion in vivo (Cohen et al., 2011; Escobar-Henriques et al., 2006; Koshiba et al., 2004). Consistent with this, absence or 50-fold overexpression of Fzo1 (Figure 7A; fzo14 and FZO1 o.e.) totally abolished respiratory growth at 30˚C (Figure 7B). In the absence of Fzo1, cryo-ET analysis revealed small mitochondria well separated from each other but mitochondrial attachment was not detected (Figure 7—figure supplement 1A), which is consistent with the essential function of mitofusins in mitochondrial anchoring (Cohen et al., 2011; Koshiba et al., 2004). On the other hand, the fusion defect caused by the overexpression of Fzo1 may result from the accumulation of Fzo1 molecules on outer membranes and an imbalance with other proteins implicated in mitochondrial fusion, such as Ugo1 and Mgm1 (Figure 7—figure sup- plement 1B). Notably, Fzo1 overexpression also induces a specific phenotype of mitochondrial aggregation (Figure 7C) in which mitochondrial puncta aggregate in one region of the cell cortex (Figure 7—figure supplement 1C). This phenotype suggests that, as well as inhibiting fusion, over- expression of Fzo1 may also promote the attachment of mitochondria to each other. Brandt et al. eLife 2016;5:e14618. DOI: 10.7554/eLife.14618 9 of 23 Research article Research article Research article Biophysics and structural biology Cell biology Figure 5. Cryo-ET time course experiment of attached mitochondria treated with GMP-PNP. (A) GMP-PNP was added at the time points indicated (dashed black lines). (B) Proportion of docked and tethered intermediates observed before and after GMP-PNP addition. Time t = 0 indicates the start of the incubation period on ice. DOI: 10.7554/eLife.14618.013 The following figure supplement is available for figure 5: Figure supplement 1. Hybrid intermediate captured upon GMP-PNP addition two minutes after centrifugation. Figure 5. Cryo-ET time course experiment of attached mitochondria treated with GMP-PNP. (A) GMP-PNP was added at the time points indicated (dashed black lines). (B) Proportion of docked and tethered intermediates observed before and after GMP-PNP addition. Time t = 0 indicates the start of the incubation period on ice. DOI: 10.7554/eLife.14618.013 The following figure supplement is available for figure 5: Figure supplement 1. Hybrid intermediate captured upon GMP-PNP addition two minutes after centrifugation. DOI: 10.7554/eLife.14618.014 Figure supplement 1. Hybrid intermediate captured upon GMP-PNP addition two minutes after centrifugation. Brandt et al. eLife 2016;5:e14618. DOI: 10.7554/eLife.14618 Fzo1 enrichment at mitochondrial contact sites Densities detected at the junctions of tethered and docked mitochondria correspond to protein complexes responsible for promoting productive attachment of outer membranes prior to their fusion. Several protein factors either within or extrinsic to the mitochondrial outer membrane may assemble into such complexes (Coonrod et al., 2007; Hoppins et al., 2009; Sesaki and Jensen, 2001; 2004). Notably, the units with a defined central density and protrusions extending towards each lipid bilayer bridged the apposed outer membranes (Figure 8A). These units were stabilized in the presence of GMP-PNP (Figure 4) and their arrangement may reflect the self-association in trans of a factor extruding from outer membranes (Figure 8B). These features thus point to a transmem- brane GTPase specialized in connecting outer membranes, raising the possibility that the observed units are indeed trans-oligomers of Fzo1 molecules. The presence and active involvement of mitofusins in mitochondrial tethering and docking is not only consistent with the established function of these DRPs in attachment and fusion of outer mem- branes (Cohen et al., 2011; Hermann et al., 1998; Ishihara et al., 2004; Koshiba et al., 2004; Legros et al., 2002; Shutt et al., 2012) but also with their documented accumulation at mitochon- drial junctions (Hoppins et al., 2009). To validate Fzo1 as a potential component of the densities found at mitochondrial contact sites, we thus devised an in situ protein labeling strategy for cryo-ET. Recent experiments to label mitochondria for cryo-ET with a purified, biotin-labelled protein import substrate conjugated with streptavidin-coated Quantum Dots have been successful (Gold et al., 2014). To biotinylate mitofusin molecules in situ, Fzo1 was C-terminally fused to the Avi tag, a 15 amino-acid peptide that is recognized and can undergo specific biotinylation by the E. coli biotin ligase BirA (Beckett et al., 1999; van Werven and Timmers, 2006). Mitochondria isolated from wild-type (FZO1) or FZO1-Avi cells were biotinylated in vitro with recombinant BirA before process- ing for outer membrane attachment assays, followed by incubation with streptavidin-coupled Q-Dots to label the Fzo1-Avi construct (Figure 8—figure supplement 1). While Q-Dots were rarely found on the surface of tagged or untagged mitochondria (Figure 8C and D, right graph), they were enriched at the junction of attached FZO1-Avi mitochondria as compared to attached FZO1 mitochondria (Figure 8D, left graph). Moreover, Q-Dots, and therefore the FZO1-Avi constructs, were located either at the periphery of docked contact sites or between tethered contact sites (Figure 8E). Biophysics and structural biology Cell biology To better characterize these perturbations, attached mitochondria were analyzed by cryo- ET. While intermediates with a docking ring were formed (Figure 7—figure supplement 2A–C), they were observed less frequently (26%) and the average contact surface was significantly smaller compared to docking intermediates observed under wild-type conditions (Table 1). In the remaining attached intermediates (74%), the surface of apposition was also reduced (4970 ± 930 nm2), with outer membranes on average 8.8 ± 0.4 nm apart (Figure 7E; blue bracket; Figure 7—figure supplement 2D–E). Importantly, numerous densities accumulated in regions of closest contact between mitochondria (Figure 7F), but the densities were disorganized and did not form globular protein repeats (Figure 7E–F). These attached mitochondria, which were clearly distinct from tethered intermediates observed under wild-type conditions, likely correspond to artefactual and abortive fusion intermediates. Video 3. 3D Rendering of tethered intermediate upon addition of GMP-PNP after two minutes, as shown in Figure 5—figure supplement 1. Outer membranes in red and orange, distinct density in blue. DOI: 10.7554/eLife.14618.015 Video 3. 3D Rendering of tethered intermediate upon addition of GMP-PNP after two minutes, as shown in Figure 5—figure supplement 1. Outer membranes in red and orange, distinct density in blue. DOI: 10.7554/eLife.14618.015 and did not form globular protein repeats (Figure 7E–F). These attached mitochondria, which were clearly distinct from tethered intermediates observed under wild-type conditions, likely correspond to artefactual and abortive fusion intermediates. These results indicate that while normal levels of Fzo1 are required for the formation of bona fide docking rings, overexpression of the mitofusin induces the formation of protein aggregates that per- turb the regulated sequence of events required to reach productive mitochondrial docking. Fzo1 overexpression inhibits mitochondrial docking DOI: 10.7554/eLife.14618.014 To verify this, in vitro fusion assays were performed using mitochondria isolated from cells overex- pressing Fzo1. As expected, in vitro outer membrane fusion was strongly inhibited but mitochondrial attachment was significantly increased, with 50% of mitochondria attached to others (Figure 7D and Figure 7—figure supplement 1D). Consistent with their compromised fusing ability, mitochondria from Fzo1-overexpressing cells were significantly smaller than controls. Further analysis indicated that Fzo1 overexpression induced a two-fold increase in mitochondrial attachment both before and after centrifugation (Figure 7—figure supplement 1E–F). Thus, mitochondria purified from Fzo1- overexpressing cells retained an increased attachment capacity, indicating that the in vivo aggrega- tion phenotype (Figure 7C) is caused, at least in part, by perturbations that are intrinsic to outer membranes. 10 of 23 Brandt et al. eLife 2016;5:e14618. DOI: 10.7554/eLife.14618 Research article Fzo1 enrichment at mitochondrial contact sites These data confirm the accumulation of mitofusins at mitochondrial contact sites and corroborate the likely contribution of Fzo1 to densities found at junctions of tethered and docked intermediates. Brandt et al. eLife 2016;5:e14618. DOI: 10.7554/eLife.14618 11 of 23 Research article Research article Biophysics and structural biology Cell biology Figure 6. Stages of outer membrane fusion. (A and B) Cryo-ET of docked mitochondria with partially fused outer membrane. (A) Slices through tomographic volume; scale bars 100 nm. The red circle highlights the region of outer membrane fusion. (B) 3D rendering. Two mitochondria are joined by one continuous outer membrane (red). The inter-membrane spaces are connected by a toroid pore of 40 nm diameter. (C and D) Fluorescence microscopy of in vitro outer membrane fusion. GMP-PNP was added at the beginning (tethering, T) or at the end (docking, D) of the 10 min incubation period. DOI 10 7554/ Lif 14618 016 Figure 6. Stages of outer membrane fusion. (A and B) Cryo-ET of docked mitochondria with partially fused outer membrane. (A) Slices through tomographic volume; scale bars 100 nm. The red circle highlights the region of outer membrane fusion. (B) 3D rendering. Two mitochondria are joined by one continuous outer membrane (red). The inter-membrane spaces are connected by a toroid pore of 40 nm diameter. (C and D) Fluorescence Figure 6. Stages of outer membrane fusion. (A and B) Cryo-ET of docked mitochondria with partially fused outer membrane. (A) Slices through tomographic volume; scale bars 100 nm. The red circle highlights the region of outer membrane fusion. (B) 3D rendering. Two mitochondria are joined by one continuous outer membrane (red). The inter-membrane spaces are connected by a toroid pore of 40 nm diameter. (C and D) Fluorescence microscopy of in vitro outer membrane fusion. GMP-PNP was added at the beginning (tethering, T) or at the end (docking, D) of the 10 min incubation period. Figure 6. Stages of outer membrane fusion. (A and B) Cryo-ET of docked mitochondria with partially fused outer membrane. (A) Slices through tomographic volume; scale bars 100 nm. The red circle highlights the region of outer membrane fusion. (B) 3D rendering. Two mitochondria are joined by one continuous outer membrane (red). The inter-membrane spaces are connected by a toroid pore of 40 nm diameter. (C and D) Fluorescence microscopy of in vitro outer membrane fusion. attachment and fusion Initially outer membranes of two attached mitochondria are tethered by globular protein repeats and membranes approach to within 6 nm (Figure 9, step 1). These tethered intermediates are seen in wild-type conditions (Figure 3) and accumulate upon addition of GMP-PNP (Figure 4). Subse- quently, GTP hydrolysis allows the fusion process to evolve progressively towards mitochondrial docking (Figure 5), as evidenced by the hybrid intermediates observed upon addition of GMP-PNP two minutes after initiation of tethering (Figure 5—figure supplement 1). Consistent with this, GTP hydrolysis by trans-complexes of atlastins precedes vesicle fusion and is essential to promote tether- ing of proteoliposomes (Liu et al., 2015; Saini et al., 2014). The docked intermediates are characterized by a docking ring of protein density that surrounds extended areas with outer membranes separated by less than 3 nm and devoid of visible densities (Figure 9, step 2). The capture of docked intermediates with partly fused outer membranes, the location of the fusion pore in the path of the docking ring undergoing disassembly and the inhibition of fusion upon treatment of docked mitochondria with GMP-PNP, demonstrates that docking is the stage that precedes merging of outer membranes (Figure 6). We do not exclude that small protein complexes, which are not visible by cryo-ET, might reside between outer membranes and participate in the fusion process. However, our observations do suggest that the docking ring of protein densi- ties is the driving force for subsequent bilayer merging. We thus propose that the fusion of bilayers is initiated by further GTP hydrolysis in the path of the docking ring where the outer membrane cur- vature is most pronounced (Figure 9, step 3). This step may trigger the disassembly of the docking ring. Tethering (Figure 9, step 4) and fusion of the inner membrane (Figure 9, step 5) mediated by OPA1/Mgm1 then completes the mitochondrial fusion process. Fzo1 enrichment at mitochondrial contact sites GMP-PNP was added at the beginning (tethering, T) or at the end (docking, D) of the 10 min incubation period. DOI: 10.7554/eLife.14618.016 DOI: 10.7554/eLife.14618.016 The following figure supplements are available for figure 6: The following figure supplements are available for figure 6: Figure supplement 1. Intermediate with fully fused outer membrane. DOI: 10 7554/eLife 14618 017 Figure supplement 1. Intermediate with fully fused outer membrane. O 0 / f 8 0 Figure supplement 2. Intermediates with partially fused outer membranes. Figure supplement 2. Intermediates with partially fused outer membranes. f 12 of 23 Brandt et al. eLife 2016;5:e14618. DOI: 10.7554/eLife.14618 Research article Biophysics and structural biology Cell biology Biophysics and structural biology Cell biology Discussion Prior to this study, the initial steps of mitochon- drial outer membrane fusion were known to involve mitofusins and to depend on GTP-bind- ing and hydrolysis (Cohen et al., 2011; Escobar- Henriques and Anton, 2013; Ishihara et al., 2004; Koshiba et al., 2004; Shutt et al., 2012). Combining an in vitro mitochondrial attachment assay with cryo-ET, we were able to dissect this process into several distinct steps. Our experi- ments on the inhibition of GTP hydrolysis allow us to temporally position each intermediate observed and thus provide the basis for a refined and comprehensive model of mitochon- drial outer membrane fusion (Figure 9). Video 4. 3D rendering of partially fused mitochondria as shown in Figure 6. Outer membranes in red, distinct density in blue. DOI: 10.7554/eLife.14618.019 Video 4. 3D rendering of partially fused mitochondria as shown in Figure 6. Outer membranes in red, distinct density in blue. DOI: 10.7554/eLife.14618.019 Mitofusins are involved in tethering, docking and fusion of outer membranes Taking into account the essential role of mitofusins in mitochondrial attachment and fusion (Hermann et al., 1998; Koshiba et al., 2004), we obtained several lines of evidence that Fzo1 con- tributes to the formation of the macromolecular assemblies we discovered at mitochondrial junctions. Overexpression of Fzo1 inhibited mitochondrial docking and fusion but stimulated the formation of artefactual tethering intermediates that are characterized by an accumulation of protein aggre- gates at mitochondrial junctions (Figure 7). This result demonstrates once more that absence of docking correlates with deficient mitochondrial fusion and implies that normal levels of mitofusins are required for the formation of the docking rings. It is also important to realize that increased lev- els of mitofusins correlate with the accumulation of protein densities at mitochondrial junctions. However, whether these aggregates correspond to abortive mitofusin oligomers remains speculative. Brandt et al. eLife 2016;5:e14618. DOI: 10.7554/eLife.14618 13 of 23 Research article Research article Biophysics and structural biology Cell biology Biophysics and structural biology Cell biology Figure 7. Overexpression of Fzo1. (A) Total protein extracts of fzo1D cells transformed with an empty vector (fzo1D), pRS314-FZO1 FZO1 (FZO1 o.e.) were analyzed by anti-Fzo1 and anti-Pgk1 immunoblotting. Fzo1 is overexpressed about 50 fold in FZO1 o.e. as conditions. (B) Serial dilutions of cells from A grown in the presence of glucose or glycerol as the sole carbon source at 30˚C. Lack Fzo1 both abolishes respiration and, therefore, growth on glycerol, consistent with inhibition of mitochondrial fusion. (C) Mitochon WT and FZO1 o.e. cells. Left: Representative morphologies. Right: Percentage of WT and FZO1 o.e. cells with indicated mitochond Error bars represent the s.d. from three independent experiments. (D) Left: TEM analysis of in vitro outer membrane fusion reactio Figure 7 continued on next page Figure 7. Overexpression of Fzo1. (A) Total protein extracts of fzo1D cells transformed with an empty vector (fzo1D), pRS314-FZO1 (WT) or pRS414-TEF- FZO1 (FZO1 o.e.) were analyzed by anti-Fzo1 and anti-Pgk1 immunoblotting. Fzo1 is overexpressed about 50 fold in FZO1 o.e. as compared to WT conditions. (B) Serial dilutions of cells from A grown in the presence of glucose or glycerol as the sole carbon source at 30˚C. Lack or overexpression of Fzo1 both abolishes respiration and, therefore, growth on glycerol, consistent with inhibition of mitochondrial fusion. (C) Mitochondrial morphology in WT and FZO1 o.e. cells. Left: Representative morphologies. Right: Percentage of WT and FZO1 o.e. cells with indicated mitochondrial morphologies. Figure 7 continued Figure 7 continued mitochondria isolated from wild-type cells or cells overexpressing Fzo1. Note that mitochondria from Fzo1 o.e. cells are smaller than from wild-type cells. Right: Effect of Fzo1 overexpression on outer membrane fusion and attachment in vitro. (E) Slices through tomographic volume of mitochondrial attached intermediates upon Fzo1 overexpression (abortive, 74%, see Table 1); outer membrane distance (blue bracket) and densities between outer membranes (red arrows) are indicated. (F) 3D rendering of two closely apposed mitochondria shown in E. DOI: 10.7554/eLife.14618.020 The following figure supplements are available for figure 7: Figure supplement 1. Absence or accumulation of Fzo1. Figure supplement 1. Absence or accumulation of Fzo1. DOI: 10.7554/eLife.14618.021 Figure supplement 2. Cryo-ET of mitochondria from Fzo1-overexpressing cells. DOI: 10.7554/eLife.14618.022 Figure supplement 2. Cryo-ET of mitochondria from Fzo1-overexpressing cells. DOI: 10.7554/eLife.14618.022 GMP-PNP that may bind to mitofusins and inhibit their GTPase activity (Amiott et al., 2009; Ishihara et al., 2004), induced the accumulation of tethered intermediates and prevented progres- sion towards the docked stage (Figure 4). Moreover, Fzo1 enrichment at mitochondrial contact sites was confirmed by Q-dot labeling (Figure 8). To our knowledge, no other GTPase has been shown to be involved in outer membrane fusion. In this context, our observations converge to propose that Fzo1 is at least a component of globular protein repeats and docking rings, possibly together with other, as yet unknown factors. In fact, the units composed of a central density with protrusions extending toward each outer membrane suggest that they may be Fzo1 trans-oligomers (Figure 8A). So far, the only structural insight into mitofusins derives from the HR2 domain of Mfn1 that has been proposed to tether outer membranes at a distance of 16 nm (Koshiba et al., 2004). This would exceed the 6 nm membrane spacing we measured in tethered intermediates. The overall morphology of the HR2 dimer can hardly account for the distinctive shape of the protein units we observed. However, the structure of the bacterial Fzo1 homolog BDLP in the open conformation (Low and Lo¨we, 2006; Low et al., 2009) and the established trans-interaction of atlastins through their GTPase domain (Klemm et al., 2011; Byrnes et al., 2013; Liu et al., 2015; Saini et al., 2014) allow us to propose an alternative model. Similar to BDLP, mitofusins bound to GMP-PNP or GTP may adopt an open conformation that, in analogy to atlastins, would promote trans-oligomerization of Fzo1 molecules through their GTPase domain. Figure 7 continued Conformational changes of the mitofusin oligomers upon GTP hydrolysis would pull the outer membranes closer together. This model is not only consistent with the shape of the protein units we visualized but also with the essential requirement of GTP hydrolysis for transition from mito- chondrial tethering to mitochondrial docking we unraveled in this study. Hence, the structure of mitofusins with or without bound GTP will be instrumental to evaluate this model. Mitofusins are involved in tethering, docking and fusion of outer membranes Error bars represent the s.d. from three independent experiments. (D) Left: TEM analysis of in vitro outer membrane fusion reactions performed with Figure 7 continued on next page Brandt et al. eLife 2016;5:e14618. DOI: 10.7554/eLife.14618 14 of 23 Research article R Biophysics and structural biology Cell biology Research article Biophysics and structural biology Cell biology In vivo vs in vitro mitochondrial outer membrane fusion An absolute prerequisite for mitochondrial fusion in vivo is that the tips of mitochondrial tubules come close enough to promote attachment between outer membranes. In yeast cells, this requires the participation of actin filaments (Simon et al., 1995; Smith et al., 1995). In the in vitro fusion sys- tem, the essential role of the cytoskeleton is replaced by centrifugation to bring mitochondria into close enough contact for fusion to proceed (Meeusen et al., 2004). However, centrifugation is not sufficient, and an incubation of at least 10 min on ice, previously suggested to promote Fzo1 associ- ation in trans, has been shown to be essential for in vitro fusion (Meeusen et al., 2004). Our results reveal that incubation on ice promotes the transition from tethering to docking, which is an active process that requires several rounds of GTP hydrolysis to progressively bring opposing outer membranes closer over an extended surface area but is not sufficient for their effective fusion. The observation that fusion starts at the edge of the docking ring and also depends on GTP hydroly- sis suggests that the transition from tethering to docking brings membranes closer over an extended area. This would induce a locally increased curvature of the lipid bilayer, which may be critical. An ultimate cycle of GTP hydrolysis in this region of local membrane curvature would therefore lead to fusion instead of bringing membranes closer together. Notably, micrographs taken from gastric mucosa of a mole, featured in Chapter 7 of Don W. Fawcett’s ’The Cell’, present a series of three pairs of mitochondria proposed to represent successive stages of mitochondrial fission (Faw- cett, 1981). The middle stage intermediate from this series and the fusion events identified in our Brandt et al. eLife 2016;5:e14618. DOI: 10.7554/eLife.14618 15 of 23 Research article Research article Biophysics and structural biology Cell biology Figure 8. Fzo1 enrichment at mitochondrial junctions. (A) Cryo-ET of tethered mitochondria. Slices through tomographic vo contact regions (red and blue boxes); Outer membranes on zooms are delimited by red bars; scale bars 10 nm. (B) Scheme density (yellow circle) and extensions (blue lines) to outer membranes (red bars) for densities detected at the junction of teth Q-Dot labelling of FZO1 and FZO1-AVI mitochondria. (C) Control with non-labelled Fzo1. Tomographic slice and zoom with membrane. In vivo vs in vitro mitochondrial outer membrane fusion (D) Q-Dots per contact area (left) or on mitochondrial surface excluding the contact area (right) for FZO1 (white) Figure 8 continued on next page Research article Biophysics and stru Figure 8. Fzo1 enrichment at mitochondrial junctions. (A) Cryo-ET of tethered mitochondria. Slices through tomographic volumes and zooms on contact regions (red and blue boxes); Outer membranes on zooms are delimited by red bars; scale bars 10 nm. (B) Scheme representing the central density (yellow circle) and extensions (blue lines) to outer membranes (red bars) for densities detected at the junction of tethered intermediates. (C–E) Q-Dot labelling of FZO1 and FZO1-AVI mitochondria. (C) Control with non-labelled Fzo1. Tomographic slice and zoom with Q-Dots on the outer membrane. (D) Q-Dots per contact area (left) or on mitochondrial surface excluding the contact area (right) for FZO1 (white) and FZO1-Avi (black). (E) Figure 8 continued on next page 16 of 23 Brandt et al. eLife 2016;5:e14618. DOI: 10.7554/eLife.14618 Research article R Research article Biophysics and structural biology Cell biology Figure 8 continued DOI: 10.7554/eLife.14618.023 The following figure supplement is available for figure 8: The following figure supplement is available for figure 8: Figure supplement 1. Biotinylation and Q-Dot labelling of Fzo1-Avi. DOI: 10.7554/eLife.14618.024 Figure supplement 1. Biotinylation and Q-Dot labelling of Fzo1-Avi. DOI: 10.7554/eLife.14618.024 study actually appear strikingly similar. This not only suggests that the sequence of events shown in this chapter might in fact correspond to successive stages of fusion but also that our model of outer membrane fusion may apply in vivo with mitochondria from mammalian cells. study actually appear strikingly similar. This not only suggests that the sequence of events shown in this chapter might in fact correspond to successive stages of fusion but also that our model of outer membrane fusion may apply in vivo with mitochondria from mammalian cells. Whereas in vitro local membrane deformation would be promoted exclusively by successive cycles of GTP hydrolysis after mitochondria become tethered through centrifugation, in vivo the transition from tethering to docking would also involve the action of the cytoskeleton. In the cell, the process of mitochondrial fusion as indicated by our in vitro studies would thus be regulated by cyto- skeletal factors. Regulation of mitochondrial fusion in vivo also involves post-translational modification of mitofu- sins (Anton et al., 2011; Cohen et al., 2008; Shutt et al., 2012). In vitro mitochondrial attachment/fusion assays Homotypic and heterotypic attachment/fusion reactions were respectively carried out with 0.5 mg of type 1 mitochondria or by mixing 0.25 mg of type 1 mitochondria with 0.25 mg of type 2 mitochon- dria. Mitochondria were then brought in vicinity by centrifugation at 10170 x g, 4˚C for 10 min. Pel- lets were left on ice for 10 min before the supernatant was replaced by Stage 1 buffer (20 mM Pipes–KOH pH 6.8, 150 mM KOAc, 5 mM Mg(OAc)2, 0.6 M sorbitol). Mitochondria were then left on ice for 30 min in attachment assays or incubated at 25˚C for 45 min in outer membrane fusion assays. Resulting attachment and fusion reactions were subsequently processed for TEM, cryo-ET or fluorescence microscopy analysis. Miscellaneous variations during in vitro mitochondrial attachment/ fusion reactions Depending on experiments, discrete variations in attachment/fusion reactions described above were used. To assess the effect of GMP-PNP on attachment of wild-type mitochondria (Figure 4), GMP- PNP (1.5 mM, Sigma-Aldrich; St Louis, MO) was added during mixing of mitochondria (prior centrifu- gation) and was kept at constant concentration during the whole attachment reactions (including in Stage 1 buffer). In contrast, 6 mM GMP-PNP were added where indicated in time course experi- ments (Figure 5). Similarly, 12 mM GMP-PNP were added at tethering or docking stages in outer membrane fusion reactions shown in Figure 6C. To evaluate effects of Fzo1 overexpression on attachment and fusion, in vitro assays were performed with mitochondria-enriched fractions pre- pared from wild-type (MCY553) or Fzo1-overexpressing cells (MCY1222) grown in dextrose medium. Yeast strains, plasmids and growth conditions Yeast strains, plasmids and growth conditions The S. cerevisiae strains and plasmids are listed in Supplementary file 1. Standard methods were used for growth, transformation and genetic manipulation of S. cerevisiae. Complete media and min- imal synthetic media [Difco yeast nitrogen base (Voigt Global Distribution Inc; Lawrence, KS), and drop-out solution] supplemented with 2% dextrose (YPD and SD) or 2% glycerol (YPG and SG) were prepared as described (Sherman et al., 1986). Mitochondrial enriched preparations Mitochondrial fractions for in vitro attachment and fusion were prepared as previously described (Ingerman et al., 2007). Cells were cultured to stationary phase in dextrose medium and then shifted to glycerol medium (or fresh dextrose medium for cells affected in respiration) to grow to a final OD 0.8–1.0. Cell walls were disrupted by incubation at 30˚C with 100 mM Tris-HCl pH 9.4 and 50 mM b-mercaptoethanol for 20 min and subsequently with 1.2 M sorbitol plus zymolyase (Zymo Research; Irvine, CA) for 30 min. The resulting spheroplasts were lysed in cold NMIB (0.6 M sorbitol, 5 mM MgCl2, 50 mM KCl, 100 mM KOAc, 20 mM Hepes pH 7.4) by douncing. After centrifugation at 4˚C of the lysate at 3000 x g for 5 min, the supernatant was further centrifuged at 10170 x g, 4˚C for 10 min to yield a pellet enriched in mitochondria. Protein concentration in mitochondria-enriched fractions was determined by Bradford assay (Bio-Rad Protein Assay; Bio-Rad Laboratories GmbH, Germany). Conclusion Our work provides a detailed dissection of the outer mitochondrial membrane fusion process in vitro and highlights the crucial involvement of Fzo1 in this system. Similar mechanisms involving atlastins or OPA1/Mgm1 are likely to be active in the fusion of ER and mitochondrial inner membranes, respectively. Future challenges include deciphering the precise composition of the complexes medi- ating outer membrane fusion, and dissecting the processes regulating their formation and function. In vivo vs in vitro mitochondrial outer membrane fusion In yeast, the efficient fusion of outer membranes requires Fzo1 ubiquitylation by the Mdm30 ubiquitin ligase and subsequent deg- radation by the proteasome (Cohen et al., 2008). While its precise function is yet to be fully charac- terized, this regulation was previously shown to take place at the stage of mitochondrial attachment (Cohen et al., 2011). It is therefore conceivable that the UPS-dependent regulation of Fzo1 partici- pates in regulating proper assembly of docking rings, which is consistent with the observation that high levels of Fzo1 inhibit mitochondrial docking (Figure 7). At this stage, we cannot exclude that ubiquitylation and degradation of Fzo1 regulates the transition from docking to effective fusion of outer membranes. Further investigations will be required to answer the fascinating question of how mitochondrial fusion is regulated. Figure 9. Model of outer membrane tethering, docking and fusion. Mitochondria (blue) are tethered or docked by protein complexes (red). DOI: 10.7554/eLife.14618.025 Figure 9. Model of outer membrane tethering, docking and fusion. Mitochondria (blue) are tethered or docked by protein complexes (red). DOI 10 7554/ Lif 14618 025 Figure 9. Model of outer membrane tethering, docking and fusion. Mitochondria (blue) are tethered or docked by protein complexes (red). DOI: 10.7554/eLife.14618.025 Brandt et al. eLife 2016;5:e14618. DOI: 10.7554/eLife.14618 17 of 23 Research article Research article Research article Biophysics and structural biology Cell biology Biophysics and structural biology Cell biology For plastic embedding, the samples were dehydrated in an ethanol gradient series (1 20 min 30%, 2 20 min 50%, 230 min 70%, 2 30 min 90%, 1 60 min 100% ethanol) followed by a switch to 1,2 propylenoxid (3 x 20 min 100%) and subsequently infiltrated using the Low Viscosity Premix Kit-Medium (Agar Sci- entific, England; 2 20 min 30%, 2 30 min 50%, 2 30 min 75%, overnight 100%, 2 2 h 100%). Polymerization was carried out at 65˚C for 16 h. Thin sections (60–70 nm) were prepared with an Ultracut S microtome (Reichert, Germany), collected on 100 mesh copper grids coated with Piolo- form FN 65 (Wacker Polymer Systems GmbH, Germany) and were double stained with 2% uranyl acetate for 2 min and lead citrate for 1 min. Sections were inspected with a transmission electron microscope (EM 208S; FEI, Germany) at 80 kV equipped with 2 x 2 k CCD camera (Gatan, Inc; Pleas- anton, CA). Ratios of attached and fused mitochondria were obtained by dividing the number of mitochondria in physical contact or those with fused outer membranes over the total number of mitochondria (n > 300). The fused intermediates shown in Figure 6—figure supplement 1 and 2 were obtained from the analysis of 87 high magnification micrographs containing one pair of attached or fused intermediates each. Electron cryo-tomography For cryo-ET, mitochondria were washed twice with 320 mM trehalose, 20 mM Tris pH 7.3, 1 mM EGTA. Samples were mixed 1:1 with fiducial gold markers (6 or 10 nm gold particles conjugated to protein A, Aurion, Netherlands) and immediately plunge-frozen in liquid ethane on Quantifoil holey carbon grids (Quantifoil Micro Tools, Germany). Single tilt series (typically ± 60˚, step size 1.5–2.0˚) were collected at 300 kV with an FEI Polara or FEI Titan Krios electron microscope equipped with a post-column Quantum energy filter and a K2 Summit direct electron detector (Gatan) at 6 mm under- focus. Magnifications were chosen to give an object pixel size of 3.5 A˚ or 3.3 A˚ , respectively. The total electron dose per tilt series was 90–120 e-/A˚ 2. If dose fractionation was used, frames were aligned using the IMOD software package (Kremer et al., 1996). Tilt series were aligned to gold fiducial markers, binned 2-fold and tomograms were reconstructed by back-projection with IMOD. A final filtering step applying non-linear anisotropic diffusion (Frangakis and Hegerl, 2001) was per- formed to increase contrast. Tomograms were manually segmented with the program Amira (FEI). Analysis of cryo-ET data y y Distances in tomographic data were analyzed using IMOD. The following parameters were measured for each contact: the radii r of mitochondria perpendicular and parallel to the contact area, the major axis la and the minor axis lb of the contact area assuming elliptical geometry and the distance between the outer membranes d. The contact area A was calculated assuming it to be elliptical, A = (plalb)/4. The normalized contact area ratio R was calculated, taking the radius r of the smaller of the two involved mitochondria into account, R = la/(2r). Biophysics and structural biology Cell biology Biophysics and structural biology Cell biology sucrose, 100 mM sodium cacodylate, pH 7.4) for 20 hr and subsequently washed twice with 100 mM sodium cacodylate, pH 7.4. To improve contrast, samples were post-fixed with 1% osmium tetrox- ide, 100 mM sodium cacodylate, pH 7.4, for 1 hr, after which samples were washed in distilled water. Then samples were embedded in 4% agar and washed with 50 mM acetate buffer pH 5.2 before they were stained with 1% uranyl acetate overnight (12 h) at 4˚C. For plastic embedding, the samples were dehydrated in an ethanol gradient series (1 20 min 30%, 2 20 min 50%, 230 min 70%, 2 30 min 90%, 1 60 min 100% ethanol) followed by a switch to 1,2 propylenoxid (3 x 20 min 100%) and subsequently infiltrated using the Low Viscosity Premix Kit-Medium (Agar Sci- entific, England; 2 20 min 30%, 2 30 min 50%, 2 30 min 75%, overnight 100%, 2 2 h 100%). Polymerization was carried out at 65˚C for 16 h. Thin sections (60–70 nm) were prepared with an Ultracut S microtome (Reichert, Germany), collected on 100 mesh copper grids coated with Piolo- form FN 65 (Wacker Polymer Systems GmbH, Germany) and were double stained with 2% uranyl acetate for 2 min and lead citrate for 1 min. Sections were inspected with a transmission electron microscope (EM 208S; FEI, Germany) at 80 kV equipped with 2 x 2 k CCD camera (Gatan, Inc; Pleas- anton, CA). Ratios of attached and fused mitochondria were obtained by dividing the number of mitochondria in physical contact or those with fused outer membranes over the total number of mitochondria (n > 300). The fused intermediates shown in Figure 6—figure supplement 1 and 2 were obtained from the analysis of 87 high magnification micrographs containing one pair of attached or fused intermediates each. sucrose, 100 mM sodium cacodylate, pH 7.4) for 20 hr and subsequently washed twice with 100 mM sodium cacodylate, pH 7.4. To improve contrast, samples were post-fixed with 1% osmium tetrox- ide, 100 mM sodium cacodylate, pH 7.4, for 1 hr, after which samples were washed in distilled water. Then samples were embedded in 4% agar and washed with 50 mM acetate buffer pH 5.2 before they were stained with 1% uranyl acetate overnight (12 h) at 4˚C. Transmission electron microscopy For analysis of attachment and outer membrane fusion intermediates by TEM, the fusion reactio were mixed with 20 volumes of fixative solution (3% paraformaldehyde, 1.5% glutaraldehyde, 2.5 18 of 23 Brandt et al. eLife 2016;5:e14618. DOI: 10.7554/eLife.14618 Research article Research article Biophysics and structural biology Cell biology Fluorescence microscopy y Fluorescence microscopy was carried out with a Zeiss Axio Observer Z1 microscope (Carl Zeiss Microscopy GmbH, Germany). For in vitro fusion assays a 100X oil immersion objective and the fol- lowing filter sets were used: 10 Alexa Fluor 489 (Excitation BP 450–490, Beam Splitter FT 510, Emis- sion BP 515–565) for GFP, mCherry (Excitation BP 542–582, Beam Splitter FT 593, Emission BP 604– 679) for mCherry. Images were acquired with an SCMOS ORCA FLASH 4.0 charge-coupled device camera (Hamamatsu Photonics K.K., Japan) and the Zen 2012 Package Acquisition/Analyse software before processing with ImageJ. Mitochondrial morphology was analyzed with a 63X oil immersion objective and an FITC filter (Filter set 44, Excitation BP 475/40, Beam Splitter FT 500, Emission BP 530/50) for GFP. Cell contours were visualized with Nomarski optics. Images were acquired with an ORCA-R2 CCD camera (Hamamatsu) and processed with ImageJ. Mitochondrial network morphology p gy Mitochondrial morphology was analyzed in cells expressing mito-GFP from the p426-TEF-mitoGFP plasmid (MC244). Strains were grown in dextrose medium to mid-log phase and fixed with 3.7% formaldehyde. Morphology phenotypes were assessed in at least 100 cells. Data reported are the mean and standard deviation (SD, error bars) of three independent experiments. Spot assays Cultures grown overnight in SD medium were pelleted, resuspended at OD600 = 1, and serially diluted (1:10) five times in water. Three microliters of the dilutions were spotted on SD and SG plates and grown for 3 days (dextrose) or 5 days (glycerol) at 30˚C. Analysis of in vitro mitochondrial fusion reactions by fluorescence microscopy py For fluorescence microscopy analysis, the fusion reactions were fixed with two volumes of 8% formal- dehyde in phosphate-buffered saline (PBS). Aliquots were immobilized on microscope slides by mix- ing 1:1 with 2% low melting point agarose (Sigma-Aldrich) in NMIB. The ratios of fused mitochondria were obtained by dividing the number of GFP and mCherry signals co-localizing with each other over the total number of OM45-GFP (obtained from strain #779) and mito-mCherry (obtained from strain #980) mitochondria (n > 1000). The levels of fused mitochondria were then determined by sub- tracting the ratios obtained for fusion reactions stopped at the mixing step (at the beginning of the reaction) from those obtained from reactions stopped at t = 45 min (at the end of the reaction). 19 of 23 Brandt et al. eLife 2016;5:e14618. DOI: 10.7554/eLife.14618 Research article Research article Biophysics and structural biology Cell biology Biophysics and structural biology Cell biology Protein extracts and immunoblotting Cells grown in SD medium were collected during the exponential growth phase (OD600 = 0.5–1). Total protein extracts were prepared by the NaOH/trichloroacetic acid (TCA) lysis technique (Volland et al., 1994). Proteins were separated by SDS-PAGE 8% and transferred to nitrocellulose membranes (AmershamTM HybondTM-ECL; GE Healthcare, UK). Primary antibodies for immunoblot- ting were mouse anti-Pgk1 (clone 22C5D8; Abcam, UK), rabbit anti-Fzo1 (Covalab, France), anti- Ugo1 (Covalab), anti-Mgm1 (kind gift from Andreas Reichert) and mouse anti-Por1 (clone 16G9E6BC4; Abcam). Primary antibodies were detected by secondary anti-mouse or anti-rabbit anti- bodies conjugated to horseradish peroxidase (HRP, Sigma-Aldrich), followed by incubation with the Clarity Western ECL kit (Bio-Rad). Immunoblotting images were acquired with a Gel DocTM XR + (Bio-Rad) and processed with the Image Lab 3.0.1 software (Bio-Rad). Brandt et al. eLife 2016;5:e14618. DOI: 10.7554/eLife.14618 Additional information Funding Funder Grant reference number Author Max-Planck-Gesellschaft Werner Ku¨ hlbrandt Fondation pour la Recherche Me´ dicale INE20100518343 Mickae¨ l M Cohen Marie Curie FP7 276912 (Mitofusion) Mickae¨ l M Cohen Labex DYNAMO ANR-11-LABX-0011- DYNAMO Mickae¨ l M Cohen Centre National de la Recherche Scientifique CNRS-INSERM ATIP-Avenir Program Mickae¨ l M Cohen The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Additional files Supplementary files . Supplementary file 1. Table of strains and table of plasmids used in the study. DOI: 10.7554/eLife.14618.026 Author contributions TB, Collected EM images, Processed and analyzed the data, Designed the experiments, Wrote the paper, Acquisition of data; LC, Performed in vitro attachment/fusion assays as well as cell and molec- ular biology experiments, Designed the experiments, Analysis and interpretation of data; WK, Wrote the paper; MMC, Designed the experiments, Wrote the paper, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents Author ORCIDs Mickae¨ l M Cohen, http://orcid.org/0000-0002-1372-680X Author ORCIDs Mickae¨ l M Cohen, http://orcid.org/0000-0002-1372-680X Author ORCIDs Mickae¨ l M Cohen, Author ORCIDs Mickae¨ l M Cohen Author ORCIDs Abutbul-Ionita I, Rujiviphat J, Nir I, McQuibban GA, Danino D. 2012. Membrane tethering and nucleotide- dependent conformational changes drive mitochondrial genome maintenance (Mgm1) protein-mediated membrane fusion. The Journal of Biological Chemistry 287:36634–36638. doi: 10.1074/jbc.C112.406769 Amiott EA, Cohen MM, Saint-Georges Y, Weissman AM, Shaw JM. 2009. A mutation associated with CMT2A neuropathy causes defects in Fzo1 GTP hydrolysis, ubiquitylation, and protein turnover. Molecular Biology of the Cell 20:5026–5035. doi: 10.1091/mbc.E09-07-0622 Anton F, Fres JM, Schauss A, Pinson B, Praefcke GJ, Langer T, Escobar-Henriques M. 2011. Ugo1 and Mdm30 act sequentially during Fzo1-mediated mitochondrial outer membrane fusion. Journal of Cell Science 124: 1126–1135. doi: 10.1242/jcs.073080 Acknowledgements g We thank Friederike Joos for expert technical assistance in preparing and imaging thin sections and Naı¨ma Belgareh-Touze´ for critical reading of the manuscript. This work was funded by the CNRS- INSERM ATIP-Avenir program, the "fondation pour la recherche me´ dicale" (INE 20100518343), a Marie Curie IRG grant (No.276912) and the labex DYNAMO (ANR-11-LABX-0011-DYNAMO) to MC and generous funding of the Max Planck Society to WK. The authors declare no competing financial interests. Quantum dot labelling Specific labelling of mitofusins on attached mitochondria was achieved using Quantum Dots coupled to streptavidin (QDot 525ITK streptavidin; Life Technologies; Carlsbad, CA), which required specific biotinylation of Fzo1. For this purpose, the FZO1 ORF placed under control of its own promoter, was fused at its 3’-end in tandem with a sequence encoding for the Avi tag, a 15 amino-acid peptide that can be recognized and undergo specific biotinylation by the E. coli biotin ligase BirA (Beckett et al., 1999; van Werven and Timmers, 2006). The resulting pFZO1-AVI plasmid (MC369) was introduced in fzo14 cells using a plasmid shuffling strategy to yield the FZO1-AVI yeast strain (MCY1155) that expresses Fzo1-Avi as the sole source of mitofusin in the cell. After verifying that Fzo1-Avi is competent for mitochondrial fusion in vivo, mitochondria were purified to promote bioti- nylation of Fzo1-Avi in vitro. Briefly, 0.5 mg of total mitochondrial enriched fractions prepared from FZO1 (MCY1154) or FZO1-Avi (MCY1155) cells were incubated at 25˚C for 60 min with 10 mg BirA and 1X biotin in biomix A + B buffer (Biotinylation kit purchased from GeneCopoeia Inc; Rockville, MD) before processing for attachment assays. Following 30 min incubation on ice in Stage 1 buffer, attachment reactions were incubated for 60 min at 4˚C with 50 nM streptavidin-coupled QDs before processing for cryo-ET analysis. 20 of 23 Brandt et al. eLife 2016;5:e14618. 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https://openalex.org/W4285509515	https://zenodo.org/records/4444094/files/Alternate%20proposal%20for%20peer%20review.pdf	English	null	An alternate Proposal for Peer Review	Zenodo (CERN European Organization for Nuclear Research)	2,021	cc-by	1,386	An Alternate Proposal for Peer Review These results motivated STScI to devise a dual- anonymization procedure that completely eliminates the names (and institutions) of the Investigators from proposals in order to focus the review on the science [8], [9]. After proposals are selected, the names of investigators and their expertise and backgrounds can be revealed and reviewed by the committee. NASA’s Science Mission Directorate has initiated a dual-anonymous peer review procedure for several programs as well.b These are admirable developments. Andrea Dupree (Center for Astrophysics \| Harvard & Smithsonian) Procedures for peer review of grant proposals and requests for telescope time have come under scrutiny because of demonstrated gender biases [1], [2], [3], [4], [5]. Studies of proposal success rates in the United States, Europe, and Canada involving major astronomical facilities both on the ground and in space reach similar conclusions: “proposals submitted by female PIs show a significantly lower probability of being allocated time”; “deficit in acceptance by female proposers is significant”; “proposals submitted by women were rated significantly worse than those submitted by men”; “significant gender-related systematic trend”; and “an overall signal favoring men.” The creation of fully anonymous proposals represents a substantial change and introduces new constraints and added responsibilities for everyone involved in the peer review process. Dual-anonymization requires pre-screening of proposals by observatory staff to ensure stylistic compliance and creditable submission. Additionally, “levelers” are required in each meeting room to carefully follow reviewer discussions and interrupt when necessary to direct the topic back to the science. In addition, segments of the “old procedures” still exist as reviewers meet to discuss and evaluate proposals together. Recognizing this problem, the Space Telescope Science Institute (STScI) has experimented with new proposal formats. After trying two different stylistic changes,a they experimented with a third variation, which required that proposals list investigators alphabetically without identifying the Principal Investigator (PI). STScI also examined their review process, which consists of an initial ranking of proposals made separately and individually by each reviewer, followed by an in-person meeting of the entire committee to continue the evaluation. Analysis of the first step, the independent individual ranking, revealed no A different, and in many ways superior, peer review procedure is suggested by a study sponsored by the National Academy of Sciences to assess the effectiveness of peer review. Perspectives Perspectives Perspectives gender bias [6]. However, gender bias did arise in the second step, the group discussion. Professional observers of the committee deliberations noted that conversations focused on the PI, the team, and the laboratory about 50% of the time [7]. 24 noirlab.edu An Alternate Proposal for Peer Review The study, by Cole and collaborators [10] of the National Science Foundation (NSF) peer review process, was carried out as part of a larger study by the National Academy of Sciences Committee on Science and Public Policy (COSPUP) [11]. 24 noirlab.edu Figure 1. The author in the Cassegrain cage of the 4m Mayall Telescope at Kitt Peak National Observatory. Figure 1. The author in the Cassegrain cage of the 4m Mayall Telescope at Kitt Peak National Observatory. The conclusions of this research lead us to suggest an alternate method of peer review and selection that has numerous advantages. Here, we outline this alternate method. Cole and collaborators studied three areas of research supported by NSF: chemical dynamics, economics, and solid-state physics. “Blue ribbon” NSF panels had previously ranked the proposals in each area. These proposals were in the traditional format in which the PI and team are identified. The same proposals were then given to a second set of equally “blue ribbon” panels for evaluation. The rankings of the proposals are shown in Figure 2, with the initial NSF rankings on the vertical axis and the new COSPUP rankings on the horizontal axis. Without an in-person meeting, proposals (with proposers identified alphabetically) are read and ranked independently by the members of the review committee, and the scores are tabulated. The “top-ranked” proposals are accepted. The lowest-ranked proposals are rejected, similar to the triage process that occurs in the evaluation of HST proposals. The remaining proposals in the middle are selected randomly until the available observing time is filled. These results are illuminating. In all 3 subject areas, both panels easily identified and agreed on the best 3–4 proposals and the worst 3–4 proposals. However, proposals in the middle show little correlation between the scores. Cole and collaborators concluded that “getting a research grant depends to a significant extent on chance.” This peer review method has many advantages: This peer review method has many advantages: • Follows procedures demonstrated to identify the best and worst proposals. The Mirror January 2021 25 Figure 2. The NSF rank (y-axis) and the corresponding rank from the COSPUP reviewers (x-axis). The asterisks mark two proposals with identical ranks. I have inserted circles to mark extremes: the “best” and the “worst.” (From [10]. Reprinted with the permission of the American Association for the Advancement of Science.) Figure 2. An Alternate Proposal for Peer Review 1981, Science, 214, 881 • Saves money and time. • Saves money and time. • Eliminates the challenge of writing “masked identity” proposals. Some have argued that good science cannot be a random choice. This new process is not random. The best proposals will be successful. The worst proposals will not advance. The process explicitly acknowledges that the final selection under most current review systems is highly dependent on the peers who happen to be around the meeting table that day. Evidence-based research demonstrates that success with the current procedures already depends to a significant extent on chance. This may be an opportune time to initiate new procedures. i b https://science.nasa.gov/researchers/dual-anonymous- peer-review An Alternate Proposal for Peer Review The NSF rank (y-axis) and the corresponding rank from the COSPUP reviewers (x-axis). The asterisks mark two proposals with identical ranks. I have inserted circles to mark extremes: the “best” and the “worst.” (From [10]. Reprinted with the permission of the American Association for the Advancement of Science.) References [1] Reid, I. N. 2014, PASP, 126, 923 [2] Patat, F. 2016, The Messenger, 165, 2 [3] Lonsdale, C. J., Schwab, F. R., & Hunt, G. 2016, arXiv:1611.04795 [4] Norman, D. 2018, private communication [5] Spekkens, K., Cofie, N., & Crabtree, D. 2018, Proc. SPIE, 10704, 107040L [6] Strolger, L. & Natarajan, P. 2019, Physics Today Commentary and Reviews, March [7] Reid, N. 2020, STScI Newsletter, 37(01) [8] Johnson, S. K. & Kirk, J. F. 2020, PASP, 132, 034503 [9] Kirk, J. F. 2020, BAAS, 52 (3) [10] Cole, S., Cole, J. R. & Simon, G. A. 1981, Science, 214, 881 [11] National Research Council. 1978. Peer Review in the National Science Foundation: Phase I of a Study. Washington, DC: The National Academies Press References [1] Reid, I. N. 2014, PASP, 126, 923 [2] Patat, F. 2016, The Messenger, 165, 2 [3] Lonsdale, C. J., Schwab, F. R., & Hunt, G. 2016, arXiv:1611.04795 [4] Norman, D. 2018, private communication [5] Spekkens, K., Cofie, N., & Crabtree, D. 2018, Proc. SPIE, 10704, 107040L [6] Strolger, L. & Natarajan, P. 2019, Physics Today Commentary and Reviews, March [7] Reid, N. 2020, STScI Newsletter, 37(01) [8] Johnson, S. K. & Kirk, J. F. 2020, PASP, 132, 034503 [9] Kirk, J. F. 2020, BAAS, 52 (3) [10] Cole, S., Cole, J. R. & Simon, G. A. 1981, Science, 214, 881 [11] National Research Council. 1978. Peer Review in the National Science Foundation: Phase I of a Study. Washington, DC: The National Academies Press • Eliminates the step in which gender bias occurs, as documented by STScI studies: the face-to-face meeting of the panels. • Allows out-of-the-box ideas to be selected. • Eliminates gender bias. • Eliminates the need for detailed review (and disqualification) by observatory staff of a non-compliant proposal. f • Eliminates the need for staff levelers to oversee discussion sessions. [8] Johnson, S. K. & Kirk, J. F. 2020, PASP, 132, 034503 • Avoids committee preferences for small proposals that distribute resources to the many rather than restrict resources to a few. [10] Cole, S., Cole, J. R. & Simon, G. A. 26 noirlab.edu Notes a In one proposal cycle, the PI and team were listed on the second page rather than the first. In another cycle, only the first initials of the PI and team were allowed. 26 noirlab.edu
https://openalex.org/W2954244088	https://europepmc.org/articles/pmc6659069?pdf=render	English	null	Accuracy of BISAP score in prediction of severe acute pancreatitis	Pakistan journal of medical sciences	2,019	cc-by	3,572	Original Article Original Article Accuracy of BISAP score in prediction of severe acute pancreatitis Anum Arif1, Farhat Jaleel2, Khalid Rashid3 ABSTRACT Objective: To determine the accuracy of BISAP score in comparison with Ranson’s score in detection of severe acute pancreatitis. Objective: To determine the accuracy of BISAP score in comparison with Ranson’s score in detection of severe acute pancreatitis. Methods: This cross sectional study was performed in Emergency department and Surgery department of Dow university hospital from January 2015 to December 2015. A total of 206 patients were included. Those diagnosed with acute pancreatitis on the basis of epigastric pain, serum amylase levels more than 300 (more than 3 times normal) and meeting the inclusion criteria were subjected to investigations for Ranson’s and BISAP scoring. BISAP score was calculated at 24 hours and Ranson’s score both at 24 and 48 hours. A score of > 3 was used to label severe acute pancreatitis according to both scoring systems. Results: In our study accuracy to predict SAP by BISAP score was 76.2 % and Ranson’s score was 82.2%. On the basis of sensitivity, Ranson’s scores predicted SAP more accurately than BISAP scores (97.4% vs. 69.2%). Regarding specificity, both scores predicted SAP almost equally (78.4% vs. 77.8%). C l i BISAP i l bl t l i di ti A t P titi i l h g pi y, p q y ( ) sion: BISAP score is a valuable tool in predicting severe Acute Pancreatitis in early hours KEYWORDS: Severe Acute pancreatitis, Ranson’s score, BISAP score. KEYWORDS: Severe Acute pancreatitis, Ranson’s score, BISAP score. KEYWORDS: Severe Acute pancreatitis, Ranson’s score, BISAP score. How to cite this: Arif A, Jaleel F, Rashid K. Accuracy of BISAP score in prediction of severe acute pancreatitis. Pak J Med Sci. 2019;35(4):1008-1012. doi: https://doi.org/10.12669/pjms.35.4.1286 How to cite this: Arif A, Jaleel F, Rashid K. Accuracy of BISAP score in prediction of severe acute pancreatitis. Pak J Med Sci. 2019;35(4):1008-1012. doi: https://doi.org/10.12669/pjms.35.4.1286 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. INTRODUCTION Pak J Med Sci July - August 2019 Vol. 35 No. 4 www.pjms.org.pk 1008 1. Dr. Anum Arif, MBBS, FCPS. Department of Surgery, Aga Khan University of Health Sciences Karachi, Pakistan. 2. Dr. Farhat Jaleel, MBBS, FCPS. Professor, Head of Surgical Unit 6, Civil Hospital Karachi, Dow University of Health Sciences, Karachi, Pakistan. 3. Dr. Khalid Rashid, MBBS, FCPS. Associate Professor, Department of Surgery, Jinnah Post-Graduate Medical Center, Jinnah Sindh Medical University, Karachi, Pakistan. Correspondence: Dr. Anum Arif, MBBS, FCPS. Department of Surgery, Aga Khan University of Health Sciences, Karachi, Pakistan. House # B-29, Popular Villas, Opposite Malir City Court, Malir, Karachi, Pakistan. E-mail: dranumarif@yahoo.com dranumarifkhan@gmail.com * Received for Publication: February 27, 2018 * Revision Received: January 28, 2019 * Accepted for Publication: April 25, 2019 INTRODUCTION Acute pancreatitis (AP) is a common disorder with substantial burden on the health-care system. AP accounts for 210,000 hospital admissions per annum in the United States.1,2 Recent studies show the incidence of AP varies between 4.9 and 73.4 cases per 100,000 worldwide.3,4 An increase in the annual incidence for AP has been observed in most recent studies. It is a complex process in which pancreatic enzyme activation causes local pancreatic damage, resulting in an acute inflammatory response.5 The individual patient’s response to pancreatic injury is highly variable and often unpredictable.5 Most of the time auto digestion of pancreas is mild and self-limiting but in 20% to 30% cases it develop severe disease that can progress to SIRS, MODS and death.6 According to Atlanta classification (2012) severe acute pancreatitis is defined as acute pancreatitis with persistent organ failure more than > 48 hours. A wide range of mortality 20% to 60% has been reported in severe acute pancreatitis.7 Pak J Med Sci July - August 20 1. Dr. Anum Arif, MBBS, FCPS. Department of Surgery, Aga Khan University of Health Sciences Karachi, Pakistan. 2. Dr. Farhat Jaleel, MBBS, FCPS. Professor, Head of Surgical Unit 6, Civil Hospital Karachi, Dow University of Health Sciences, Karachi, Pakistan. 3. Dr. Khalid Rashid, MBBS, FCPS. Associate Professor, Department of Surgery, Jinnah Post-Graduate Medical Center, Jinnah Sindh Medical University, Karachi, Pakistan. Correspondence: Dr. Anum Arif, MBBS, FCPS. Department of Surgery, Aga Khan University of Health Sciences, Karachi, Pakistan. House # B-29, Popular Villas, Opposite Malir City Court, Malir, Karachi, Pakistan. INTRODUCTION E-mail: dranumarif@yahoo.com dranumarifkhan@gmail.com * Received for Publication: February 27, 2018 * Revision Received: January 28, 2019 * Accepted for Publication: April 25, 2019 1. Dr. Anum Arif, MBBS, FCPS. Department of Surgery, Aga Khan University of Health Sciences Karachi, Pakistan. Acute pancreatitis (AP) is a common disorder with substantial burden on the health-care system. AP accounts for 210,000 hospital admissions per annum in the United States.1,2 Recent studies show the incidence of AP varies between 4.9 and 73.4 cases per 100,000 worldwide.3,4 An increase in the annual incidence for AP has been observed in most recent studies. It is a complex process in which pancreatic enzyme activation causes local pancreatic damage, resulting in an acute inflammatory response.5 The individual patient’s response to pancreatic injury is highly variable and often unpredictable.5 Most of the time auto digestion of pancreas is mild and self-limiting but in 20% to 30% cases it develop severe disease that can progress to SIRS, MODS and death.6 According to Atlanta classification (2012) severe acute pancreatitis is defined as acute pancreatitis with persistent organ failure more than > 48 hours. A wide range of mortality 20% to 60% has been reported in severe acute pancreatitis.7 2. Dr. Farhat Jaleel, MBBS, FCPS. Professor, Head of Surgical Unit 6, Civil Hospital Karachi, Dow University of Health Sciences, Karachi, Pakistan. 1008 Anum Arif et al. with other causes of hyperamylasemia and with carcinoma pancreas were excluded from study. Bedside Index of Severity of Acute Pancreatitis (BISAP) was calculated. It includes 5 variables. Blood urea nitrogen levels > 25 mg/dl, impaired mental status or GCS < 15, SIRS, age > 60 years and pleural effusion on imaging. Each variable was granted a score of 1. All patients were subjected to investigations including CBC, LDH, SGOT, RBS, BUN and Chest x-ray. Age SIRS, GCS was recorded to assess the BISAP score and Ranon’s score at 24 hours. Patients were admitted and further evaluated with investigations required for assessment of 48 hours Ranson’s score which included CBC (to asses fall in Hematocrit), BUN, serum calcium, ABGs and fluid deficit. Final agreement between the scores was evaluated at 48 hours. All demographics and outcome variables were entered into the proforma. Outcome variable of this study were age, duration of symptoms, Ranson score and BISAP score. All patients enrolled were diagnosed and classified as mild, moderately severe and SAP according to latest Atlanta classification. INTRODUCTION SAP was characterized by persistent organ failure for more than 48 hours. A cut off score ≥ 3 was taken to categorize SAP according to Ranson and BISAP score at 24 and 48 hours. Many scoring systems have been developed for the detection of severe acute pancreatitis including Ranson’s score, APACHE-II score, CTSI score, MOSS and GLASGOW score.8-10 The Ranson’s score has been used over three decades. It is moderately accurate in classifying patients in terms of severity, but has the disadvantage of requiring a full 48 hours to be completed, missing a potentially valuable early therapeutic window.8,11 BISAP score is a newly developed scoring system containing data that can be evaluated at the time of admission which are accurate in predicting patient’s outcome within 24 hours.12 The international studies comparing both showed varied results. One of the study shows that the sensitivity of severity acute pancreatitis predicted by BISAP was 74.2%, specificity 68.3% positive predictive value 63.4% and negative predictive value 77.8%6 whereas in another study sensitivity is 38.6%, specificity 93.2%, positive predictive value 59.1% and negative predictive value 85.6%.7 Ranson’s score requires lots of variables raising cost of complete diagnosis and management whereas BISAP score has less variables which are cost effective and can be done in emergency setting. As the above mentioned studies results are variable, the rationale of this study was to determine BISAP score in emergency setting. If results prove to be in favor of BISAP score than it can help in early diagnosis of severe acute pancreatitis, preventing complications and overall mortality can be reduced. Statistical Analysis: All data was entered in Statistical Package for Social Science (SPSS) software, Version 16. Mean and standard deviation were calculated for quantitative variables. Frequency and percentages were computed for qualitative variables. Effect modifiers like age, gender, baseline pain were controlled by stratification. Associations between SAP and age and gender were measured using Chi-square analysis. The objective of this study was to determine the accuracy of BISAP score in comparison with Ranson’s score in detection of severity of acute pancreatitis in a tertiary care hospital. Pak J Med Sci July - August 2019 Vol. 35 No. 4 www.pjms.org.pk 1009 METHODS Further, to see the accuracy for both scoring systems in predicting SAP, Receiver Operating Characteristics (ROC) analysis was also performed. Overall accuracy in predicting SAP, sensitivity, specificity, positive predictive value (+PV), negative predictive value (-PV) were calculated. To check the strength of agreement between standard classified diagnosis using Atlanta classification and Ranson’s and BISAP scores, Kappa coefficient of agreement was also calculated. This cross sectional study was performed in Emergency department and Surgery department of Dow university hospital from January 2015 to December 2015 after the approval from Research and Training monitoring cell of CPSP. A total of 206 patients were included in the study. The sample size was calculated by taking prevalence 25% and 6%. Informed and written consents were taken from all patients. Inclusion criteria contained both males and females, age range between 20 to 50 years, patient presenting within 48 hours of onset of pain ( VAS > 5), raised serum amylase levels (more than or equal to 300 IU) within 48 hours of epigastric or upper abdominal pain. Patients who refused to participate in the study, those presenting after 48 hours of onset of pain, patients To observe the accuracy for both systems, Area under the curves (AUC) with 95% confidence intervals (CI) were also calculated, and curves were plotted against standard diagnosis by Atlanta classification taken as reference line. All the analyses were performed by using the Statistical Package for Social Science (SPSS) Pak J Med Sci July - August 2019 Vol. 35 No. 4 www.pjms.org.pk 1009 BISAP score in prediction of severe acute pancreatitis Fig.1: Receiver operation characteristic (ROC) curve of Ranson’s scores in predicting SAP. Fig.2: Receiver operation characteristic (ROC) curve of BISAP scores in predicting SAP. Fig.1: Receiver operation characteristic (ROC) curve of Ranson’s scores in predicting SAP. Fig.1: Receiver operation characteristic (ROC) curve of Ranson’s scores in predicting SAP. Fig.2: Receiver operation characteristic (ROC) curve of BISAP scores in predicting SAP. Fig.2: Receiver operation characteristic (ROC) curve of BISAP scores in predicting SAP. Fig.2: Receiver operation characteristic (ROC) curve of BISAP scores in predicting SAP. Fig.1: Receiver operation characteristic (ROC) curve of Ranson’s scores in predicting SAP. software, Version 16, and Receiver Operating Characteristics (ROC) analysis was performed using software STATA version 8.1. For all analysis p-value < 0.05 was takes as significant. Surgery department of Dow University Hospital on the patient fulfilling the inclusion criteria after taking informed consent. METHODS According to Atlanta classification, diagnosed patients with severe AP were found 18.9% (n=39), and patients with mild and moderately severe AP were 81.1% (n=167). Table-I: Describes the characteristics of study participants. The observed BISAP and Ranson’s scores distribution was also reported in Table-I. Pak J Med Sci July - August 2019 Vol. 35 No. 4 www.pjms.org.pk 1010 RESULTS The study included a total of 206 patients of Acute Pancreatitis (AP) from Emergency department and Table-I: Description of patient’s characteristics (n=206). Table-I: Description of patient’s characteristics (n=206). Study variables mean ± SD Age in years 35.25 ± 8.29 Duration of symptoms (hrs.) 10.93 ± 9.94 Baseline pain score (VAS) 6.63 ± 1.31 n (%) Severity of pain Moderate 110 (53.4%) Severe 96 (46.6%) Age groups 20 - 29 61 (29.6%) 30 - 39 74 (35.9%) 40 - 50 71 (34.5%) Sex Male 81 (39.3%) Female 125 (60.7%) BISAP score 1 56 (27.2%) 2 86 (41.7%) 3 34 (16.5%) 4 30 (14.6%) Ranson’s score 1 65 (31.6%) 2 67 (32.5%) 3 20 (9.7%) 4 28 (13.6%) 5-7 26 (12.6%) BISAP: bedside index of severity in acute pancreatitis. Table-I: Description of patient’s characteristics (n=206). Study variables mean ± SD Age in years 35.25 ± 8.29 Duration of symptoms (hrs.) 10.93 ± 9.94 Baseline pain score (VAS) 6.63 ± 1.31 n (%) p Cutoff of ≥3 score values was taken as criteria for SAP. For BISAP, 31.10% (n=64) patients were found as SAP, whereas 35.90% (n=74) patients Table-II: Characteristics of patients having score ≥ 3 (n=206). Table-II: Characteristics of patients having score ≥ 3 (n=206). Characteristics BISAP score ≥ 3 p-value No Yes n (%) n (%) Age groups 20 - 29 35 (24.6) 26 (40.6) 0.017 30 - 39 50 (35.2) 24 (37.5) 40 - 50 57 (40.1) 14 (21.9) Sex Male 52 (36.6) 29 (45.3) 0.237 Female 90 (63.4) 35 (54.7) Ranson score ≥ 3 No Yes n (%) n (%) p-value Age groups 20 - 29 24 (18.2) 37 (50.0) < 0.01 30 - 39 53 (40.2) 21 (28.4) 40 – 50 55 (41.7) 16 (21.6) Sex Male 43 (32.6) 38 (51.4) < 0.01 Female 89 (67.4) 36 (48.6) Comparison between Ranson’s and BISAP score in predicting SAP is shown in Table-III. Anum Arif et al. Table-III: Comparison of BISAP with Ranson’s score in predicting SAP at ≥ 3 (n=206). RESULTS Accuracy Sensitivity Specificity +PV -PV AUC (95% CI) Measure of agreement Kappa (95% CI) Ranson ≥ 3 82.0% 97.4% 78.4% 51.3% 99.2% 0.92 (0.87 - 0.97) 0.56 (0.44 - 0.67) < 0.001 BISAP ≥ 3 76.2% 69.2% 77.8% 42.2% 91.5% 0.73 (0.64 - 0.82) 0.37 (0.23 - 0.50) < 0.001 BISAP: bedside index of severity in acute pancreatitis, SAP: severe acute pancreatitis, +PV: positive predictive value, -PV: negative predictive value, AUC: Area under curve, CI: confidence intervals. Table-III: Comparison of BISAP with Ranson’s score in predicting SAP at ≥ 3 (n=206 study conducted by Cho JH et al. according to which sensitivity of Ranson and BISAP score was 85.7 % and 61.9 % respectively.15 This is in contrast to BISAP score sensitivity of 58.33% reported by Kim BG et al.16 had SAP condition according to Ranson’s score. Characteristics of patients with score ≥3 are shown in Table-II. Comparison between Ranson’s and BISAP score in predicting SAP is shown in Table-III. With regard to mortality, 10 patients needed ICU admission, five had sepsis, four with pancreatic necrosis and one with MODS. Out of them there were 6 mortalities and all had Ranson score of > 3 and 4 had BISAP score of > 3. DISCUSSION Accurate and quick prediction of SAP is important in order to decrease mortality rate which is around 20% to 60%.7,11 Many scoring systems have been developed to determine the severe acute pancreatitis early so that better care can be provided to patients and mortality can be decreased. An ideal scoring system should be simple, safe, cheap and less time consuming. BISAP score is one of the newer scoring systems to predict the severity of acute pancreatitis. It has got 5 variable that can be done quickly in emergency department within 24 hours. In this study we determined the accuracy of BISAP score in comparison with Ranson score in predicting the SAP. The accuracy of BISAP score for predicting SAP is 76.2 %. Kappa value is 0.34 showing slight agreement between the the two whereas accuracy of Ranson’s score for predicting SAP is 82.0 % kappa value 0.56 showing fair agreement between the two. This is in comparison with the study reported by Khana AK et al6 as in his study accuracy of BISAP and Ranson;s score was 70.8 % and 80.6 % respectively. Slight different results are observed in study conducted by Kim B G et al. in which accuracy of BISAP and Ranson’s score for predicting severe acute pancreatitis was 84% and 94 % respectively which is far greater than accuracy of serum procalcitonin reported in the same study as 58%.16 Female predominance is noted in most of the studies which is confirmed in our study too 6,8,13 whereas no difference is reported between either sex in certain studies.7,14 Out of these SAP was seen in 54.7% and 48.6% of females according to BISAP and Ranson’s score respectively. Limitations of the study: It’s a single center study with small sample size. A multicenter validation study is required to confirm our results and second our observations of BISAP score in severe acute pancreatitis. Mean age of patients with acute pancreatitis was 35.25 ± 8.29 years (range 20 – 50) with 35.9% of the population in age range between 30 to 39 years. However majority of the patients fall in 4th and 5th decade of life according to Shabbir S et al13 and between 21-30 years according to Khanna AK et al.6 No difference of age group was reported by Chen L et al.7 REFERNCES 10. Mofidi R, Patil PV, Suttie SA, Parks RW. Risk assessment in acute pancreatits. Br J Surg. 2009;96:137-150. 1. Papachristou GI, Clermont G, Sharma A. Risk and markers of severe acute Pancreatitis Gastroenterol Clin North Am. 2007;36:277-296. doi: 10.1016/j.gtc.2007.03.003. 11. Ranson JH, Pasternack BS. Statistical methods for quantifying the severity of clinical acute pancreatitis. J Surg Res. 1977;22(2):79-91. 2007;36:277-296. doi: 10.1016/j.gtc.2007.03.003. 2. Forsmark CE, Baillie J. AGA Institute technical review on acute pancreatitis. Gastroenterol. 2007;132:2022-2044. doi: 10.1053/j.gastro.2007.03.065. 12. Ranson JH, Rifkind KM, Roses DF. Objective early identification of severe acute pancreatitis. Am J Gastroenterol. 1974;61(6):443-451. j g 3. Peery AE, Dellon ES, Lund J. Burden of gastrointestinal diseases in the United States: 2012 Update. Gastroenterol. 2012;143(5):1179-1187. doi: 10.1053/j.gastro.2012.08.002. 13. Shabir S, Jamal S, Khaliq T, Khan ZM. Comparison of BISAP with Ranson;s score in determining the severity of severe acute pancreatitis. J Coll Physicians Surg Pak. 2015;25(5) 328-331. j g 4. Tenner S, Baillie J, DeWitt J, Vege SS. American College of Gastroenterology Guideline: Management of Acute Pancreatitis. Am J Gastroenterol. 2013;108(9):1400-1415. doi: 10.1038/ajg.2013.218 ( ) 14. Singh VK, Wu BU, Bollen TL, Repas K, Maurer R, Johannes RS, et al. A prospective evaluation of the bedside index of severity of acute pancreatitis score in assessing mortality and intermediate markers of severity in acute pancreatitis. Am J Gastroenterol. 2009;104:966-971. doi :10.1038/ajg.2009.28. 5. Papachristou GI, Muddana V, Yadav D,O’ Connell M, Sanders MK, Slivka A, et al. Comparison of BISAP, Ranson’s, APACHE-II, and CTSI Scores in predicting organ Failure, complications, and mortality in acute pancreatitis. Am J Gastroenterol. 2010;105(2):435-441. doi: 10.1038/ajg.2009.28. 15. Cho JH, Kim TN, Chung H, Kim KH. Comparison of scoring systems in predicting severity of acute pancreatitis. World J Gastroenterol. 2015;21(8):2387-2397. doi: 10.3548/wjg.v21.i8.2387. 6. Khanna AK, Meher S, Prakash S, Tiwary SA, Singh U, Srivastava A, et al. Comparison of Ranson, Glasgow, MOSS, SIRS, BISAP, APACHE II, CTSI score, CRP, procalcitonin in predicting severity, organ failure, pancreatic necrosis and mortality in acute pancreatitis. HPB Surg. 2013;2013:367581. doi: 10.1155/2013/367581 jg 16. Kim BG, Noh MH, Ryu CH, Nam HS, Woo SM, Ryu SH et al. A comparison of the bisap score and serum procalcitonin fir predicting the severity of acute pancreatitis. Korean J Intern Med. 2013;28(3):322-329. doi: 10.3904/kjim.2013.28.3.322. CONCLUSION BISAP score is a valuable tool in predicting severity of severe acute pancreatitis being simple, easy and cost effective. The assessment is completed in 24 hours that allows early decision making and prompt management. Accuracy of BISAP and Ranson’s score is comparable. In our study, out of 206 patients, total number of 18.9% (n=39) patients has SAP according to Atlanta classification. We found that 38 out of 39 patients had SAP according to Ranson score therefore sensitivity of 97.4% whereas 27 out of 39 patients has SAP according to BISAP score therefore sensitivity of BISAP score was 69.2%. It is comparable to a Acknowledgement: Naureen Shaukat for technical help and writing assistance. Pak J Med Sci July - August 2019 Vol. 35 No. 4 www.pjms.org.pk 1011 Pak J Med Sci July - August 2019 Vol. 35 No. 4 www.pjms.org.pk 1012 Authors Contribution: 7. Chen L, Lu G, Zhou Q, Zhan Q. Evaluation of the BISAP score in predicting severity and prognosis of acute pancreatitis in Chineese patients. Int Surg. 2013;98;6-12. doi: 10.9738/0020- 8868-98.1.6. AA: Conceived, designed, did data collection, statistical analysis and manuscript writing. AA: Conceived, designed, did data collection, statistical analysis and manuscript writing. 8. Zhang J, Shahbaz M, Fang R, Liang B, Gao C, Gao H, et al. Comparison of the BISAP scores for predicting the severity of acute pancreatitis in Chinese patients according to the latest Atlanta classification. 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https://openalex.org/W2159595711	https://europepmc.org/articles/pmc3944673?pdf=render	English	null	Kawasaki disease in a girl with turner syndrome: a remarkable association	The Italian Journal of Pediatrics/Italian journal of pediatrics	2,014	cc-by	3,028	* Correspondence: stefano.stagi@yahoo.it 1Department of Health Sciences, University of Florence, Anna Meyer Children’s University Hospital, Florence, Italy Full list of author information is available at the end of the article Abstract We describe a girl with Turner syndrome, a genetic disorder of the X chromosome in a phenotypic female at increased risk of autoimmune and immunological diseases, who developed Kawasaki disease at the age of four years. Given the possible relationship between these two disorders, we recommend suspecting Kawasaki disease in patients with Turner syndrome who present with persistent fever of unknown origin and who are not responsive to antibiotic therapy. Attention should be given to this phenomenon, as patients with Turner syndrome are themselves at higher risk of cardiovascular defects. Further studies are needed to better clarify this issue. Open Access Open Access Introduction cardiovascular, and renal abnormalities. This syndrome is also associated with autoimmune diseases such as diabetes, coeliac disease, and rheumatologic and thyroid disorders [7]. Kawasaki disease (KD) is a febrile systemic vasculitis mainly affecting young children and complicated by coronary ar- tery aneurysms in approximately 25% of untreated patients [1]. Immunological abnormalities have been widely de- scribed in the acute phase of the disease [1]. Extensive immunological changes may be out of the normal range of responses to either viral or bacterial antigens [2], as has also been hypothesised for the mechanism of several auto- immune diseases [3]. Although patients with KD rarely de- velop a second immunological disorder, a study in a large cohort of affected children reported a higher incidence of coeliac disease than in the general population, strengthen- ing the possible link between KD and other autoimmune disorders [4]. Moreover, KD has been described in asso- ciation with a variety of immunodeficiency diseases, in- cluding chronic granulomatous disease, Wiskott-Aldrich syndrome, and hyperimmunoglobulin (Ig) E syndrome, conditions that are in turn associated with certain auto- immune diseases [5]. In the literature, only one case of a boy with mosaic 45, XY/45, XO who developed KD has been reported [8]. We describe a girl with TS who developed KD, and we hypothesise a possible relationship between these two pathologies. Kawasaki disease in a girl with turner syndrome: a remarkable association efano Stagi1*, Stefania Losi1, Francesco Chiarelli3, Maurizio de Martino1 and Fernanda Falcini2 © 2014 Stagi 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 credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Stagi et al. Italian Journal of Pediatrics 2014, 40:24 http://www.ijponline.net/content/40/1/24 Stagi et al. Italian Journal of Pediatrics 2014, 40:24 http://www.ijponline.net/content/40/1/24 ITALIAN JOURNAL OF PEDIATRICS Central Ltd. This is an Open Access article distributed under the terms of the Creative /creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and ed the original work is properly credited. The Creative Commons Public Domain mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, Stagi et al. Italian Journal of Pediatrics 2014, 40:24 http://www.ijponline.net/content/40/1/24 Stagi et al. Italian Journal of Pediatrics 2014, 40:24 http://www.ijponline.net/content/40/1/24 level of 12.3 g/dL, a white blood cell count of 11.90 × 109 cells/L, a fibrinogen level of 573 mg/dL, a sodium level of 142 mEq/L, an ALT level of 164 IU/mL (nv = 10–40), and a gamma GT level of 78 IU/ml (nv < 40 IU/mL). Microbiological evaluation for bacterial and viral infec- tions, including adenovirus, cytomegalovirus, parvovirus, herpes, and Epstein-Barr virus infections; Staphylococcus infection; and group A Streptococcus infection, yielded negative results. Throat and nasopharyngeal swabs for adenovirus culture were negative. Chest x-ray and abdom- inal ultrasound were unremarkable. Electrocardiography was normal, and 2D echocardiography showed mildly dilated coronary arteries associated with a bicuspid aortic valve, along with a moderate ascending aortic coarctation. (AGA) IgA and IgG, anti-endomysium (EmA) IgA, anti- transglutaminase (tTG) IgA, perinuclear antineutrophil cytoplasmic antibody (p-ANCA), anti-Saccharomyces cere- visiae antibodies (ASCAs), anticardiolipin (aCL) antibodies (ACAs), anti-thyroperoxidase (TPO), and antithyroglobu- lin (Tg) antibodies were negative. Antinuclear antibodies (ANAs) were positive, with a titre of 1:160. level of 12.3 g/dL, a white blood cell count of 11.90 × 109 cells/L, a fibrinogen level of 573 mg/dL, a sodium level of 142 mEq/L, an ALT level of 164 IU/mL (nv = 10–40), and a gamma GT level of 78 IU/ml (nv < 40 IU/mL). Microbiological evaluation for bacterial and viral infec- tions, including adenovirus, cytomegalovirus, parvovirus, herpes, and Epstein-Barr virus infections; Staphylococcus infection; and group A Streptococcus infection, yielded negative results. Throat and nasopharyngeal swabs for adenovirus culture were negative. Chest x-ray and abdom- inal ultrasound were unremarkable. Electrocardiography was normal, and 2D echocardiography showed mildly dilated coronary arteries associated with a bicuspid aortic valve, along with a moderate ascending aortic coarctation. During follow-up, at 5 years and 1 month old, the child developed transient low positivity for EmA and tTG, whereas at 8 years and 3 months old, she developed auto- immune thyroiditis, with negativity for AGA, EmA, tTG, p-ANCA, ASCAs, and ACAs. Case report A 4 d A 4-year-and-1-month-old Caucasian girl was admitted to our department with a 7-day history of persistent high fever, reaching 39°C despite antibiotic therapy, along with pharynx redness and a maculopapular rash on her trunk. Her past medical history revealed that she was born at the 33rd week of a 2nd dizygotic pregnancy. At 3 months of age, she was operated on for intestinal occlusion, and during the first year of life, she underwent two surgical interventions for a diaphragmatic hernia. Her neurological and psychological development was slightly delayed. Turner syndrome (TS) is a genetic disorder (1 in 2000– 2500 live-born female infants) resulting from the absence of an X chromosome or the presence of an abnormal X chromosome in a phenotypic female [6]. TS is charac- terised by certain typical features, such as growth retard- ation; gonadal insufficiency with infertility; and skeletal, At admission, she was extremely irritable, miserable, and pale. Her heart rate was 120/min; respiratory rate, 34/min; and brachial blood pressure, 80/50 mmHg. Her weight was 11.800 kg (< 3rd centile), and her height was 93.6 cm (3rd centile). Extensive laboratory tests revealed an erythrocyte sedi- mentation rate (ESR) of 61 mm/h (nv < 15), a C-reactive protein (CRP) level of 9.71 mg/dL (nv < 0.5), a haemoglobin Page 2 of 4 Page 2 of 4 Discussion On day 2 after admission, the girl developed non- exudative conjunctivitis, cervical lymphadenopathy, and mucositis, prompting us to diagnose KD (Figure 1a, b, and c). After intravenous immunoglobulin (IVIG; 2 g/Kg) and aspirin (50 mg/Kg in three divided doses) administration, her fever promptly dropped, and in the 2nd week, her platelet count reached 873 × 109. On day 15 after the onset of fever, the typical peeling of her digits supported a diagnosis of KD. TS is a disorder characterised by an increased risk of several autoimmune diseases, such as thyroid diseases, coeliac disease, inflammatory bowel disease, and rheuma- tologic and neurological disorders [6]. The mechanisms of this susceptibility are unknown. Non-random X chromo- some inactivation has been hypothesised as being involved in the development of autoimmunity, and X chromosome monosomy has also been proposed as a common etiologic mechanism for certain autoimmune diseases [7,9]. In our patient, the association of TS and KD might be purely coincidental. However, another patient, with mosaic 45, XY/45, XO and who developed KD and mildly dilated coronary arteries, has been described in the literature [8]. Thus, we assume that TS might be potentially complicated by a higher risk of developing KD. In parallel, due to the presence of dysmorphisms (cubitus valgus, a short fourth metacarpal, hyperconvex nails, and a high-arched palate), neonatal and heart malformations, and frequent episodes of serious otitis media, a genetic syndrome was assumed. Karyotype analysis revealed that the patient had a 45, XO karyotype, confirming the diag- nosis of TS (Figure 1d). Furthermore, it is interesting to note that our patient developed transient positivity for EmA and tTG and sub- sequently developed autoimmune thyroiditis, a condition A second step of laboratory tests was performed to ex- clude the presence of autoimmune diseases. Anti-gliadin Figure 1 Main characteristics of the patient during Kawasaki disease. a) Bilateral, non-exudative conjunctivitis; b) diffuse erythematous maculopapular rash; c) mucositis and glossitis; d) 45, X karyotype. Figure 1 Main characteristics of the patient during Kawasaki disease. a) Bilateral, non-exudative conjunctivitis; b) diffuse erythematous maculopapular rash; c) mucositis and glossitis; d) 45, X karyotype. Stagi et al. Italian Journal of Pediatrics 2014, 40:24 http://www.ijponline.net/content/40/1/24 Page 3 of 4 that is very common in TS. Consent Written informed consent was obtained from the par- ents of the patient for publication of this case report and accompanying images. A copy of the written con- sent is available for review by the Editor-in-Chief of this journal. 14. Noto N, Okada T, Karasawa K, Ayusawa M, Sumitomo N, Harada K, Mugishima H: Age-related acceleration of endothelial dysfunction and subclinical atherosclerosis in subjects with coronary artery lesions after kawasaki disease. Pediatr Cardiol 2009, 30:262–268. 15. Cohen Tervaert JW: Translational mini-review series on immunology of vascular disease: accelerated atherosclerosis in vasculitis. Clin Exp Immunol 2009, 156:377–385. 15. Cohen Tervaert JW: Translational mini-review series on immunology of vascular disease: accelerated atherosclerosis in vasculitis. Clin Exp Immunol 2009, 156:377–385. References Peeples E, Varman M, Yaghmour A, Chatterjee A: Kawasaki disease in a young boy with a neck mass. Consult Pediatr 2011, 10:276–280. 8. Peeples E, Varman M, Yaghmour A, Chatterjee A: Kawasaki disease in a young boy with a neck mass. Consult Pediatr 2011, 10:276–280. 9. Jørgensen KT, Rostgaard K, Bache I, Biggar RJ, Nielsen NM, Tommerup N, Frisch M: Autoimmune diseases in women with turner’s syndrome. Arthritis Rheum 2010, 62:658–666. 9. Jørgensen KT, Rostgaard K, Bache I, Biggar RJ, Nielsen NM, Tommerup N, Frisch M: Autoimmune diseases in women with turner’s syndrome. Arthritis Rheum 2010, 62:658–666. Hence, KD could be another immunologic disorder that is potentially associated with TS. Because of the possible congenital and postnatal cardiovascular prob- lems typical of TS, more attention must be given to pa- tients with the syndrome who present with a prolonged fever of unknown origin that is refractory to broad- spectrum antibiotic treatment, along with high levels of inflammation. 10. Falcini F, Trapani S, Turchini S, Farsi A, Ermini M, Keser G, Khamashta MA, Hughes GR: Immunological findings in kawasaki disease: an evaluation in a cohort of Italian children. Clin Exp Rheumatol 1997, 15:685–689. 10. Falcini F, Trapani S, Turchini S, Farsi A, Ermini M, Keser G, Khamashta MA, Hughes GR: Immunological findings in kawasaki disease: an evaluation in a cohort of Italian children. Clin Exp Rheumatol 1997, 15:685–689. 11. Gupta M, Johann-Liang R, Bussel JB, Gersony WM, Lehman TJ: Elevated IgA and IgM anticardiolipin antibodies in acute kawasaki disease. Cardiology 2002, 97:180–182. 11. Gupta M, Johann-Liang R, Bussel JB, Gersony WM, Lehman TJ: Elevated IgA and IgM anticardiolipin antibodies in acute kawasaki disease. Cardiology 2002, 97:180–182. 12. Venkatraman R, Singh S, Minz RW: Study of the autoantibody profile after the acute phase of kawasaki disease in a cohort of children from North India. Rheumatol Int 2006, 26:693–696. 12. Venkatraman R, Singh S, Minz RW: Study of the autoantibody profile after the acute phase of kawasaki disease in a cohort of children from North India. Rheumatol Int 2006, 26:693–696. 13. Carlson M, Airhart N, Lopez L, Silberbach M: Moderate aortic enlargement and bicuspid aortic valve are associated with aortic dissection in turner syndrome: report of the international turner syndrome aortic dissection registry. Circulation 2012, 126:2220–2226. 13. Authors’ contributions SS performed the endocrinological evaluation and wrote the paper. SL performed the gynaecological evaluation and participated in writing the paper. FC performed the endocrinological evaluation and participated in writing the paper. FF performed the rheumatologic evaluation. MdM participated in the rheumatologic evaluation and in writing the paper. All authors read and approved the final manuscript. SS performed the endocrinological evaluation and wrote the paper. SL performed the gynaecological evaluation and participated in writing the paper. FC performed the endocrinological evaluation and participated in writing the paper. FF performed the rheumatologic evaluation. MdM participated in the rheumatologic evaluation and in writing the paper. All authors read and approved the final manuscript. Funding Thi g This research did not receive any specific grant from any funding agency in the public, commercial, or not-for-profit sector. This association may also be of great concern because patients with TS (nearly 30%) may have and/or develop cardiovascular anomalies, such as aortic malformations and aneurysms, and particularly coronary artery disease (CAD), which is one of the most common causes of morbidity and mortality in TS and in KD [6]. Therefore, early cardiac imaging and an echocardiographic follow- up should be mandatory for TS patients [13]. References Carlson M, Airhart N, Lopez L, Silberbach M: Moderate aortic enlargement and bicuspid aortic valve are associated with aortic dissection in turner syndrome: report of the international turner syndrome aortic dissection registry. Circulation 2012, 126:2220–2226. Author details 1D f 1Department of Health Sciences, University of Florence, Anna Meyer Children’s University Hospital, Florence, Italy. 2Department of Internal Medicine, Rheumatology Section, Transition clinic, University of Florence, Florence, Italy. 3Department of Paediatrics, University of Chieti, Chieti, Italy. Received: 17 November 2013 Accepted: 20 January 2014 Published: 28 February 2014 CAD seems to be a common cardiac complication in both KD and TS, although the potential pathogenetic mechanisms are still unknown, and different sites of coronary arteries may be involved. Discussion Moreover, our recent data have emphasised a possible link between KD and other autoimmune disorders, indicating a higher incidence of coeliac disease than in the general population [4] and strengthening past data on the concurrence of autoanti- bodies in both the acute and the convalescent phases of KD, such as aCL [10,11], ANCAs [10], ANAs, and anti- thyroid microsomal antibodies [12]. 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The authors declare that there are no conflicts of interest that could be perceived as prejudicing the impartiality of the research reported. Page 4 of 4 Page 4 of 4 Stagi et al. Italian Journal of Pediatrics 2014, 40:24 http://www.ijponline.net/content/40/1/24 Stagi et al. Italian Journal of Pediatrics 2014, 40:24 http://www.ijponline.net/content/40/1/24 17. Furuno K, Yuge T, Kusuhara K, Takada H, Nishio H, Khajoee V, Ohno T, Hara T: CD25 + CD4+ regulatory T cells in patients with kawasaki disease. J Pediatr 2004, 145:385–390. 18. Gambineri E, Bianchi L, Losi S, Gelli MG, Stagi S, Moriondo M, Azzari C: Turner’s syndrome and autoimmunity: role of FOXP3 and regulatory T-cells. Clin Immunol 2006, 119(Suppl 1):S83. doi:10.1186/1824-7288-40-24 Cite this article as: Stagi et al.: Kawasaki disease in a girl with turner syndrome: a remarkable association. Italian Journal of Pediatrics 2014 40:24. 18. Gambineri E, Bianchi L, Losi S, Gelli MG, Stagi S, Moriondo M, Azzari C: Turner’s syndrome and autoimmunity: role of FOXP3 and regulatory T-cells. Clin Immunol 2006, 119(Suppl 1):S83. doi:10.1186/1824-7288-40-24 Cite this article as: Stagi et al.: Kawasaki disease in a girl with turner syndrome: a remarkable association. Italian Journal of Pediatrics 2014 40:24. 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https://openalex.org/W3102814978	https://link.springer.com/content/pdf/10.1007/978-3-030-56316-5_10.pdf	English	null	The Role of Demographic Policies in the Internationalization of Romanian Higher Education	Springer eBooks	2,020	cc-by	5,946	The Role of Demographic Policies in the Internationalization of Romanian Higher Education Robert Santa and Cezar Mihai Haj R. Santa (B) National University of Political Studies and Public Administration, Bucharest, Romania e-mail: robi.santa@gmail.com C. M. Haj Executive Agency for Higher Education, Research, Development and Innovation Funding Bucharest, Romania e-mail: cezarhaj@gmail.com © The Author(s) 2020 A. Curaj et al. (eds.), European Higher Education Area: Challenges for a New Decade, https://doi.org/10.1007/978-3-030-56316-5_10 1 Introduction In recent years, several European countries have tuned their policies pertaining to international students to their need for immigration reform and the recruitment of highly skilled, highly educated professionals into their economies. Europe has been lagging behind other developed regions when it comes to attracting highly educated labour from abroad, despite being one of the regions with the steepest demographic downturns in the world. Governments have been trying to correct this either by making it easier for highly skilled immigrants to move to Europe (via a multitude of schemes such as the EU-backed ‘Blue Card’) or by allowing international graduates to become long-term residents in an expedited fashion. A growing shortage of skilled workers and the role of higher education in tackling this issue have also been emerging as an important topic in the Romanian public debate, and immigration legislation has been revised and tuned to European practices. This paper aims to analyse the implementation of recent legal changes that now facilitate the employment of non-EU graduates of Romanian universities. It will try to explore the extent to which the law is already implemented, the way in which it has been internalized and used by universities to communicate to non-EU students or in their student recruitment activities, but also to look at how inter-institutional cooperation functions in light of recent legal changes. The paper is exploratory in nature and tracks the implementation of Romania’s new immigration legislation at a very early stage, just a year from the time of adoption. Nevertheless, from a policy analysis perspective, this is useful in order to identify weak spots on the road between legislative decisions and institutional practices. 131 132 R. Santa and C. M. Haj R. Santa and C. M. Haj R. Santa and C. M. Haj Avoiding any major controversies, the Romanian Parliament discretely modiﬁed immigration legislation in 2018,1 trying to overhaul high thresholds for access to permanent residency. Prior to this, becoming a permanent resident in Romania as a non-EU citizen was more difﬁcult and blocked at several choking points. On the one hand, a higher minimum wage was regulated for foreigners, on the other, a ﬁxed quota and stern enforcement of employment preference for EU citizens represented further obstacles, though the latter provision is still formally in place. 1Law text (Romanian) available online at: https://lege5.ro/Gratuit/gmydqobqgeza/legea-nr- 247-2018-pentru-modiﬁcarea-si-completarea-unor-acte-normative-privind-regimul-strainilor- in-romania. 2https://ind.nl/en/news/Pages/New-directive-improves-mobility-within-the-EU-for-researchers- and-students-from--%E2%80%98third-countries%E2%80%99.aspx. 3Eurostat, retrieved in October 2019 and available at: https://ec.europa.eu/eurostat/web/products- eurostat-news/-/DDN-20190523-1. 1 Introduction Changes in the new legislation included a provision that enabled foreign graduates in Romanian uni- versities to seek employment for up to nine months after graduation, as an alternative to the six months awarded for the resolution of administrative issues following stud- ies. The legislation was spearheaded by the need to align Romanian legislation with the provisions of European Directive (EU) 2016/801. The purpose of the Directive is, in turn, to harmonise the conditions for admission and authorisation at EU level and foster mobility for students and researchers. The Directive governs the condi- tions for third-country nationals for admission and authorisation as a researcher (and family members), student, trainee or volunteer in the context of European volunteer service.2 These new approaches are not unique to Romania and should be seen in light of similar policy adaptation across Europe. These changes address the need of many governments to compensate for the ageing population of various European countries, the need for ﬁscal sustainability and the desire to make immigration ﬁscally valuable. 4Eurostat, retrieved in October 2019 and available at: https://ec.europa.eu/eurostat/statistics- explained/index.php?title=Migrant_integration_statistics_-_education#Educational_attainment. 5Eurostat, retrieved in October 2019 and available at: https://ec.europa.eu/eurostat/statistics- explained/index.php/Migrant_integration_statistics_%E2%80%93_labour_market_indicators# Employment_rates. 6OECD data for PISA 2015, retrieved in October 2019 and available at: https://www.oecd- ilibrary.org/education/pisa-2015-results-volume-i/immigrant-background-student-performance- and-students-attitudes-towards-science_9789264266490-11-en. 2 Background WhiletheEUistryingtoexpandtheshareofpersonsaged30–34whohavecompleted a form of tertiary education to 40%, non-EU immigration in many countries weighs down such goals. With a few exceptions, notably the UK with its high share of educated migrants, European countries tend to have immigrant populations with low levels of education. For example, according to Eurostat data, almost 35% of non-EU immigrants had at most lower secondary education (ISCED 0–2), double the rate among Europeans without a migrant background. The share of tertiary education graduates among migrants was lower than the rate for natives and EU immigrants.3 Tertiary level The Role of Demographic Policies in the Internationalization … 133 attainment would be even lower in the post-Brexit EU27, as Britain (and indeed, Ireland) tended to be outliers via their attraction of a highly educated migrant popu- lation.4 attainment would be even lower in the post-Brexit EU27, as Britain (and indeed, Ireland) tended to be outliers via their attraction of a highly educated migrant popu- lation.4 The education level of immigrants seems to have a cascading effect in society, impacting other metrics. For example, one can easily notice that Britain has a smaller gap between non-EU migrant employment rates and the respective rate for natives.5 Ireland is in a similar position. Also, the gap in PISA test scores between immigrant and non-immigrant students is lower in countries with a more educated migrant population. In the case of Europe, this again leads to smaller differences in the United Kingdom,6 though it should be noted that—despite having a large number of migrants with ISCED 0–2 education—countries such as Spain and Italy also display moderate differences in results based on migration background. Research has already identiﬁed the key role of immigration policies in shaping the success of immigrants and their children in educational settings (e.g. Entorf and Minoiu 2004). All of these issues are, from a demographic standpoint, important for European countries. All EU members, sans exception, have below-replacement fertility levels and have had them for decades. This means that the eventual decline in the number of people working will have to be compensated either by raising the productivity of the dwindling domestic workforce (for example via greater automation), by immigration or (as is most likely) by a combination of both. International students have become a target for increasingly generous ‘waivers’ offered upon graduation in order to look for employment. 7Exceptions do exist, such as the recent UK proposal on using a points-based system to assess immigration decisions after 2021. 8Dutch Statistics CBS, retrieved in October 2019 from: https://www.cbs.nl/en-gb/news/2019/30/ indian-knowledge-migration-has-doubled. 9French Statistics INSEE, retrieved in October 2019 from: https://www.insee.fr/fr/statistiques/ 3640742#titre-bloc-6. 10For example, family reuniﬁcation. 2 Background While Britain brieﬂy reversed a pre-2012 policy on allowing students to seek employment, it has since reverted to it, offering graduates a generous two-year period to seek employment (Adams 2019). Sweden has also introduced similar policies in order to tackle short- ages of skilled workers (The Local 2019). Such policies also exist in countries such as the Netherlands, Denmark, Germany and, indeed, Romania since 2018. These policies have a fairly simple principle: they enable international students to try and apply for employment in the country they study after graduation. The host country, especially if it has not asked the students to pay for the full cost of their tuition, or if they study in a ﬁeld that sees skills shortages, is directly interested to at least offer the graduates a chance to extend their stay. The host country solves several issues related to immigration and integration by selecting graduates from domestic universities. First of all, there is a head-start on integration, even though it has to be said that many contemporary programs are taught in a foreign language (usually English). Secondly, issues such as diploma recognition and sector-speciﬁc internship experience are often solved before employment. Lastly, when the point 134 R. Santa and C. M. Haj of immigration is tertiary education, the state waives most prior integration costs (language tutoring, pre-tertiary education) and quickly starts receiving the net ﬁscal beneﬁt of having one more highly skilled resident in the tax system. All of these beneﬁts contrast with the more problematic integration of children with an immigrant background in general. Dronkers and de Heus (2012), as well as Dronkers and van der Velden (2013) point to a complex web of factors that inﬂuence educational performance among immigrant children in general, with factors such as religion, country of origin and community structures playing a role in education outcomes. With immigrants arriving as international students, the point of entry already includes a fairly high barrier deﬁned by previous academic success. Still, this modus operandi has some limitations. Policies aimed at recruiting stu- dents as skilled workers have a different logic than points-based systems, such as those developed by Australia and Canada. 10For example, family reuniﬁcation. 2 Background Most European countries use neither explicit quotas nor formally quantiﬁed systems of grading the merit of individual applications for residency.7 Employment and immediate labour market needs seem to be key concerns for policy-makers, in line with prior European efforts of recruiting ‘guest workers’. Immediate needs take priority over long-term concerns with inte- gration, and this could be seen as reﬂective of the lack of cultural awareness of what being a ‘country of immigration’ entails. Policies aimed at facilitating immigration by international graduates are already impacting the makeup of immigrant contingents that are awarded residency in some of the countries that use them. The Netherlands, for example, now receives a steady ﬂow of Indian immigrants, which often top annual non-EU, non-refugee immigra- tion.8 Efforts to reduce immigration via family reuniﬁcation that have preceded the recent international student boom mean that such inﬂows now dwarf immigration from previously dominant countries of origin (such as Morocco and Turkey). France has also seen its immigrants become increasingly educated,9 as have other countries inside the EU. The impact of the adoption of policies aimed at attracting a greater share of those highly skilled might be difﬁcult to gauge for a while, especially when concerning indirect networked migration,10 as the 2015 refugee crisis has seen a big inﬂow of migrants that were not screened before arrival in Europe. That means that the overall sociodemographic proﬁle of the total immigrant population might not improve in the short term. While Romania has been—until recently—aloof of these efforts, the debate around attracting international students has intensiﬁed. After 2009, the number of students fell abruptly, especially in the private sector and in the fee-paying subsector in public universities (CNFIS 2014). At the same time, the one chronic problem of The Role of Demographic Policies in the Internationalization … 135 unemployment and underemployment began gradually being reversed, with unem- ployment being as low as 3.9% in September 2019,11 below the EU average. Short- ages in high-skills sectors could be potentially problematic in any national effort to completely close the middle-income trap. Romania is in a very poor position, as Eurostat places it in the very last spot when it comes to tertiary education attainment. 11Eurostat, retrieved in October 2019 and available at: https://ec.europa.eu/eurostat/statistics- explained/index.php/Unemployment_statistics#Recent_developments. 12Eurostat, retrieved in October 2019 and available at: https://ec.europa.eu/eurostat/statistics- explained/index.php/Europe_2020_indicators_-_education#Increasing_attainment_at_tertiary_ level. 2 Background Less than 25% of people aged 30–34 have a higher education diploma as of 2018, and the number has even declined year-on-year.12 Romania is thus one of the few EU countries that risk failing to meet their Europe 2020 targets for tertiary education attainment. In these conditions, Romania is at a tipping point in its need to attract a greater number of highly skilled graduates. It displays a mix of demographic contraction, low share of highly educated people among its own citizenry, rapid economic and wage growth and low unemployment. Legal efforts to facilitate highly skilled immigration now exist, and the ensuing trickle-down effect has now been set in motion by deﬁning a legislative framework, though it is yet to be seen whether and how it will be used. 3 Methodology The present paper used a three-fold approach in analysing the relevant topic. On the one hand, it analysed the legislative tools that govern education-centred immigration policies in both greater Europe and in Romania. This was necessary to frame recent legislative changes in Romania into what is a wider policy practice in Europe. The second tool was a brief desk research covering materials and articles related to inter- nationalization efforts, including the argumentation used for the adoption of current legislation. The third tool was the use of interviews with key institutional representa- tives in Romania, to see the degree to which policy changes have been internalized by universities and are being used as part of Romania’s offer to international students. Of these instruments, semi-structured interviews were arguably the most impor- tant given that the paper tackles a very recent issue that has not yet been documented in academic literature or even in statistics bulletins. Due to some difﬁculties in estab- lishing interviews with central authorities, the ﬁrst four interviews were taken with representatives of universities that were deemed representative for the scope of this paper. These included three public and one private university. Three of the universi- ties were based in Bucharest, while one was regional. The ﬁfth interview was with central level representatives of the authority responsible with immigration, while a sixth was taken with the representative of a human resources company. The inter- views, with two exceptions, were either with two persons or included follow-up 136 R. Santa and C. M. Haj phone calls. This was due to the need, in bigger universities, to ask questions from both persons involved in decision-making and staff involved with the practical and administrative side of managing admission for international students. Thus, in total, 10 individuals were interviewed for this article. It should be noted that some criteria were used in selecting universities. These had to have a signiﬁcant (by Romanian standards) number of international students. Medical universities were excluded as these have traditionally attracted international students due to factors such as cost, numerus clausus in the home country or the value of Romanian diplomas in the context of professional regulation. Similarly, the universities were screened to avoid those that have an overwhelmingly Moldovan- origin international student body, as linguistic ties and legal facilities mean that Moldovan students are not international stricto sensu. 4 Internationalization in Romania Internationalization has been the object of attention for education and policy researchers over the past few years, while its importance in higher education dis- course and political practice has been rising. As universities have seen fewer and fewer domestic students due to the poor quality of secondary education and due to demographic factors, internationalization has also presented a greater level of interest for universities. Deca et al. (2015) noted that internationalization efforts in Romania started off in a largely ad hoc manner, with no national strategy and with many policy changes deter- mined by the need to comply with Bologna Process requirements or policy require- ments associated with EU accession. These included the adoption of the European Credit Transfer and Accumulation System (ECTS), the use of the diploma supple- ment and more participation in EU mobility programmes, but did not preclude the continuation of traditional partnerships such as those associated with Agence Uni- versitaire de la Francophonie (AUF) membership (ibid.). They also point to several structural obstacles existing in the way of internationalization efforts, including poor data collection, the lack of a national strategy and limited use of institutional strate- gies. g These deﬁciencies are also visible when looking at existing statistics. Romania remainsafairlymarginaldestinationforinternationalstudents.Thiscan,forexample, beseenwithEuropeanmobilities,with2.5timesmoreRomaniansleavingthecountry than other Europeans arriving to study in local universities (UEFISCDI 2018). But the number of international students who undertake their studies in Romania outside the ﬁeld of medicine, and who do not beneﬁt from ethno-preferential access is small. There is no research with regard to the degree to which employability was a factor in determining existing students to choose Romania. Such research does however exist for more general international student populations. When Medina and Duffy (1998) deﬁned ﬁve main directions for branding for universities seeking to promote themselves internationally, graduate career prospects were one of these directions. 137 The Role of Demographic Policies in the Internationalization … In their paper, graduate career prospects referred to employment prospects per se, expected income and employer attitudes towards said graduates. Rajika Bhandari (2018) noted that Indian and Chinese students (the main US intakes) reported con- cerns about employment opportunities, especially when enrolling at graduate level. 4 Internationalization in Romania 41 of university campus administrators in the United States had, in fact, reported that concerns over the limited number of H1B work visas (which offer temporary employment to skilled foreign nationals) were a factor in the decline in the number of international students applying to study in the country (ibid). An earlier study by Binsardi and Ekwulugo (2003) found that immigration and admission procedures ranked second after educational standards/qualiﬁcation recog- nition among motivations offered by international students who had chosen to attend universities in Britain. Employment was third, ahead of costs, culture and lifestyle. The impact of talent retention is, of course, quite positive for the countries of des- tination, which reap the rewards of having a greater number of graduates within their overall populations. Varghese (2008:24) noted that employment prospects for internationally mobile students are high and that while this premium is greater in developing countries (often the countries of origin), many do stay, giving as an exam- ple the large share of Chinese and Indian students in the US tech sector. It should be noted that while employability and employment prospects are a potential hook for international students, they are not necessarily a key driver for internationalization efforts by institutions. Altbach and Knight (2006) do not list the provision of employment for national labour markets as an institutional objective for internationalization. Ultimately, universities themselves beneﬁt from international- ization mainly while the students are present. As stated above, data shows that progress in attracting international students remains limited. Despite increased efforts to promote Romania as an international student destination, the number of newly arriving international students has been rising slowly. Furthermore, once Moldovans (who, due to the common language, are an atypical group of international students) are taken out of the tally, we actually see the past few years witnessing a slight decline in the number of study visas issued to non-Moldovan non-EU citizens (Table1). Nevertheless,withinthebodyofstudentsawardedRomanianstudyvisas,therehas been some diversiﬁcation. While Israeli, Tunisian, Iraqi and Nigerian students seem to have witnessed a steep decline in the past few years (the latter two nationalities with a steep drop between 2015 and 2016), there has been a steady rise in the number of ‘other’ students coming from non-traditional destinations. These have risen from 28.3% in 2015 to 36.5% in 2018 among non-Moldovan arrivals. Of the big traditional countries of origin for international students, Turkey has seen a signiﬁcant rise in total arrivals. 4 Internationalization in Romania R. Santa and C. M. Haj 138 Table 1 International student admissions (source: IGI) Citizens of 2015 2016 2017 2018 Total Moldova 1612 1720 1849 2202 7383 Israel 655 692 641 479 2467 Turkey 443 509 586 591 2129 Morocco 255 260 277 256 1048 Tunisia 355 234 200 173 962 Serbia 215 256 201 196 868 Ukraine 115 138 141 183 577 Iraq 226 132 96 107 561 Syria 126 96 113 112 447 Nigeria 246 53 67 75 441 Other 1039 1149 1175 1249 4612 Total-MD 3675 3519 3497 3421 14112 Total 5287 5239 5346 5623 21495 Table 1 International student admissions (source: IGI) 5 Findings Lastly, students graduating in Romanian universities were awarded the chance to stay for nine months to seek employment. It should be noted, however, that Romanian legislation does limit the absolute number of visas issued across categories. As such, there is an absolute cap that is placed on the number of foreign workers, currently at around 30.000 persons per year (Interview 5). This additional legislation authorizes the government to regulate the cap on a year-by-year basis, though interviewees from the immigration authority noted that this cap is not set in stone, and the total number of new admissions can be extended. The other restriction to the formally open legislation is the requirement for prior- itization of Romanian and European Union citizens. This is common across most of Europe as part of anti-social-dumping regulations that aim to limit employers from recruiting foreign workers and limiting wages. Nevertheless, law 247/2018 also toned down existing restrictions. For example, it lowered minimum wage requirements for non-EU citizens. Romanian minimum wage is now sufﬁcient to employ a non-EU foreigners while before 2018 the ﬂoor was higher. The changes in legislation are likely to have a more limited effect on tertiary graduates, as they usually have a higher level of income to begin with. It should be noted that while the new legislation explicitly regulated seeking employment as a valid reason for a visa extension, graduates had been able to ﬁnd employment under the previous law (Interviews 4, 5). Even though legislation did not explicitly permit seeking employment upon graduation, immigration ofﬁcials noted that the six-month extension offered to students in order to ﬁnish graduation formalities were in some cases used for this purpose. Nevertheless, the pre-Law 247 immigration regimen was often restrictive. One university (Interview 2) complained that, in practice, students had been struggling with visa extensions should they need a deadline extension for ﬁnal thesis projects. Labour shortages seemed to be acknowledged by most interviewees as a societal reality that is likely to affect Romania’s long-term development. And, in the informal setting of the interviews, the respondents often acknowledged the importance of uni- versities in attracting highly skilled foreign workers in the context of the demographic crisis. Employing skilled foreign workers has indeed been a long-time demand by employers, who often complain about labour shortages and currently use corporate networks or foreign agencies to recruit non-EU labour (Interview 6). 5 Findings Our initial research effort looked at existing legal documents and the arguments that they used. The Law 237/2018 was a catch-all overhaul law for Romania’s immi- gration and residency legislation, creating new immigration pathways, simplifying others, reducing the requirements necessary to employ non-EU staff and facilitat- ing international mobility in research, education and au pair childcare work. These changes brought Romanian legislation in line with European practices, but the law itself went beyond the scope of European Directive (EU) 2016/801. Among the new provisions introduced or perfected by the Law, the most mean- ingful from the standpoint of education include: 1. A deﬁnition was now provided for what an international student was (both ter- tiary and pre-tertiary). A similar deﬁnition was provided for international interns (“stagiar”). These deﬁnitions did not change de facto practices but enabled better alignment with EU and additional legislation; 2. The concept of educational project was introduced and used as a criterion in awarding certain types of visas; 3. Punitive clauses were introduced to limit access to residency in Romania for foreigners who had committed various crimes and misdemeanours, including criminal acts, breaches of migration and employment legislation in Romania and other EU states; 4. The criteria for being awarded an international study visa was updated (though in practice remained broadly similar to prior conditions); 5. Additional criteria linked to income and assurance were inserted, in order to both ensure that international students can afford their studies; 139 The Role of Demographic Policies in the Internationalization … 6. Provisions were introduced to facilitate the international mobility of non-EU citizens studying in another EU country; 6. Provisions were introduced to facilitate the international mobility of non-EU citizens studying in another EU country; 6. Provisions were introduced to facilitate the international mobility of non-EU citizens studying in another EU country; 6. Provisions were introduced to facilitate the international mobility of non-EU citizens studying in another EU country; 7. There was an overhaul of criteria used to award visas to non-EU researchers, and to ease intra-EU mobility for non-EU researchers; 7. There was an overhaul of criteria used to award visas to non-EU researchers, and to ease intra-EU mobility for non-EU researchers; 8. Lastly, students graduating in Romanian universities were awarded the chance to stay for nine months to seek employment. 8. 5 Findings In fact, leg- islative and executive authorities had already been addressing this issue before the adoption of Law 247/2018. For example, the overall cap on foreign workers has been raised in the past few years consistently, and it is current policy to raise it should the demand for workers exceed supply (Interview 5). However, up until now, this cap has mostly been used for recruitment in the hospitality and construction industries (Interview 5, 6). 140 R. Santa and C. M. Haj R. Santa and C. M. Haj However, none of the academic responders had resorted to using employment prospects as a hook or a prominent feature of their public discourse targeting potential international students. Universities would often tout the cost-effectiveness of their programmes (Interviews 1, 3), the lifestyle offered by living in a major European capital (Interview 2) or a mix between the two (Interview 4). Respondents usually seemed to consider membership of the European Union as a major selling point, as this would enable easy recognition of awarded degrees for employment purposes (elsewhere in the European Union). This, of course, is not entirely unexpected given the recent nature of the topic of immigration in public discourse in Romania. And, while immigration has been limited for the most part and is broadly a very recent phenomenon, emigration of both graduates and non-graduates has been a massiﬁed trend which has resulted in over 3.500.000 Romanian citizens living in other European Union countries. Nevertheless, there has also been a sharp increase in the number of immigrants living in the country in recent years, though this in itself is still largely an effect of circular migration by Romanian citizens moving back-and-forth from/to EU countries and a small but rapidly rising contingent of foreigners. As Eurostat data indicates (see Table2) the highest share of foreign-born residents inRomaniaisgivenbycountrieswithRomaniandiasporas,eitherethnicormigratory. This points to a fairly low level of authentically foreign permanent or long-duration immigration to the country and could be a factor in explaining why the idea of targeting non-nationals for employment purposes has yet to catch on. There is a rapidly growing number of non-nationals who are employed on a temporary basis, but these are not skills-selected but are awarded visas based on existing (and often short-term) needs in the labour market (Interviews 5, 6). This non-familiarity with the very topic of immigration can also be seen in inter-institutional cooperation, and how respondents related to it. 5 Findings While Bucharest- Table 2 Residents in Romania by country of birth (source: Eurostat) Country/year 2013 2018 Country/year 2013 2018 Romania 19,862,852 19,013,651 Russia 4,952 7,189 Moldova 59,670 199,703 Greece 4,085 6,864 Italy 22,486 62,914 China 2,978 5,473 Spain 18,827 47,311 USA 2,360 4,888 Ukraine 8,743 24,570 Israel 1,665 3,660 United Kingdom 2,604 21,050 Syria 2,295 3,358 Germany 3,759 20,168 Belgium 54 3,269 France 3,780 15,867 Ireland 3,780 2,632 Bulgaria 11,163 10,543 Serbia 1,529 2,465 Hungary 5,795 8,648 Austria 121 2,084 Turkey 5,057 7,901 Iraq 1,136 2,045 The Role of Demographic Policies in the Internationalization … 141 based universities tended to appreciate their cooperation with immigration authorities (Interviews 2, 3, 4), they mainly valued its role in facilitating visas and informing students on their rights, status changes etc. The only regional university interviewed had a less fortunate track-record in cooperating with regional immigration authorities (Interview 1). This contrasted with the attitude of the responders from the immigra- tion authority, which seemed to consider employment as a priority in awarding visas. It should be noted that respondents who became familiar with recent legal changes during the interview process expressed openness to using employment prospects as a bigger part of their marketing and branding efforts. gg p g g A major point of criticism within inter-institutional cooperation was the process of awarding ﬁrst-time entry visas for students. Due to the timing of the Romanian admission process (just 2–3 months before courses commence), the tradition of summer holidays in embassies and the limited capacity in consular ofﬁces, many students arrived in Romania after course started, with universities reporting delays ranging from over a month (Interview 2) to as long as three (Interview 4). There were also reports of countries where the rate of rejected visa applications was high enough to discourage future applicants (Interview 4). Among other ﬁndings of the interviews, there seemed to be a trend towards simplifying bureaucratic processes (a decision is often communicated to students using scanned ﬁles as opposed to physical dossiers), as well as an effort to better accommodate international students during their stay. The needs of international students reported by the interviewees were diverse, ranging from the provision of foreign language administrative services to—in an extreme example—protection from radicalization efforts. 5 Findings One university complained that accreditation processes are not conducive to the development of study programmes in foreign languages, placing signiﬁcant burdens on universities that try to develop English or French language versions of their existing study offer (Interview 2). 6 Conclusions Romanian authorities have, in recent years, simpliﬁed many of the immigration- related restrictions that previously made attracting international students more oner- ous than in many other European countries. This has included better alignment to European regulations, more leniency in processing admission dossiers and indeed greater leeway for international students graduating in Romania to stay and seek employment within the country. Administrative bodies tasked with implementing legislation seem proactive in implementing legislation to the advantage of international students, though the recent nature of the current legal framework does not offer scope for a quantitative analysis based on the number of issued visas and variations by category. Nevertheless, most Bucharest-based respondents deemed central level institutions as being supportive in their efforts to attract international students. 142 R. Santa and C. M. Haj R. Santa and C. M. Haj R. Santa and C. M. Haj On the other hand, the intra-institutional dialogue still seemed problematic. Most universities did not seem entirely familiar with the impact of recent legislative changes but were overall keen to use them in the future in order to better market themselves abroad. However, other state bodies were less conducive to greater open- ness. The late timeline of admissions, as currently regulated by law, means that students are pressed to obtain visas in a very short amount of time. Bureaucratic burdens remain and are indicative of a lack of inter-institutional trust, with certain policy priorities not reﬂected in the operational practices of embassies, for example. As a broad conclusion, it can be said that the updated legislative framework is, at the moment, limited in its overall impact on internationalization of Romanian higher education by the permanence of certain barriers. Chief among them is the scheduling of admissions and the limited capacity of overseas Romanian embassies to process dossiers in order to award visas, though domestic bureaucratic issues also exist. The present article should warrant a follow-up once statistics are compiled for the ﬁrst few years in the implementation of Law 247/2018, in order to determine if a statistically signiﬁcant rise in international graduates seeking employment in the country occurs. From a chronological point of view, and going beyond the ﬁndings of this paper, the new reforms can be seen as a new waypoint on the road to aligning Roma- nian higher education policies to those found in much of the rest of Europe. 6 Conclusions This started with the adoption of most Bologna tools, greater levels of mobility and greater research cooperation, but policy alignment is now crossing the boundary between education and immigration legislation in line with recent European practice. How- ever, the extensive transformation brought about by Bologna and European Union membership is still incomplete. As in many other countries, de facto practices in the higher education system are anchored as much in older and deep-rooted traditions as they are in newer policy initiatives. List of interviews Interview Responders Interview 1 Public university. Conducted via phone in two stages. Two responders Interview 2 Public university. Conducted face-to-face. Two responders Interview 3 Private university. Conducted face-to-face. One responder Interview 4 Public university. Conducted face-to-face with phone follow-up. Two responders Interview 5 Public authority dealing with immigration. Face-to-face interview with two responders Interview 6 Representative of human resources company. Telephone interview List of interviews Interview Responders Interview 1 Public university. Conducted via phone in two stages. Two responders Interview 2 Public university. Conducted face-to-face. Two responders Interview 3 Private university. Conducted face-to-face. One responder Interview 4 Public university. Conducted face-to-face with phone follow-up. Two responders Interview 5 Public authority dealing with immigration. Face-to-face interview with two responders Interview 6 Representative of human resources company. Telephone interview References Adams, R. (2019, September 10th). UK work visas for foreign graduates to be extended to two years, The Guardian. Retrieved from: https://www.theguardian.com/education P. G. Altbach and J. Knight, (2006), The Internationalization of Higher Education: Motivations and Realities, The NEA 2006 Almanac of Higher Education pp. 27–36 A Binsardi, F. Ekwulugo, (2003), International marketing of British education: research on th students’ perception and the UK market penetration CNFIS – National Council for Higher Education Funding (2014). Raportul public annual: Starea ﬁnant˘arii înv˘am˘amântului superior românesc si m˘asurile de optimizare ce se impun. Bucharest: CNFIS. Deca L., E Egron-Polak, CR.Fit, (2015). Internationalisation of Higher Education in Romanian National and Institutional Contexts, p. 138, Springer Dronkers, J. and de Heus, M. (2012) Immigrants’ Children Scientiﬁc Performance in a Double Com- parative Design: The Inﬂuence of Origin, Destination, and Community. Discussion Paper Series 13/12. Centre for Research and Analysis of Migration Department of Economics, University College London. Dronkers, J. and van der Velden, R. (2013) Positive but also Negative Effects of Ethnic Diversity in Schools on Educational Performance? An Empirical Test Using PISA Data. In Windzio, M., Integration and Inequality in Educational Institutions. Dordrecht: Springer Science. Entorf, H. and Minoiu, N. (2004). PISA Results: What a Difference Immigration Law Makes. Discussion Paper. IZA DP No. 1021. Bonn: Institute for the Study of Labor (IZA). Medina, J. F. & Duffy, M. F. (1998) Standardisation vs globalisation: a new perspective of brand strategies, Journal of Product and Brand Management, Vol. 7 No. 4, pp. 173–178. Rajika Bhandari, (2018). Attracting and Retaining Global Talent: International Graduate Students in the United States, International Higher Education, no. 93 d he Local (2019, Sept 23rd). Student job-hunters to get to stay one year in Sweden after uni, The Local. Retrieved from: https://www.thelocal.se/ The Local (2019, Sept 23rd). Student job-hunters t Local. Retrieved from: https://www.thelocal.se/ Local. Retrieved from: https://www.thelocal.se/ UEFISCDI, (2018) Public Policy Center, Internationalizarea înv˘at˘amântului superior. Policy brief Bucuresti: UEFISCDI. Varghese, N.V. (2008). Globalization of higher education and cross-border student mobility. Paris: International Institute for Educational Planning. Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. The Role of Demographic Policies in the Internationalization … The Role of Demographic Policies in the Internationalization … 143 References The images or other third party material in this chapter are included in the chapter’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.
https://openalex.org/W4302334741	https://zenodo.org/record/1479097/files/7117ijbb01.pdf	English	null	Modern Era of Medical Field : E-Health	Zenodo (CERN European Organization for Nuclear Research)	2,018	cc-by	4,856	MODERN ERA OF MEDICAL FIELD: E-HEALTH Sandeepak Bhandari Aleksandras Stulginskis University, Akademija, Kaunas, Lithuania. ABSTRACT E-Health is alluded to as utilizing of information and communication technologies (ICT) in restorative field to control treatment of patients, research, and wellbeing training and checking of general wellbeing. The reason for this paper is thusly to investigate an institutionalized system for E-Health challenges confronted by e-wellbeing A rundown of both e-wellbeing difficulties are given and a proposed structure is likewise accommodated E-Health and could give direction in the execution of e-wellbeing To understand the motivation behind the paper, an inductive substance examination procedure was taken after. The fundamental outcomes were that in spite of the fact that the difficulties exceeds the advantages in the gave records, there is still trust that through appropriate ICT arrangements the advantages of e-wellbeing can develop all the more quickly. This can prompt to enhanced e-wellbeing administration conveyance and nationals in nations can all profit by this. KEYWORDS Introduction, ICT,EHR,PHR, Proposed architecture and Challenges. International Journal on Bioinformatics & Biosciences (IJBB) Vol.7, No.1, March 2017 International Journal on Bioinformatics & Biosciences (IJBB) Vol.7, No.1, March 2017 1. INTRODUCTION E-Health is the use of information and communication technologies (ICT) for health. The E- Health unit works with partners at the global, regional and country level to promote and strengthen the use of information and communication technologies in health development. It is the means to deliver responsive healthcare tailored to the needs of the citizen. The Electronic Health Record (EHR) is a fundamental building block of all of these applications. The EHR allows the sharing of medical records between care providers across disciplines, institutions and geographic boundaries-Health can be used in different ways by: • The citizen/patient utilizes e Health when he looks for data on the web, utilizes self- administration instruments, takes an interest in electronic groups, and demands a moment feeling. • Primary Care includes the use of ICT by the Primary Health Care Team (PHCT) for patient management, medical records and electronic prescribing. Healthcare professionals can also call upon e Health for their Continuing Medical Education. • Home Care incorporates mind administrations which are conveyed by home care experts through broadcast communications to a patient in the home. • Home Care incorporates mind administrations which are conveyed by home care experts through broadcast communications to a patient in the home. DOI:10.5121/ijbb.2017.7101 1 1 International Journal on Bioinformatics & Biosciences (IJBB) Vol.7, No.1, March 2017 International Journal on Bioinformatics & Biosciences (IJBB) Vol.7, No.1, March 2017 • Hospitals may call upon ICT for planning coordination, persistent organization, research facility data, radiology, drug store, nursing, electronic informing between the doctor's facility and other human services performing artists for correspondence of clinical and authoritative information, and telemedicine and second conclusions, in any claim to fame. In 1999 a national study of telemedicine in Australia led to the promotion of the concept of 'e- health', the health sector's equivalent of 'e-commerce'. A new study explored the view that, with the convergence of technologies and the consequent increase in ability to perform multiple functions with those technologies, it is unwise to promote telemedicine in isolation from other uses of technologies in health-care [1]. In 1999 a national study of telemedicine in Australia led to the promotion of the concept of 'e- health', the health sector's equivalent of 'e-commerce'. 2.2 EMR /HER EMR stands for Electronic Medical Record and EHR stands for Electronic Health Record. Both terms have same importance and utilized for same reason. It alludes to the systematized gathering of patient and populace electronically-put away wellbeing data in an advanced organization. These records can be shared crosswise over various human services settings. Records are shared through system associated, venture wide data frameworks or other data systems and trades. EHRs may incorporate a scope of information, including socioeconomics medicinal history, drug and sensitivities, inoculation status, research center test outcomes, radiology pictures, key signs, individual insights like age and weight, and charging data. 2.1 ICT ICT stands for Information and communication technologies. It is an amplified term for data innovation (IT) which focuses on the part of brought together correspondences and the mix of media communications (phone lines and remote signs), PCs and in addition fundamental endeavour programming, middleware stockpiling, and varying media frameworks, which empower clients to get to, store, transmit, and control data. 2. TERMS USED IN E-HEALTH. . S US N . • ICT • EMR/HER • TRANSMURAL CARE • PHR • BORN DIGITAL 2 1 I 1. INTRODUCTION A new study explored the view that, with the convergence of technologies and the consequent increase in ability to perform multiple functions with those technologies, it is unwise to promote telemedicine in isolation from other uses of technologies in health-care [1]. International Journal on Bioinformatics & Biosciences (IJBB) Vol.7, No.1, March 2017 International Journal on Bioinformatics & Biosciences (IJBB) Vol.7, No.1, March 2017 It is types of record used by e-health to store information and data in digitized from (0, 1). It is types of record used by e-health to store information and data in digitized from (0, 1). 2.4 PHR PHR stands Personal health record (PHR) is an electronic application used by patients to maintain and manage their health information in a private, secure, and confidential environment. PHRs are overseen by patients. It incorporates data from an assortment of sources, including social insurance suppliers and patients themselves. It help patients safely and secretly store and screen wellbeing data, for example, slim down arrangements or information from home checking frameworks, and in addition understanding contact data, determination records, medicine records, sensitivity records, vaccination histories, and a great deal more. 2.3 Transmural Care Intra and Extra mural, refers to transmural care. It can be characterized as deal with patient some time recently, amid and after the doctor’s facility remains. This element makes contrast between ordinary wellbeing and e-wellbeing and this component of e-wellbeing is truly advantageous for patient as they will get take mind even after treatment. 2 2 3. RELATED WORK The rate at, and degree to, which such changes are acknowledged will be resolved to a limited extent by outside strengths that impact the expenses of creating and actualizing biomedical applications and the capacity of clinicians, patients, and 3 International Journal on Bioinformatics & Biosciences (IJBB) Vol.7, No.1, March 2017 the human services framework to accumulate the potential advantages. Creators compress a few worldwide strengths that are influencing biomedical figuring and that will decide the degree to which PCs are acclimatized into restorative practice: (1) new improvements in PC equipment and programming; (2) a continuous increment in the quantity of experts who have been prepared in both clinical drug and biomedical informatics; and (3) progressing changes in social insurance financing intended to control the rate of development of therapeutic consumptions. The new equipment innovations have made capable PCs economical and along these lines accessible to doctor's facilities, to offices inside clinics, and even to individual doctors. The wide determination of PCs of all sizes, costs, and abilities makes PC applications both alluring and available. Innovative advances in data stockpiling gadgets are encouraging the reasonable stockpiling of a lot of information, in this manner enhancing the plausibility of information escalated applications, for example, the all-computerized radiology division. Institutionalization of equipment and advances in system innovation are making it less demanding to share information and to incorporate related data administration works inside a doctor's facility or other medicinal services association. In this paper [4] authors discussed and research related to EHRs and presents several methodologic challenges. They evaluating the impact of an EHR are often conducted in complex, operational environments that do not accommodate blinding of study subjects to the intervention and often do not allow for creation of a simultaneous control arm. What's more, the effect on patients is aberrant and passed on through clinicians who are the immediate framework clients. Along these lines, while the measurements of EHR effect are regularly tolerant based parameters, the examination of this effect needs to mull over the truth that the patient-level measures are not autonomous of the clinician utilizing the EHR. The way that the EHR has various levels of effect has repercussions both for deciding the unit of examination in these reviews and for deciding how contemplate subjects ought to be randomized. 3. RELATED WORK In this paper[1], author discuss that a national study of telemedicine in Australia led to the promotion of the concept of ‘e-health’, the health sector's equivalent of ‘e-commerce’. A new study explored the view that, with the convergence of technologies and the consequent increase in ability to perform multiple functions with those technologies, it is unwise to promote telemedicine in isolation from other uses of technologies in health-care. The major sources of information for the study were the presentations and discussions at five national workshops held to discuss the findings of the original report on telemedicine. Nineteen case studies were identified. The case studies showed that with the convergence of technologies telehealth is becoming part of e-health. The cost-effectiveness of both telehealth and telemedicine improves considerably when they are part of an integrated use of telecommunications and information technology in the health sector. In this paper [2,] authors discuss that E-Health research is at an early stage of development. E-- Health research and the information collected from such research are complex. On the off chance that outlined, created, and utilized ideally, eHealth applications can possibly connect wellbeing variations, encourage investigation of populace level information to empower fitting of social insurance conveyance, and speed the interpretation of revelations into practice—progresses that parallel progressive advancements in biomedical science as atomic focusing of medications and the mapping of the human genome. Like other biomedical advances, eHealth applications have the potential for both advantages and damages. The last incorporate the possibility to imperil understanding protection, increment wellbeing abberations, lead patients far from successful medications, and extend the computerized separate. The dormant force of this blossoming medium requires analysts from various segments (industry, government, and the scholarly world) to team up on how best to tackle the specialized capacities of developing data advances to bolster the social and social substances in which individuals work and live, while improving the framework capacity to address the wellbeing needs of people In this paper [3] authors emphasize the myriad ways in which computers are used in biomedicine to ease the burdens of information processing and the means by which new technology promises to change the delivery of health care. 3. RELATED WORK Understanding of effect is likewise intricate in that the EHR can influence the human services framework in an assortment of ways. Thusly, EHR affect should be translated with regards to different partner bunches for whom the effect may have distinctive repercussions (e.g., tolerant versus payer points of view). In this paper [4] authors discussed and research related to EHRs and presents several methodologic challenges. They evaluating the impact of an EHR are often conducted in complex, operational environments that do not accommodate blinding of study subjects to the intervention and often do not allow for creation of a simultaneous control arm. What's more, the effect on patients is aberrant and passed on through clinicians who are the immediate framework clients. Along these lines, while the measurements of EHR effect are regularly tolerant based parameters, the examination of this effect needs to mull over the truth that the patient-level measures are not autonomous of the clinician utilizing the EHR. The way that the EHR has various levels of effect has repercussions both for deciding the unit of examination in these reviews and for deciding how contemplate subjects ought to be randomized. Understanding of effect is likewise intricate in that the EHR can influence the human services framework in an assortment of ways. Thusly, EHR affect should be translated with regards to different partner bunches for whom the effect may have distinctive repercussions (e.g., tolerant versus payer points of view). All the more for the most part, EHR designers, evaluators, and clients need a wide vision of EHRs as supporting general wellbeing, investigate, individual wellbeing administration (especially interminable malady administration), and clinical care. They ought to likewise bolster the advancement of a national wellbeing data foundation as a system for empowering information administration for general wellbeing and research past the fringes of an individual association. Specialists who depend on individual particular information for their investigations to make new learning that advances medicinal services conveyance, general wellbeing, and individual wellbeing administration need to eloquenthow currentarrangements hinder their capacity to work and in this way moderate potential headways that could influence the soundness of natives and the productivity of the wellbeing framework. It is likely that enactment will be expected to address this specific issue. 4 International Journal on Bioinformatics & Biosciences (IJBB) Vol.7, No.1, March 2017 5 4. 3. RELATED WORK PROPOSED DESIGNED FRAMEWORK FOR E-HEALTH Figure1: Proposed Framework for E-health USER LAYER/USER INTERFACE LAYER Online ServicesOffline/Emergency Services HEALTH LAYER Dentists, Laboratories, Pharmacies, Medical Specialists, Hospitals, Private Insures, Government ELECTRONIC LAYER Information and Communication Technologies (ICT) SHARED DATABASE EMR & PHR 4. PROPOSED DESIGNED FRAMEWORK FOR E-HEALTH Online ServicesOffline/Emergency Services Services Figure1: Proposed Framework for E-health ELECTRONIC LAYER Information and Communication Technologies (ICT) DATABASE EMR & PHR Figure1: Proposed Framework for E-health 5 International Journal on Bioinformatics & Biosciences (IJBB) Vol.7, No.1, March 2017 Different creators characterized and composed structure for E-Health in various routes from alternate points of view and every one of them have their own needs. In this examination, another, straightforward and viable structure proposed for E-wellbeing called three layers engineering/system of E-Health. In this system every layer draws in with different layers specifically or in a roundabout way. The system is basic and straight so that work of every layer can be see effectively and work of every layer never given way with another. Another critical property of this structure is that Independent. Its imply that if there is have to roll out improvements one layer of system of E-Health that won't influence alternate layers and every other layer ought to work ordinarily. Three layers system of E-Health comprises taking after layers with shared database. Different creators characterized and composed structure for E-Health in various routes from alternate points of view and every one of them have their own needs. In this examination, another, straightforward and viable structure proposed for E-wellbeing called three layers engineering/system of E-Health. In this system every layer draws in with different layers specifically or in a roundabout way. The system is basic and straight so that work of every layer can be see effectively and work of every layer never given way with another. Another critical property of this structure is that Independent. Its imply that if there is have to roll out improvements one layer of system of E-Health that won't influence alternate layers and every other layer ought to work ordinarily. Three layers system of E-Health comprises taking after layers with shared database. • USER LAYER/USER INTERFACE LAYER • HEALTH LAYER • ELECTRONIC LAYER 4.1 User Layer/User Interface Layer The first layer of three layers framework of E-Health is USER LAYER/USER INTERFACELAYER It is layer by which client collaborate with E-Health. This layer goes about as front end of E-wellbeing. This layer ought to be outlined in a manner that each kind and level of client can utilize and take the upsides of administrations gave by E-wellbeing. To make this layer more compelling great GUI(Graphical User Interface) can be utilized to make it easy to use. In this system, two sorts of administrations gave by client layer to be specific Online Services and Offline/Emergency Services. In Online administration client can connect with E- wellbeing and check the diverse administrations gave by E-wellbeing and one can get to his/her Personal wellbeing record (PHR) after production of record on E-wellbeing and get data in regards to his/her wellbeing reports, Medical test and some more. At some point there is crisis circumstance for client like mischance so it is critical for E-wellbeing that Emergency administrations ought to be given to handle this sort of circumstance. Crisis administrations like arrangement through Telephone call, Ambulance benefit by giving crisis number to client without use of system since Offline Service. 4.2 Health Layer The middle and intermediate layer is HEALTH LAYER. This layer go about as transitional between USER LAYER and E-LAYER (third layer).This layer is centertherapeutic layer i.e. this layer is in charge of wellbeing administrations gave to client who collaborate with E-Health by client. This layer utilize the administrations gave by Electronic layer and give administrations to the User layer. This layer comprise different medicinal administrations like Dentists, Laboratories, Pharmacies, Medical Specialists, Hospitals. This layer is additionally in charge of patients care amid, prior and then afterward the treatment it is called transmural mind. As opposed to restorative administrations to the patient there ought to be extra and critical upheld gave to the patients/clients, if there should arise an occurrence of, there is some kind of problem with patients amid treatment by giving therapeutic guarantees through Private Insures organizations or Government. Like User layer this layer likewise access to shared database and transfer all the data and information identified with the administrations, booked for arrangement, specialist's points of interest and other critical data required by the clients/patients ought to be additionally available to them. This layer is likewise in charge of the bona fide administrations 6 International Journal on Bioinformatics & Biosciences (IJBB) Vol.7, No.1, March 2017 ought to be given to the patients; it incorporates the all-around qualified and experienced specialists and legitimate treatment by utilizing present day innovations. 4.3 Electronic Layer The third and final layer of E-Health is ELECTRONIC LAYER. It is the layer on which E- Health is based. It is the layer which is in charge of digitization of wellbeing framework by utilizing Information and Communication Technologies (ICT).It is the layer which make, create and dealt with the common databased which is utilized by different layers . Data and correspondences advancements (ICTs) can assume a basic part in enhancing human services for people and groups. By giving new and more proficient methods for getting to, conveying, and putting away data, ICTs can connect the data isolates that have risen in the wellbeing part in creating nations—between wellbeing experts and the groups they serve and between the makers of wellbeing exploration and the specialists who require it. Through the advancement of databases and different applications, ICTs likewise give the ability to enhance wellbeing framework efficiencies and avoid therapeutic mistakes. For instance, a physician in a remote rural hospital is initially unable to diagnose a patient with a complex array of symptoms. However, using his MEDLINE search training and the hospital’s Internet connection, he is able to diagnose and successfully treat the patient for a tropical disease the patient picked up while traveling abroad. 5. CHALLENGES FOR E-HEALTH E-Health is exceptionally hard to characterize in correct way. Distinctive creators, diverse Organization and boards of trustees characterized E-Health in different ways-wellbeing term first time proposed, by national investigation of telemedicine in Australia in 1999.Later different creators, scientist characterized it in their own specific manners. E-wellbeing is digitization type of wellbeing to give wellbeing administrations online by actualizing Information and Communication Technologies (ICT) in restorative field but there are number of challenges for E- Health, it includes: • Privacy • Privacy • Effective Medical treatment • Acceptance of E-Health • Money related Barriers • Trouble learning and utilizing the product • Institutionalizing of health information systems • Lack of appropriate software • Difficult to handle EMR/HER • Effective Medical treatment • Acceptance of E-Health • Institutionalizing of health information systems • Lack of appropriate software • Difficult to handle EMR/HER 5.2 Effective Medical Treatment E-wellbeing is utilization of data and correspondence advances in medicinal field to give successful wellbeing administrations to patients on the web. It is one of the greatest difficulties confronted by the E-Health due to absence of fitting programming and innovations for the diverse medical issues. As opposed to procedures required for restorative issue, in some cases there is absence of specialists who can utilize the product or E-Health fittingly. 5.1 Privacy Privacy can be characterized as security of individual information on the web. It is a wide term that alludes to an assortment of variables, systems and advances used to ensure touchy and private information, correspondences, and inclinations. Protection is compulsory in this day and age. Everybody need to keep his/her secret information and protection secure from other.So.it is vital 7 7 International Journal on Bioinformatics & Biosciences (IJBB) Vol.7, No.1, March 2017 that E-Health ought to outlined in a way that it is fit for keep security of clients, patients, specialists and everybody who is a piece of E-wellbeing specifically or indirectly.So.it is one of the enormous difficulties enemy E-Health to give protection who are the piece of E-wellbeing. High and most recent security strategies ought to be utilized to give protection. 5.3 Acceptance Of E-Health E-Health is first time proposed in 1999, so a large portion of the general population doesn’t have mindfulness with respect to E-Health. So huge numbers of the general population don't acknowledge E-Health in restorative field and willing to utilize E-Health to get treatment on ,in some country places it is extremely hard to actualize data and correspondence innovations in medicinal field due to ignorance of data advances, required venture for foundation in E-Health. In creating nations it is exceptionally hard to actualize E-Health on account of inaccessibility of web, specialists and individuals don't willing to acknowledge E-Health as a result of ignorance. 5.4 Money Related Barriers E-health required research, foundation, specialists and expert individuals which prompted to immense venture and required financed bolster. So it is impractical for each nation now to execute E-Health in medicinal field. For example, in creating nations there is absence of get to administrations online in view of nonattendance of web .To give offices of web gigantic venture is required which prompt to enormous capital prerequisite. 5.5 Trouble Learning And Utilizing The Product E-Health programming is extremely perplexing and exceptionally uncommon individuals are specialists in them and it is extremely hard to learn them, on the grounds that the advancements changes quick. So E-Health is constantly endured in light of nonappearance of specialists. On other in the event that somebody to learn programming the learning expense is excessively costly on the grounds that few individuals know about that. On the off chance that specialists are accessible however at some point patients don't consent to acknowledge the utilization of programming or item for wellbeing reason. 5.6 Institutionalizing Of Health Information Systems The human services conveyance framework today utilizes a wide range of data frameworks from various merchants, both inside a solitary association and over different associations. For instance, a healing center may have a lab framework from one merchant, a drug store framework from another seller, and a patient care documentation framework from a third merchant. Doctors associated with the healing facility likewise have diverse frameworks in their workplaces, yet 8 International Journal on Bioinformatics & Biosciences (IJBB) Vol.7, No.1, March 2017 require access to information from the doctor's facility on their patients. In human services, principles give a typical dialect and set of desires that empower interoperability amongst frameworks as well as gadgets. In a perfect world, information trade construction and models ought to allow information to be shared between clinician, lab, doctor's facility, drug store, and patient paying little heed to application or application merchant keeping in mind the end goal to enhance human services conveyance. 5.7 Lack Of Appropriate Software In E-Health, there is absence of fitting programming for legitimate treatment. It is troublesome for specialists to create fitting programming in light of the fact that these delicate products are exceptionally mind boggling. There are different classes of programming which incorporates Diagnostic programming Public wellbeing and bio observation, Dental administration and patient record, Electronic wellbeing or restorative record ,Health framework administration, imaging/perception and Medical data frameworks Health programming required part of research with the goal that data is accessible in regards to the issue and arrangement then it is just conceivable to build up the product. 5.8 Difficult to Handle EMR/EHR. EMR stand for Electronic medical record, it is used to store all data and information related to E- Health. EHR contains huge amount of data so it is very hard to handle this large amount of data. E-Health requires that it handle EMR in such a way it is easy to update the data in EMR and retrieval of data from EMR. Cottage Med, Free MED, Gaia EHR, GNU med and GNU Health are the examples of HER. FUTURE WORK Could incorporate inquiring about in e-medical advantages and difficulties are experienced E-wellbeing intercessions should likewise be possible to minimize e-wellbeing challenges and to expand the advantages of e-wellbeing. International Journal on Bioinformatics & Biosciences (IJBB) Vol.7, No.1, March 2017 International Journal on Bioinformatics & Biosciences (IJBB) Vol.7, No.1, March 2017 6 . CONCLUSION The purpose of this paper is to explore the concept of E-health, challenges faced by E-Health and a proposed three layer architecture/framework for E-Health. E-Health is first timeproposed in 1999.E-health is very beneficial for medical field if it is implemented successfully. The difficulties that ought to be very respected before actualizing e-wellbeing incorporate budgetary obstructions, absence of IT and clinical assets, the trouble of learning and utilizing e-wellbeing programming, work force costs, institutionalization of Health Information Systems, time challenges, the usage of e-wellbeing in provincial regions (availability), information protection, interoperability, manageability, information quality, ease of use and the move from paper to electronic wellbeing records.E-Health gives the equivalent medicinal offices similarly for all individuals. Distinctive creators, association characterized E-Health in various ways. A three layer design/system for E-Health is proposed. This structure is straightforward, free, and successful for E-Health. It contains three layers to be specific client/UI layer, wellbeing layer and Electronic layer and a common database. 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https://openalex.org/W2047803773	https://zenodo.org/records/2088307/files/article.pdf	English	null	A Basis for a Durable Peace Between Germany and England	The annals of the American Academy of Political and Social Science/The Annals	1,917	public-domain	2,160	113 their birth to secure equality of rights and therefore must rest on justice. With the success of the Allies it is not only fair to presume, but most probable, that there will be no menacing autocratic powers after the termination of the present war. The democratic nations will be preponderant and they will have learned the lesson to be vigilant, so that for the first time in history the leading powers of the world being democratic will be privileged to enter into a partnership that will give security, under a league of democracies, for the perpetuation of freedom and the equal rights of all its con- stituents, great and small. Under the domination of autocratic nations the international relationship of the world was in an anar- chistic state. But under the league of democratic nations the international relationship of the world can and doubtless will be secured upon the broad and lasting foundation of international justice. at UNIV OF ILLINOIS URBANA on March 10, 2015 ann.sagepub.com Downloaded from BY WILLIAM C. BULLITT, Philadelphia. I shall not attempt to deal with the problems of durable peace in general but shall try to concentrate attention on one of those zones of hostility and hatred in which a conflagration is likely to arise and to wreck a durable peace after it has apparently been made. i There are, of course, many such zones in the world. There is the zone in the Pacific where the interests of the United States and Japan conflict. There is the zone in the Balkans where the inter- ests of Russia and Austria conflict; but I wish to call your attention to the zone in the North Sea, where the hatred of Germany and England concentrates. And I shall try to explain the source of that hatred and a method by which it may be eliminated. I do t think that th h tilit f G d E l d I do not think that the hostility of Germany and England springs primarily from commercial and industrial rivalry. I do not think that England’s hatred of Germany springs primarily from her wrath at the violation of Belgium and the atrocities com- at UNIV OF ILLINOIS URBANA on March 10, 2015 ann.sagepub.com Downloaded from 114 mitted in Belgium and France. II do not think it springs from envy of the growth of Germany’s power in the past decade. I do not think that Germany’s hatred of England springs primarily from envy of the vast British Colonial Empire or from the belief which is widespread in Germany, unbelievable as it may seem on this side of the water, that England started and organized the present war. g g p None of these things, to my mind, is at the bottom of the hostility between Germany and England. It lies much deeper; in the thing which is usually at the bottom of a great hatred- fear. Fear on the part of Germany, that the British fleet will starve her to death; fear, on the part of England, that the German submarines will starve her to death. How legitimate are these fears is shown vividly by the condi- tion of affairs in both those countries today-Germany on the verge of starvation; England afraid that in six months, if the submarine campaign goes on, she will be on the verge of starvation. But these fears are not simply things of today. at UNIV OF ILLINOIS URBANA on March 10, 2015 ann.sagepub.com Downloaded from I don’t think you can realize unless you have gone to bed hungry in Berlin during the war, how intensely every class in Ger- many, from the top of the Foreign Office to the end of the minority Socialist party, is determined that in some way there must come out of this war something which will eliminate the danger of being cut off from overseas supplies. The German Conservatives have their solution. They say, &dquo;All we have to do is to build a bigger fleet than England or simply destroy England altogether.&dquo; Fortunately, that is more easily dreamed than accomplished. For until England is willing to com- mit suicide, she will retain her present naval supremacy. She lives partly on her banking, to be sure, but vitally on the earnings of her shipping, on her imports of raw products, on her exports of finished products. Furthermore, her relationship with her colonies imposes on her the obligation of defending them, and this she ac- complishes, not by maintaining fleets in their waters, but by a con- centration of force in the North Sea, which is at once the base of defense and attack for the whole world. But this very supremacy in the North Sea, which England must maintain, means a perpetual latent control of German com- merce. This is the vicious circle of fear which produces the hatred and enmity between England and Germany. So long as the fleets of each threaten the merchantmen of the other, so long will there be fear and hatred and war between them. h id f h i i hi i The President of the United States perceived this a long time ago, and in January, 1915, in order to attempt to reconcile Germany and England, he sent an emissary to both those countries to propose what I consider one of the wisest plans that has ever been put for- ward by the great man, for I believe he, who is our President, is a great man. Th i f th P id t d d h The emissary of the President was ordered to propose that Germany and England and all the other nations in the world should agree that even in time of war, all merchantmen, both belligerent and neutral, should be unhindered in their passage except when carrying contraband, and that contraband should be confined strictly to munitions of war. BY WILLIAM C. BULLITT, Philadelphia. They are inherent in the economic life and geographical position of those two great industrial nations, cooped together in the same corner of Europe. Germany, today, scarcely less than England, is dependent upon the sea for her life. She has ceased to produce enough food to sup- port her people. She may be able to live through the present war with closed frontiers, but her agriculture has already been raised to a very high state of development. It is not susceptible of much greater development, and with her normal increase of population in ten years she will be utterly unable to live with closed frontiers. Her life will be in the gun muzzles of the British fleet.. Moreover, she earns her livelihood largely by importing raw materials, turning them into finished products, and exporting the finished products, and for this entire process she must have security on the sea. Fur- thermore, the fear that she will be cut off by the British fleet from her supplies of food and raw products is kept constantly in front of her by the fact that every German ship that goes to the ocean must pass by the door of England. Her ships can reach the open ocean only by way of the Channel or the North Sea, which is in truth but another channel, varying from three to four hundred miles in width, which can be controlled almost as easily by the fleet based on the Orkneys as the Channel is controlled by the fleet based on Ports- mouth. 115 at UNIV OF ILLINOIS URBANA on March 10, 2015 ann.sagepub.com Downloaded from This would mean that even in time of war the merchantmen of England and Germany would come un- hindered into port, that there would be no starvation of civilian populations, that there would be no threat of such starvation. 116 And I believe that it would mean that the fear which is at the bot- tom of the hostility between those nations would be eliminated and that in time, perhaps a decade or two, their mutual interest in the peaceful development of the undeveloped portions of the earth would lead to their cooperation and ultimately to their friendship. p y p The leaders of the German army and navy and of the Con- servative parties met the President’s proposal with a most em- phatic &dquo;No!&dquo; They said, &dquo;We will not give up our great offensive weapon, the submarine, by which some day we shall be able to starve England into submission.&dquo; But on the other hand, the Socialists, the Radicals, and Von Jagow, who was at that time the head of the Foreign Office, assented to the President’s proposal. They said, &dquo; We are willing to agree to give up our weapon of of- fense if we can make sure that we shall never have to suffer again the food shortage which is sucking the blood of our children, our wives and our parents.&dquo; And although these men are not in control of Germany today, there has been every indication in the past few months that they will be in control of Germany when the war closes, and I believe that in the peace conference Germany will stand firmly behind the President’s proposal. When the President’s emissary reached England, he met al- most exactly the same reception as in Germany. The Conserva- tives said, &dquo;No, never! We will never give up the means by which we killed Napoleon, by which we are killing Germany today. We will never give up the commercial blockade!&dquo; But the labor leaders, the Socialists, and particularly the group of Liberals led by Lord Loreburn, accepted the suggestion, and Sir Edward Grey himself was inclined very strongly in that direction. Then the sinking of the Lusitan,ia killed all hope of immediate reconcilement between Germany and England; and the subsequent career of the President’s proposal I have not time to trace. at UNIV OF ILLINOIS URBANA on March 10, 2015 ann.sagepub.com Downloaded from i But the fact is that when the peace conference comes, the President’s proposal will again be pushed by the representative of the United States. And I believe that England can be brought to back this proposal, although the sentiment there today, I imagine, would be against it. I believe that she will accept it ultimately for the reason that the submarine in the next six months will bring home to her what it means to fear starvation, what it means to be afraid that not only yourself, but also your children and your parents will not have food. at UNIV OF ILLINOIS URBANA on March 10, 2015 ann.sagepub.com Downloaded from 117 Furthermore, it has come to be generally recognized in the British Foreign O~ce, that England has been able to carry out her blockade of Germany, not merely because of her fleet, but also because we have been willing to acquiesce in that blockade because we believe, on the whole, that her cause has been just and that her triumph will be to the interest of the whole world. Furthermore, England knows that the submarine is still a relatively undeveloped weapon, and that no one can tell how fatal to merchant shipping the.super-submarine of the future may become. p I therefore believe, that if the President has the united support of America on this proposition, it will go through, particularly in view of a recent addition which the government has made to it. The addition is this: that although the right to stop merchantmen in time of war should be taken away from any individual state, it should be reserved to all the nations of the world acting collectively through the League to Enforce Peace. In other words, the league would carry the pistol which would be denied to any individual state. This addition will remove the chief objection of the British Conservatives; which is that the German army, if this plan should be adopted, would dictate the course of events in Europe; for the League to Enforce Peace would hold in its hands a counterpoise to balance the power of the German army. It is, I believe, the duty of all Americans who are interested in a durable peace to back the President in this proposal, because I see no other way whereby the hatred between Germany and Eng- land can be abolished, and unless that hatred can be done away with, unless the roots of it can be cut, while the League to Enforce Peace may prolong peace, it will never establish a peace which can be considered durable. h if hi h ld if h Furthermore, if this proposal should be adopted, if the starva- tion of civilian populations should be taken out of war, a great step forward will have been made in the establishment of decent inter- national mores. And after all, we are entering this war for one purpose and one only-that better international mores may be established on the earth. at UNIV OF ILLINOIS URBANA on March 10, 2015 ann.sagepub.com Downloaded from
https://openalex.org/W2892100861	https://tspace.library.utoronto.ca/bitstream/1807/90554/1/viruses-10-00487-v2.pdf	English	null	Viruses of Eukaryotic Algae: Diversity, Methods for Detection, and Future Directions	Viruses	2,018	cc-by	17,269	Received: 7 August 2018; Accepted: 7 September 2018; Published: 11 September 2018 Abstract: The scope for ecological studies of eukaryotic algal viruses has greatly improved with the development of molecular and bioinformatic approaches that do not require algal cultures. Here, we review the history and perceived future opportunities for research on eukaryotic algal viruses. We begin with a summary of the 65 eukaryotic algal viruses that are presently in culture collections, with emphasis on shared evolutionary traits (e.g., conserved core genes) of each known viral type. We then describe how core genes have been used to enable molecular detection of viruses in the environment, ranging from PCR-based ampliﬁcation to community scale “-omics” approaches. Special attention is given to recent studies that have employed network-analyses of -omics data to predict virus-host relationships, from which a general bioinformatics pipeline is described for this type of approach. Finally, we conclude with acknowledgement of how the ﬁeld of aquatic virology is adapting to these advances, and highlight the need to properly characterize new virus-host systems that may be isolated using preliminary molecular surveys. Researchers can approach this work using lessons learned from the Chlorella virus system, which is not only the best characterized algal-virus system, but is also responsible for much of the foundation in the ﬁeld of aquatic virology. Keywords: eukaryotic algal virus; algal-NCLDV; Picornavirales; phytoplankton Keywords: eukaryotic algal virus; algal-NCLDV; Picornavirales; phytoplankton Viruses of Eukaryotic Algae: Diversity, Methods for Detection, and Future Directions Samantha R. Coy 1 , Eric R. Gann 1 , Helena L. Pound 1 , Steven M. Short 2 and Steven W. Wilhelm 1,* 1 The Department of Microbiology, The University of Tennessee, Knoxville, TN 37996, USA; srose16@tennessee.edu (S.R.C.); egann@tennessee.edu (E.R.G.); hpound@tennessee.edu (H.L.P.) 2 The Department of Biology, The University of Toronto Mississauga, Mississauga, ON L5L 1C6, Canada; steven.short@utoronto.ca 1 The Department of Microbiology, The University of Tennessee, Knoxville, TN 37996, USA; srose16@tennessee.edu (S.R.C.); egann@tennessee.edu (E.R.G.); hpound@tennessee.edu (H.L.P.) 2 The Department of Biology, The University of Toronto Mississauga, Mississauga, ON L5L 1C6, Canada; steven.short@utoronto.ca 1 The Department of Microbiology, The University of Tennessee, Knoxville, TN 37996, USA; srose16@tennessee.edu (S.R.C.); egann@tennessee.edu (E.R.G.); hpound@tennessee.edu (H.L.P.) 2 The Department of Biology, The University of Toronto Mississauga, Mississauga, ON L5L 1C6, Canada; steven.short@utoronto.ca * Correspondence: wilhelm@utk.edu; Tel.: +1-865-974-0665 * Correspondence: wilhelm@utk.edu; Tel.: +1-865-974-0665 viruses viruses viruses viruses 1. Introduction Viruses infecting eukaryotic algae are extremely diverse. They have been reported with DNA or RNA genomes in various architectures (linear, circular, double-stranded, single-stranded, segmented) and sizes (4.4 to 638kb) [1]. Some viruses accomplish infection with just a few viral genes at their disposal, while others maintain a gene arsenal nearly 100 times that size. Viruses infecting algae inﬂuence large ecological and biogeochemical processes. They direct the evolution of hosts through predator-prey selection and genetic exchange, consequently inﬂuencing algal ﬁtness, population dynamics, and ultimately, microbial community structure. Infection can also alter the composition and distribution of organic matter in the environment (a process referred to as the aquatic ”viral shunt” [2]) and inﬂuence particle size-distribution, nutrient cycling, and biological system activity (e.g., respiration [3]). While algal viruses are important members in many aquatic environments, their contribution to these processes at the global scale primarily arises when they infect and lyse abundant bloom-forming algae. This includes harmful bloom formers and ecosystem scale specialists like coccolithophores that form blooms large enough to be observed from outer space [4]. Viruses 2018, 10, 487; doi:10.3390/v10090487 www.mdpi.com/journal/viruses www.mdpi.com/journal/viruses Viruses 2018, 10, 487 Viruses 2018, 10, 487 2 of 27 It is a relatively recent realization that algal viruses are ecologically signiﬁcant. In fact, the whole history of algal virus research has occurred primarily in just the last half century (Figure 1). While there have been sporadic observations of virus infection of algae cultures since the early 1970s [5,6], the importance of algal viruses in natural systems was brought into the limelight by a series of observations of virus-like-particles associated with important bloom-forming algae [7–9]. These ﬁndings inspired questions about the identity and evolutionary relationships within these virus-host systems. Such questions, however, required viruses to be isolated and genetically characterized. 3 of 27 3 of Viruses 2018, 10, 487 uses 2018, 10, x Figure 1. Timeline of eukaryotic algal virus research with important milestones highlighted. Colored bars represent the annual citations and publications generated f a Web of Science Citation Report using the field tag TS = (algal virus) for all databases. The search was conducted on 8 May 2018 at 11:00 a.m. Citation Report results w visualized as heatmaps using custom R scripts. Electron micrograph image [10] and electrophoretic gel [11] reprinted by permission. Network analysis [12] reprinted un authority of Creative Commons. Figure 1. Timeline of eukaryotic algal virus research with important milestones highlighted. gure 1. Timeline of eukaryotic algal virus research with important milestones highlighted. Colored bars represent the annual citations and publications generated fro Web of Science Citation Report using the field tag TS = (algal virus) for all databases. The search was conducted on 8 May 2018 at 11:00 a.m. Citation Report results we sualized as heatmaps using custom R scripts. Electron micrograph image [10] and electrophoretic gel [11] reprinted by permission. Network analysis [12] reprinted und uthority of Creative Commons. Figure 1. Timeline of eukaryotic algal virus research with important milestones highlighted. Colored bars represent the annual citations and publications generated from a Web of Science Citation Report using the ﬁeld tag TS = (algal virus) for all databases. The search was conducted on 8 May 2018 at 11:00 a.m. Citation Report results were visualized as heatmaps using custom R scripts. Electron micrograph image [10] and electrophoretic gel [11] reprinted by permission. Network analysis [12] reprinted under authority of Creative Commons. 1. Introduction Colored bars represent the annual citations and publications generated from a Web of Science Citation Report using the ﬁeld tag TS = (algal virus) for all databases. The search was conducted on 8 May 2018 at 11:00 a.m. Citation Report results were visualized as heatmaps using custom R scripts. Electron micrograph image [10] and electrophoretic gel [11] reprinted by permission. Networ analysis [12] reprinted under authority of Creative Commons. gure 1. Timeline of eukaryotic algal virus research with important milestones highlighted. Colored bars represent the annual citations and publications generated from Web of Science Citation Report using the field tag TS = (algal virus) for all databases. The search was conducted on 8 May 2018 at 11:00 a.m. Citation Report results wer ualized as heatmaps using custom R scripts. Electron micrograph image [10] and electrophoretic gel [11] reprinted by permission. Network analysis [12] reprinted unde thority of Creative Commons. Figure 1. Timeline of eukaryotic algal virus research with important milestones highlighted. Colored bars represent the annual citations and publications generated from a Web of Science Citation Report using the ﬁeld tag TS = (algal virus) for all databases. The search was conducted on 8 May 2018 at 11:00 a.m. Citation Report results were visualized as heatmaps using custom R scripts. Electron micrograph image [10] and electrophoretic gel [11] reprinted by permission. Network analysis [12] reprinted under authority of Creative Commons. Viruses 2018, 10, 487 4 of 27 One of the ﬁrst algal-virus systems to achieve “model” status were the double-stranded DNA (dsDNA) viruses that infect the unicellular, ex-symbiotic, green alga Chlorella [13]. The Chlorella virus-host model system remains the best characterized of all the algae-virus models, with genomes [14–18], transcriptomes [19,20], and proteomes [14] documented in the literature. Indeed, it was sequencing of the DNA polymerase B (polB) genes from Chlorella viruses PBCV-1 and NY-2A [21], and later from Micromonas pusilla virus SP1, that revealed a conserved amino acid sequence distinct from other known polB protein sequences. This observation enabled the development of degenerate PCR primers that selectively ampliﬁed these algal-virus polB genes [11,22]. The sequences of these PCR amplicons supported a unique monophyletic viral clade, now recognized as the family Phycodnaviridae of the Nucleocytoplasmic Large dsDNA Viruses (NCLDV). For a while the Phycodnaviridae was thought to be home to all of the large dsDNA algal viruses: perhaps even dominating the overall algal virus community. 1. Introduction This perspective changed when sequencing of new isolates demonstrated that their “core” genes were more closely related to genes from the protist-infecting “giant viruses” of family Mimiviridae [23–25]. In general, algal-infecting viruses are recognized as members of one of these two families, though future work may challenge the monophyletic nature of these groups. For example, clustering of the Phycodnaviridae is at times disrupted when homologs from other cellular or viral families are included in phylogenetic reconstructions [26–28] There have also been increasing reports of single-stranded DNA (ssDNA) viruses, mostly infecting diatoms, RNA viruses (Table 1) [29,30], and even parasites of these large algal viruses known as virophage [31,32]. The most informative reports on these systems have come from metagenomic and metatranscriptomic datasets that can detect the presence and activity of a wide range of DNA and RNA viruses. In turn, the known diversity of eukaryotic algal viruses has greatly expanded, at times even yielding putative full-length viral genome assemblies [12]. Perhaps most promising is the possibility of predicting virus-host relationships in silico [12,33,34], whereas traditional methods have relied on virus isolation from a relative few cultivated algae. Shotgun -omics further create the opportunity to identify virus-host pairs from environmental data and place them in semi-quantitative ecological context. Indeed, these studies may even serve as preliminary assessments of the future cultivation requirements for isolating new virus-host systems. This burgeoning scientiﬁc frontier necessitates a review on the known diversity of eukaryotic algal viruses, the molecular toolkit available for in situ studies on their ecology, and the direction aquatic virology is taking to adapt to these methodologies. 2. Diversity of Cultured Virus-Host Systems The diversity of algal viruses mirrors that of their hosts, bearing in mind that the name “algae” does not denote a common evolutionary relationship. Indeed, algae have been observed in freshwater, marine, and terrestrial systems, in unicellular, colonial, or multicellular forms, and in disparate taxonomic lineages. Nevertheless, the diversity of algae can be depicted using an existing taxonomic framework that includes seven “supergroups” consisting of Excavata, Amoebozoa, Opisthokonta, Archaeplastida, the SAR group (Stramenophila, Alveolata, and Rhizaria), and a series of non-delineated, “cryptic” organisms collectively referred to as the Incerta sedis [35]. Beyond this framework, the manner in which certain taxa are placed within eukaryotic phylogeny varies in the literature and is a subject of ongoing scientiﬁc debate. We adapted the schematic phylogeny presented by the TARA Oceans group [36] to illustrate the diversity of marine eukaryotic plankton, their relative abundance based on TARA Oceans 18S rDNA gene surveys, and lineage association with viruses that have been isolated and are maintained in lab cultures (Figure 2 and Table 1). This framework demonstrates that marine eukaryotic algae are known to occupy all but the Amoebozoa and Opisthokonta supergroups. Algae-infecting viruses have been isolated using hosts spanning almost all abundant planktonic lineages, though many are single systems or instances without genomic information to deﬁne viral phylogenetic placement (e.g., TampV). Although Pyramimonadales and Raphidophyceae were not abundant in the TARA Oceans 18S dataset, select species in these groups are known bloom-formers [37–40] making the available algal-virus system for these lineages ecologically 5 of 27 Viruses 2018, 10, 487 informative. Viruses have also been isolated on important non-planktonic species, such as brown and red macroalgae (Phaeophyceae and Rhodophyceae). Abundant lineages without an algae-infecting virus include photosynthetic Dictyochophyceae, the Prasino Clade 7 group, the Chryso/Synuro group, and the Apicomplexans—though some of the highly represented lineages could be attributed to non-photosynthetic members. Establishing well characterized host-virus systems in these lineages could be very useful for bloom-forming algae of these lineages. For example, it would be appealing to isolate a Pseudochattonella (Dictyochophyceae) infecting virus, as the host alga is responsible for ﬁsh kills. In 2016 Pseudochattonella was responsible for a massive ﬁsh kill in Peru amounting to an economic loss of ~$800 million dollars [41]. In another interesting, albeit more complicated example, survival of the red-tide, bloom-forming ciliate Mesodinium rubrum depends on ingestion of photosynthetic cryptophytes to obtain necessary organelles (e.g., plastid, mitochondria, nucleus) [42]. 2. Diversity of Cultured Virus-Host Systems Viruses infecting cryptophyte prey may compete with this grazer, thus serving as an important control on the frequency and duration of red tides. Such broad trophic effects have been shown in studies on Emiliania huxleyi, where viral-infected cells are ingested by zooplankton at different rates than non-infected cells [43,44]. 6 of 27 6 of 26 Viruses 2018, 10, 487 Vi 2018 10 Figure 2. (a) Schematic phylogeny adapted from de Vargas et al. demonstrating known virus- interactions with eukaryotic alga lineages. The phylogeny was originally constructed on recognized eukaryotic plankton lineages that were detected in TARA Oceans datasets, which included hits to all aquatic algal containing lineages. We collapsed the original tree to highlight these lineages in the context of their current phylogenetic placement. Green lines denote lineages with photosynthetic algal representatives, whereas the text color indicates whether all or only some representatives are phototrophic-green or black text, respectively; (b) Yellow boxes denote the top ten most abundant, planktonic, phototroph-associated lineages based on 18S rDNA surveyed in the TARA Oceans study. Asterisks denote lineages that were artificially grouped for simplicity, and their full descriptions can be found at http://taraoceans.sb-roscoff.fr/EukDiv/; (c) Red boxes denote algal-lineages that have an isolated algae-infecting virus in culture collection, though these are not all marine systems. The virus isolates are listed in Table 1. Figure 2. (a) Schematic phylogeny adapted from de Vargas et al. demonstrating known virus-interactions with eukaryotic alga lineages. The phylogeny was originally constructed on recognized eukaryotic plankton lineages that were detected in TARA Oceans datasets, which included hits to all aquatic algal containing lineages. We collapsed the original tree to highlight these lineages in the context of their current phylogenetic placement. Green lines denote lineages with photosynthetic algal representatives, whereas the text color indicates whether all or only some representatives are phototrophic-green or black text, respectively; (b) Yellow boxes denote the top ten most abundant, planktonic, phototroph-associated lineages based on 18S rDNA surveyed in the TARA Oceans study. Asterisks denote lineages that were artiﬁcially grouped for simplicity, and their full descriptions can be found at http://taraoceans.sb-roscoff.fr/EukDiv/; (c) Red boxes denote algal-lineages that have an isolated algae-infecting virus in culture collection, though these are not all marine systems. The virus isolates are listed in Table 1. Figure 2. (a) Schematic phylogeny adapted from de Vargas et al. demonstrating known virus- interactions with eukaryotic alga lineages. 2. Diversity of Cultured Virus-Host Systems The phylogeny was originally constructed on recognized eukaryotic plankton lineages that were detected in TARA Oceans datasets, which included hits to all aquatic algal containing lineages. We collapsed the original tree to highlight these lineages in the context of their current phylogenetic placement. Green lines denote lineages with photosynthetic algal representatives, whereas the text color indicates whether all or only some representatives are phototrophic-green or black text, respectively; (b) Yellow boxes denote the top ten most abundant, planktonic, phototroph-associated lineages based on 18S rDNA surveyed in the TARA Oceans study. Asterisks denote lineages that were artificially grouped for simplicity, and their full descriptions can be found at http://taraoceans.sb-roscoff.fr/EukDiv/; (c) Red boxes denote algal-lineages that have an isolated algae-infecting virus in culture collection, though these are not all marine systems. The virus isolates are listed in Table 1 Figure 2. (a) Schematic phylogeny adapted from de Vargas et al. demonstrating known virus-interactions with eukaryotic alga lineages. The phylogeny was originally constructed on recognized eukaryotic plankton lineages that were detected in TARA Oceans datasets, which included hits to all aquatic algal containing lineages. We collapsed the original tree to highlight these lineages in the context of their current phylogenetic placement. Green lines denote lineages with photosynthetic algal representatives, whereas the text color indicates whether all or only some representatives are phototrophic-green or black text, respectively; (b) Yellow boxes denote the top ten most abundant, planktonic, phototroph-associated lineages based on 18S rDNA surveyed in the TARA Oceans study. Asterisks denote lineages that were artiﬁcially grouped for simplicity, and their full descriptions can be found at http://taraoceans.sb-roscoff.fr/EukDiv/; (c) Red boxes denote algal-lineages that have an isolated algae-infecting virus in culture collection, though these are not all marine systems. The virus isolates are listed in Table 1. 7 of 27 Viruses 2018, 10, 487 Table 1. Algal viruses currently in culture collection. Table 1. Algal viruses currently in culture collection. Table 1. Algal viruses currently in culture collection. Host Algae Type Size (kbp or knt) Code References Chlorophyceaea Tetraselmis spp. 2. Diversity of Cultured Virus-Host Systems dsDNA 668 TetV Schvarcz et al., 2018 [45] Tetraselmis striata dsDNA 31 Tsv-N1 Pagarete et al., 2015 [46] Trebouxiophyceae Chlorella variabilis NC64A dsDNA 287–369 PBCV-1 Jeanniard et al., 2013 [17] Chlorella variabilis Syngen 2-3 dsDNA 327 OSy-NE5 Quispe et al., 2017 [18] Chlorella heliozoae SAG 3.83 dsDNA 288–327 ATCV-1 Jeanniard et al., 2013 [17] Micratinium conductrix Pbi dsDNA 302–329 CVM Jeanniard et al., 2013 [17] Mamiellophyceae Ostreococcus lucimarinus dsDNA 182–196 OlV1 Derelle et al., 2015 [47] Ostreococcus tauri dsDNA 184–192 OtV5 Weynberg et al., 2011 [48] Ostreococcus mediterraneus dsDNA 193 OmV1 Derelle et al., 2015 [47] Bathycoccus sp. RCC1105 dsDNA 187–198 BpV Moreau et al., 2010 [49] Micromonas pusilla CCMP1545 dsDNA 186–195 MpV-02T Martinez Martinez et al., 2015 [50] Micromonas pusilla LAC38 dsDNA 173–205 MpV1 Finke et al., 2017 [51] Micromonas pusilla LAC38 dsRNA 25.5 MpRV Brussaard et al., 2004 [52] Micromonas polaris dsDNA 191–205 MpoV Maat et al., 2017 [53] Pyramimonadales Pyramimonas orientalis dsDNA 560 PoV Sandaa et al., 2001 [54] Rhodophyta Chondrus crispus dsRNA 6 CcV Rousvoal et al., 2016 [55] Dinophyceaea Heterocapsa circularisquama dsDNA 356 HcDNAV Ogata et al., 2009 [56] Heterocapsa circularisquama ssRNA 4.4 HcRNAV Tomaru et al., 2004 [57] Heterocapsa pygmea dsDNA ND HpygDNAV Kim et al., 2012 [58] Gymnodinium mikimotoi ND ND GM6/GM7 Onji et al., 2003 [59] Bacillariophyta Chaetoceros cf. gracilise ND ND CspNIV Bettarel et al., 2005 [60] Chaetoceros salsugineum ssDNA 6 CsalDNAV* Nagasaki et al., 2005 [61] Chaetoceros setoensis ssDNA 5.8 CsetDNAV* Tomaru et al., 2013 [62] Chaetoceros socialis f. radians ssRNA 9.4 CsfrRNAV Tomaru et al., 2009b [63] Chaetoceros lorenzianus ssDNA 5.8 ClorDNAV* Tomaru et al., 2011 [64] Chaetoceros tenuissimus ssDNA 5.6 CtenDNAV-I* Tomaru et al., 2011 [65] Chaetoceros tenuissimus ssDNA 5.6 CtenDNAV-II* Kimura and Tomaru 2015 [66] 8 of 27 Viruses 2018, 10, 487 Table 1. Cont. Host Algae Type Size (kbp or knt) Code References Chaetoceros tenuissimus ssRNA 9.4 CtenRNAV Shirai et al., 2008 [67] Chaetoceros tenuissimus, Chaetoceros spp. ssRNA 9.6 CtenRNAV-II Kimura and Tomaru 2015 [66] Chaetoceros spp. SS628-11 ssDNA 5.5 Csp07DNAV* Kimura et al., 2013 [68] Chaetoceros spp. TG07-C28 ssDNA ND Csp05DNAV Toyoda et al., 2012 [69] Chaetoceros debilis ssDNA ND CdebDNAV Tomaru et al., 2008 [70] Chaetoceros sp. SS08-C03 ssRNA 9.4 Csp03RNAV Tomaru et al., 2013 [71] Chaetoceros cf. 2. Diversity of Cultured Virus-Host Systems wighamii ssDNA 7-8 CwNIV Eissler et al., 2009 [72] Asterionellopsis glacialis ssRNA 9.5 AglaRNAV Tomaru et al., 2012 [73] Thalassionema nitzschioides ssDNA 5.5 TnitDNAV Tomaru et al., 2012 [73] Rhizosolenia setigera ssRNA 11.2 RsetRNAV Nagasaki et al., 2004 [74] Skeletonema costatum ND ND ScosV Kim et al., 2015 [75] Stephanopyxis palmeriana ND ND SpalV Kim et al., 2015 [76] Pelagophyceae Aureococcus anophagefferens dsDNA 370 AaV Moniruzzaman et al., 2014 [23] Phaeophyceae Ectocarpus fasciculatus dsDNA 340 EfasV Kapp et al., 1997 [77] Ectocarpus siliculosus dsDNA 320 EsV Kapp et al., 1997 [77] Feldmannia irregularis dsDNA 180 FirrV Kapp et al., 1997 [77] Feldmannia simplex dsDNA 220 FlexV Kapp et al., 1997 [77] Feldmannia species dsDNA 170 FsV Henry and Meints 1992 [78] Hincksia hinckiae dsDNA 240 HincV Kapp et al., 1997 [77] Myriotrichia clavaeformis dsDNA 320 MclaV Kapp et al., 1997 [77] Pilayella littoralis dsDNA 280 PlitV Maier et al., 1998 [79] Raphidophyceae Heterosigma akashiwo dsDNA ND HaV Nagasaki et al., 1997 [80] Heterosigma akashiwo dsDNA 180 O1s1 Lawrence et al., 2006 [81] Heterosigma akashiwo ssRNA 9.1 HaRNAV Tai et al., 2003 [82] Heterosigma akashiwo ND ND HaNIV Lawrence et al., 2001 [83] Haptophyta Emiliania huxleyi dsDNA 415 EhV Castberg et al., 2002 [84] Phaeocystis globosa dsDNA 466 PgV-16T (Group I) Baudoux et al., 2005 [85] Phaeocystis globosa dsDNA 177 PgV-03T (Group II) Baudoux et al., 2005 [85] Phaeocystis globosa dsDNA 176 PgV-102P Wilson et al., 2006 [86] Phaeocystis pouchetii dsDNA 485 PpV Jacobsen et al., 1996 [87] 9 of 27 Viruses 2018, 10, 487 Table 1. Cont. Host Algae Type Size (kbp or knt) Code References Chrysochromulina breviﬁlum, Chrysochromulina strobilus dsDNA ND CbV Suttle and Chan 1995 [88] Chrysochromulina ericina dsDNA 510 CeV Sandaa et al., 2001 [54] Chrysochromulina parva dsDNA 485 CpV Mirza et al., 2015 [89] Haptolina ericina, Prymnesium kappa dsDNA 530 HeV-RF02 Johannessen et al., 2015 [90] Prymnesium kappa, Haptolina ericina dsDNA ND PkV-RF01 Johannessen et al., 2015 [90] Prymnesium kappa dsDNA 507 PkV-RF02 Johannessen et al., 2015 [90] Prymnesium parvum dsDNA ND PpDNAV Wagstaff et al., 2017 [91] Cryptophyta Teleaulax amphioxeia ND ND TampV Nagasaki et al., 2009 [92] Table 1. Summary of all reported eukaryotic algal viruses that have been isolated. A range of genome sizes (kbp or knt) represents multiple virus strains associated with the same host species, and in this case, only the type virus is reported under the code column. Table 1. Summary of all reported eukaryotic algal viruses that have been isolated. A range of genome sizes (kbp or knt) represents multiple virus strains associated with the same host species, and in this case, only the type virus is reported under the code column. Asteriks denote original names for some of the diatom ssDNA viruses, which have since been renamed and placed into genera of the family Bacilladnaviridae (Chaetoceros setoensis DNA virus = Diatodnavirus; Chaetoceros salsugineum DNA virus 1 = Chaetoceros protobacilladnavirus 1; Chaetoceros sp. DNA virus 7 = Chaetoceros protobacilladnavirus 2; Chaetoceros lorenzianus DNA virus = Chaetoceros protobacilladnavirus 3; Chaetoceros tenuissimus DNA viruses type I and II = Chaetoceros protobacilladnavirus 4). ND = Not detected or reported. 2. Diversity of Cultured Virus-Host Systems Asteriks denote original names for some of the diatom ssDNA viruses, which have since been renamed and placed into genera of the family Bacilladnaviridae (Chaetoceros setoensis DNA virus = Diatodnavirus; Chaetoceros salsugineum DNA virus 1 = Chaetoceros protobacilladnavirus 1; Chaetoceros sp. DNA virus 7 = Chaetoceros protobacilladnavirus 2; Chaetoceros lorenzianus DNA virus = Chaetoceros protobacilladnavirus 3; Chaetoceros tenuissimus DNA viruses type I and II = Chaetoceros protobacilladnavirus 4). ND = Not detected or reported. Table 1. Summary of all reported eukaryotic algal viruses that have been isolated. A range of genome sizes (kbp or knt) represents multiple virus strains associated with the same host species, and in this case, only the type virus is reported under the code column. Asteriks denote original names for some of the diatom ssDNA viruses, which have since been renamed and placed into genera of the family Bacilladnaviridae (Chaetoceros setoensis DNA virus = Diatodnavirus; Chaetoceros salsugineum DNA virus 1 = Chaetoceros protobacilladnavirus 1; Chaetoceros sp. DNA virus 7 = Chaetoceros protobacilladnavirus 2; Chaetoceros lorenzianus DNA virus = Chaetoceros protobacilladnavirus 3; Chaetoceros tenuissimus DNA viruses type I and II = Chaetoceros protobacilladnavirus 4). ND = Not detected or reported. Viruses 2018, 10, 487 Viruses 2018, 10, 487 10 of 27 Eukaryotic algal viruses in culture collections have been isolated from ~60 alga species (Table 1). Most of these are lytic, dsDNA viruses of the NCLDV group with a narrow, known host-range. The abundance of NCLDVs would imply that these are an ecologically relevant algal-virus type in the virus community, but whether or not these are the dominating type is unclear. This would certainly contrast with plant viromes which are dominated by RNA viruses. It is also possible that NCLDVs are more easily detected and isolated, thus explaining why only dsDNA viruses have been isolated from water samples that putatively contained other types of viruses. For example, electron micrographs of bloom-associated Emiliania huxleyi cells have been observed to simultaneously contain both small (50–60 nm) and large (185–200 nm) intracellular VLPs [93]. Similar observations been made in Pyramimonas orientalis [94], but currently only one type of dsDNA virus has been isolated for this algae [54]. It is possible that these viruses compete for algal infection, but they may also represent a case of virus-infecting virophage that are already known to co-occur with Mimiviridae [95,96], and perhaps even Phycodnaviridae [31,33] viruses. 2. Diversity of Cultured Virus-Host Systems Observations of co-occurring viruses are not limited to microscopy either; network analysis of metatranscriptomic data has linked the brown alga Aureococcus anophagefferens to its known dsDNA virus AaV as well as to uncharacterized ssDNA viruses [12], although the mechanism of this linkage (either direct, or via a co-occurring microbial host of the virus) remains elusive. In short, algae may be infected by many types of viruses, potentially at the same time, and the numerically dominant virus type may not always represent that which is in the culture collection. To date, there are four algal species that are known to be infected by diverse viruses comprised of different nucleic acid types. These include Heterosigma akashiwo, Chaetoceros tenuissimus, Micromonas pusilla, and Heterocapsa circularisquama, and in all cases the different virus types infect the same host strain [97]. The coexistence of Heterosigma akashiwo viruses HaRNAV and HaDNAV is especially intriguing given these viruses exhibit opposite infection dynamics; the RNA virus has a high viral production rate, but a slower lytic cycle, whereas the DNA virus quickly replicates but produces fewer particles [81]. It was hypothesized that coexistence could be maintained through variable host densities and viral decay rates, thus representing viruses that may have evolved as r- or k- strategists as has been proposed for Heterocapsa viruses [98], but is certainly not supported enough to be extrapolated as an explanation for all co-occurring viruses. Even virus isolates of the same nucleic acid type and species can exhibit considerable diversity. This can be extreme in some cases, where dsDNA viruses infecting the same algal host, which would be expected to cluster phylogenetically, are afﬁliated with NCLDV viral families Mimiviridae or Phycodnaviridae (e.g., Phaeocystis globosa Virus Groups I and Groups II [25,99]. It is possible that eukaryotic algae may commonly be infected by viruses of diverse replication strategies, and evolutionary histories, but the extent of this, as well as the factors that may allow this, needs more thorough investigation. 2.1. dsDNA Viruses Infecting Eukaryotic Algae Most dsDNA viruses infecting algae are members of the NCLDV group, with the proposed exception of Tsv-N1 [46]. Algal-NCLDV viruses have large genomes that encode hundreds of protein coding genes. Their evolutionary relationship has been inferred by core genes conserved across NCLDVs [100], placing them into either the family Phycodnaviridae or as extended members of the family Mimiviridae. Algal viruses of the latter group have recently been given the proposed distinction of Mesomimivirinae [101], but for our purposes we will maintain the Mimiviridae description. The one exception to these two family assignments is HcDNAV, which shares closer similarity to the family Asfarviridae [56]. To date, the NCLDV core gene compliment has been reduced to just a few genes (e.g., D5R packaging ATPase, D13L major capsid protein, and B family DNA polymerase), implying that the genetic diversity is huge among this group. Indeed, a genomic comparison among Phycodnaviridae members PBCV-1 (Chloroviruses), EsV-1 (Phaeoviruses), and EhV-86 (Coccolithoviruses) yielded only 14 conserved homologs from a pool of ~1000 genes [102]. A more comprehensive look at these diverse genes can be found in genus-speciﬁc reviews of the Phycodnaviridae [17,47,51,103–105]. Viruses 2018, 10, 487 Viruses 2018, 10, 487 11 of 27 11 of 27 It is anticipated that any single algal host can be permissive to many closely related virus variants, whereby phylogenetic comparisons of their core genes will reveal distinct clades (e.g., Micromonas pusilla and Chlorella variabilis viruses) with differences in latent phases, burst sizes, and genome size [17]. In closely related viruses this is best resolved using concatenated alignments of marker protein sequences. At the same time, the origin of some of these genes is often attributed to gene transfer events. Many algal NCLDVs have acquired non-ancestral genes, but the majority of these appear to come from difference sources: Prasinoviruses acquire most of these from their host, Chlorovirus non-ancestral genes mostly derive from bacteria [106], and Aureococcus anophagefferens Virus (AaV) encodes a more even mixture of host, bacterial, archaeal, and viral genes [23]. At the same time, it is worth noting that the origin of some genes could be difﬁcult to ascertain if only a limited subset of viral (and host) homologs have been sequenced and annotated in public databases. 2.2. ssDNA Viruses Infecting Eukaryotic Algae To date, the only ssDNA alga-infecting viruses that have been isolated are those which infect diatoms (Bacillariophyceae). In total, diatoms are a collective of an estimated 12,000–30,000 species, representing one of the most abundant phytoplankton groups in freshwater and marine environments [108]. Most diatom-virus systems currently in culture are those infecting the cosmopolitan genus Chaetoceros. These isometric virus particles are ~35 nm in diameter and house circular, ssDNA genomes ranging from ~5.5–6.0 kb [66]. The genomes generally encode four open reading frames consisting of an endonuclease (Rep), a major capsid protein, and two ORFs with unknown function. The capsid and replication initiating endonuclease are used in phylogenetic analyses. Three new members (whose genomes are ~4.5–4.7 kb) were recently reported from a de novo assembly of metagenomic reads from the mollusk Amphibola crenata and from sediment within an estuary in New Zealand [109]. Phylogenetic analysis of the capsid proteins suggest this gene is a recent acquisition from ssRNA viruses, which is interesting, though not without precedent [110,111]. These metagenome assembled viruses have resulted in the taxonomic reclassiﬁcation of diatom viruses into the family Bacilladnaviridae that includes cultured diatom viruses noted in Table 1 with asterisks [112]. Many other ssDNA viruses are being detected in omics datasets [12], though resolving their speciﬁc host is an ongoing challenge. 2.1. dsDNA Viruses Infecting Eukaryotic Algae Regardless, it has been suggested that viruses whose hosts are in closer association with bacteria tend to encode more putative non-ancestral genes, and that these genes cluster near the terminal ends of the viral genome [107]. However, while the Chlorella algae is an endosymbiont of Paramecium that is certainly in close proximity to bacteria, the non-ancestral genes carried by the virus are evenly dispersed across its genome [17]. In contrast, AaV displays terminal clusters of non-ancestral genes [23], but its host is a free-living photo/osmotroph. In either case, the biological implication of such high viral gene diversity, and how it is generated, is unclear. It may help the virus acquire its speciﬁc needs for infection but has also been proposed to allow viruses to infect multiple hosts. 2.3. RNA Viruses Infecting Eukaryotic Algae Algae-infecting viruses with single (ss) and double-stranded (ds) RNA genomes have also been isolated and characterized, although most attention has been focused on the ssRNA isolates. Both virus groups encode an RNA-dependent RNA polymerase (RdRP), as well as proteases and helicases that can be used to infer distant evolutionary relationships. Most information on dsRNA algal viruses has been derived from the original isolation papers describing the evolutionary relationships of the isolates. MpRV, a dsRNA virus of Micromonas pusilla, forms its own genus within the family Reoviridae (unassigned order) and has been proposed to be the ancestral line of the Reoviridae based on its placement between clades that demonstrate turreted or non-turreted virions [113]. The other dsRNA virus isolate is Chondrus crispus virus (CcV), a toti-virus like entity. CcV represents an extraordinary case of a putative quasispecies virus that was accidentally discovered when a small band of dsRNA (~6 kb) was observed during host genomic preparation for sequencing [55]. Similar dsRNA bands 12 of 27 12 of 27 Viruses 2018, 10, 487 have been observed in extracts from all algal life phases, geographic locations, and in extracts from other red algae, though virus-like-particles and host lysis was not observed. The CcV system may represent either a latent or chronic (i.e., particle production below the limit of detection) viral infection that is ubiquitous among red algae, similar to known latent dsDNA viral infections of brown algae by Phaeoviruses [114]. Since both Chondrus crispus and Micromonas pusilla are ecologically important algae, characterization of their relationship with these viruses is important and perhaps reﬂective of a need to search for more dsRNA viruses associated with algae. ssRNA viruses have received considerably more attention since their hosts are common marine phytoplankton with some species capable of forming harmful blooms [39,115,116]. Most of the alga-infecting ssRNA viruses are members of the order Picornavirales (Figure 3), with a few contradictions that are awaiting a taxonomic re-evaluation based on molecular data. The viruses infecting Heterocapsa and Heterosigma are the sole members of the families Alvernaviridae (unassigned order) and Marnavirdiae (order Picornavirales), respectively [109,112], while the genus Bacillarnavirus (order Picornavirales) includes formal members Chaetoceros socialis forma radians RNA virus, Chaetoceros tenuissimus RNA virus 01, and Rhizosolenia setigera RNA virus 01. Other diatom viruses Csp03RNAV, AglaRNAV, and CtenRNAV type II are putative members of Bacillarnavirus based on phylogenetic relationships of replicase or structural proteins [1]. 2.3. RNA Viruses Infecting Eukaryotic Algae The diatom viruses are generally thought to be highly species speciﬁc based on host-range experiments, with the exception of CtenRNAV type II which can infect four Chaetoceros sp. in addition to Chaetoceros tenuissimus [66]. These viruses and their hosts represent ecologically important systems that may reveal much on the persistence, co-existence, and competition of diatom viruses. Foxtail Mosaic virus Indian Citrus Ringspot virus Heterocapsa circularsquama virus Turkey Coronavirus Bat Coronavirus Turkey Astrovirus 1 Phytophthora infestans RNA virus 1 Barley Yellow Mosaic virus Tomato Ringspot virus Heterosigma akashiwo RNA virus Acute Bee Paralysis virus Cricket Paralysis virus Triatoma virus Black Queen Cell virus Varroa Destructor virus Antheraea pernyi Ifavirus Sacbrood virus Rhizosolenia setigera virus Marine RNA virus JP-A Chaetocerus tenuissimus RNA virus II Marine RNA virus SF-1 Marine RNA virus JP-B Beihai Picorna-like virus 63 Aurantiochytrium ssRNA virus 01 Wenzhou Picorna-like virus 24 98 91 52 98 100 97 66 83 91 100 90 52 99 99 99 98 99 80 100 Phylogenetic Group Picornavirales Order Secoviridae Marnaviridae Dicistroviridae Ifaviridae Unclassifed Tymovirales Order Nidovirales Order Unclassifed Order Bovine Diarrhea virus Asterionellopsis glacialis virus Chaetocerus tenuissimus RNA virus II Figure 3. Diversity of single-stranded RNA viruses depicted based on phylogeny of RNA-dependent RNA polymerase (Rdrp NCBI CDD:01699) reference sequences downloaded from NCBI RefSeq database (see in Supplemental Table S1). Sequences were aligned and trimmed in Mega7 [117] and an unrooted maximum likelihood phylogeny was created using PhyML 3.0 with LG model [118]. Empirical equilibrium frequencies were used with aLRT SH-like statistics for branch support. Phylogenetic groups are color coded with algal viruses denoted by a star. Viral isolates from metagenomic assemblies are in red text. Figure 3. Diversity of single-stranded RNA viruses depicted based on phylogeny of RNA-dependent RNA polymerase (Rdrp NCBI CDD:01699) reference sequences downloaded from NCBI RefSeq database (see in Supplemental Table S1). Sequences were aligned and trimmed in Mega7 [117] and an unrooted maximum likelihood phylogeny was created using PhyML 3.0 with LG model [118]. Empirical equilibrium frequencies were used with aLRT SH-like statistics for branch support. Phylogenetic groups are color coded with algal viruses denoted by a star. Viral isolates from metagenomic assemblies are in red text. 3.1. PCR Applications for Estimating Viral Diversity and Dynamics 3.1. PCR Applications for Estimating Viral Diversity and Dynamics Developing algal-virus model systems in the lab can inform much on the biology and ecology of algal viruses, but dependence on these systems is a limiting step. The ability to determine viral Viruses 2018, 10, 487 13 of 27 geographic distributions, population ﬂuctuations, and diversity ultimately depends on analysis of environmental samples. Microscopic methods [119], ﬂow cytometry [120–122], and infectivity assays (e.g., most probable number, plaque assay [13]) have been used to answer these questions, but these approaches lack taxonomic resolution and/or the relatively quick processing time that molecular techniques provide. To date, the principal molecular method for studying environmental algal viruses has been based on PCR ampliﬁcation of conserved marker genes. Most of this work has focused on algal NCLDVs using polB [11] and the NCLDV major capsid protein (mcp) as gene targets [123]: subsets of this community have been further examined using primers that speciﬁcally target the extended, algal Mimiviridae major capsid protein (AMmcp) [124]. For reference, the potential ampliﬁcation ranges of these primers are mapped against a phylogeny of sequenced virus isolates (Figure 4). There has been discussion on ampliﬁcation bias of polB primers based on observations that environmental datasets tend to amplify prasinoviruses, even though these may be environmentally abundant viral types [98]. The gene ampliﬁed by this primer set has also been suggested to be a poor marker for resolving within algal virus genera. For example, there are two distinct groups of Phaeocystis globosa infecting viruses, and these groups phylogenetically cluster into different families [1]. Diversity may be better assessed using genome ﬂuidity measurements of the pan-genome [125], but this would work better for describing viruses with full-genome sequences. Indeed, marker gene primer sets remain useful for elucidating environmental diversity of algal NCLDVs. Phycodnaviridae polB (AVS1 and AVS2) mcp (mcpF and mcpR) mcp (AMmcpF and AMmcpR) Mimiviridae Ostreococcus tauri virus 2 Ostreococcus tauri virus 1 Micromonas pusilla virus 12T Bathycoccus spp. virus Dishui Lake Phycodnavirus 1 Yellowstone Lake Phycodnavirus 1 Yellowstone Lake Phycodnavirus 2 Paramecium bursaria Chlorella virus FR483 Acanthocystis turfacea Chlorella virus 1 Paramecium bursaria Chlorella virus 1 Only Syngen Nebraska Virus 5 Paramecium bursaria Chlorella virus AR158 Paramecium bursaria Chlorella virus NY2A Heterosigma akashiwo virus 01 Chrysochromulina breviflium virus PW1 Chrysochromulina parva virus BQ1 Ectocarpus siliculosus virus 1 Feldmannia spp. 3.1. PCR Applications for Estimating Viral Diversity and Dynamics virus Emiliania huxleyi virus 86 Emiliania huxleyi virus 201 Pyramimonas orientalis virus Tetraselmis virus 1 Aureococcus anophageferens virus Organic Lake Phycodnavirus 1 Organic Lake Phycodnavirus 2 Chrysochomulina ericina virus Prymnesium kappa virus Phaeocystis globosa virus 16T Phaeocystis pouchetti virus Heterocapsa circularisquama DNA virus Figure 4. Phylogenetic tree depicting the evolutionary relationships of algal NCLDVs based on amino acid alignment (ClustalW) of the core gene, DNA polymerase B (see in Supplemental Table S2). The tree was built using the maximum likelihood method based on the JTT matrix-based model with 200 iterations in MEGA7 [117]. Viruses belong either to the family Phycodnaviridae or are recognized “extended members” of the family Mimiviridae. The recently discovered dinoﬂagellate infecting virus, Heterocapsa circularisquama DNA virus, was used to root the tree and shows little similarity to other algal NCLDVs despite being a large DNA virus. Viruses in red text denote metagenome assembled viral genomes, meaning their association with an alga host is putative. Colored dots to the right indicate the viruses can be putatively PCR ampliﬁed by the respective PCR primer set based on ≥90% match between each primer and its respective target binding site. This equates to ≤2 primer mismatches, which has been shown to be capable of producing a PCR reaction, albeit at lower efﬁciency (for RT-qPCR) [126]. The same study shows that three or more mismatches in the same primer completely inhibit a PCR reaction, and is an observation that aligns with failed PCR reactions reported for Ectocarpus siliculosus virus 1 and Feldmannia spp. virus [11]. PCR ampliﬁcation predictions were done using motif searches in CLC Genomics and the software De-MetaST-BLAST [127]. Phycodnaviridae polB (AVS1 and AVS2) mcp (mcpF and mcpR) mcp (AMmcpF and AMmcpR) Mimiviridae Ostreococcus tauri virus 2 Ostreococcus tauri virus 1 Micromonas pusilla virus 12T Bathycoccus spp. virus Dishui Lake Phycodnavirus 1 Yellowstone Lake Phycodnavirus 1 Yellowstone Lake Phycodnavirus 2 Paramecium bursaria Chlorella virus FR483 Acanthocystis turfacea Chlorella virus 1 Paramecium bursaria Chlorella virus 1 Only Syngen Nebraska Virus 5 Paramecium bursaria Chlorella virus AR158 Paramecium bursaria Chlorella virus NY2A Heterosigma akashiwo virus 01 Chrysochromulina breviflium virus PW1 Chrysochromulina parva virus BQ1 Ectocarpus siliculosus virus 1 Feldmannia spp. 3.1. PCR Applications for Estimating Viral Diversity and Dynamics virus Emiliania huxleyi virus 86 Emiliania huxleyi virus 201 Pyramimonas orientalis virus Tetraselmis virus 1 Aureococcus anophageferens virus Organic Lake Phycodnavirus 1 Organic Lake Phycodnavirus 2 Chrysochomulina ericina virus Prymnesium kappa virus Phaeocystis globosa virus 16T Phaeocystis pouchetti virus Heterocapsa circularisquama DNA virus polB (AVS1 and AVS2) mcp (mcpF and mcpR) mcp (AMmcpF and AMmcpR) Figure 4. Phylogenetic tree depicting the evolutionary relationships of algal NCLDVs based on amino acid alignment (ClustalW) of the core gene, DNA polymerase B (see in Supplemental Table S2). The tree was built using the maximum likelihood method based on the JTT matrix-based model with 200 iterations in MEGA7 [117]. Viruses belong either to the family Phycodnaviridae or are recognized “extended members” of the family Mimiviridae. The recently discovered dinoﬂagellate infecting virus, Heterocapsa circularisquama DNA virus, was used to root the tree and shows little similarity to other algal NCLDVs despite being a large DNA virus. Viruses in red text denote metagenome assembled viral genomes, meaning their association with an alga host is putative. Colored dots to the right indicate the viruses can be putatively PCR ampliﬁed by the respective PCR primer set based on ≥90% match between each primer and its respective target binding site. This equates to ≤2 primer mismatches, which has been shown to be capable of producing a PCR reaction, albeit at lower efﬁciency (for RT-qPCR) [126]. The same study shows that three or more mismatches in the same primer completely inhibit a PCR reaction, and is an observation that aligns with failed PCR reactions reported for Ectocarpus siliculosus virus 1 and Feldmannia spp. virus [11]. PCR ampliﬁcation predictions were done using motif searches in CLC Genomics and the software De-MetaST-BLAST [127]. Figure 4. Phylogenetic tree depicting the evolutionary relationships of algal NCLDVs based on amino acid alignment (ClustalW) of the core gene, DNA polymerase B (see in Supplemental Table S2). The tree was built using the maximum likelihood method based on the JTT matrix-based model with 200 iterations in MEGA7 [117]. Viruses belong either to the family Phycodnaviridae or are recognized “extended members” of the family Mimiviridae. The recently discovered dinoﬂagellate infecting virus, Heterocapsa circularisquama DNA virus, was used to root the tree and shows little similarity to other algal NCLDVs despite being a large DNA virus. Viruses in red text denote metagenome assembled viral genomes, meaning their association with an alga host is putative. 3.1. PCR Applications for Estimating Viral Diversity and Dynamics Colored dots to the right indicate the viruses can be putatively PCR ampliﬁed by the respective PCR primer set based on ≥90% match between each primer and its respective target binding site. This equates to ≤2 primer mismatches, which has been shown to be capable of producing a PCR reaction, albeit at lower efﬁciency (for RT-qPCR) [126]. The same study shows that three or more mismatches in the same primer completely inhibit a PCR reaction, and is an observation that aligns with failed PCR reactions reported for Ectocarpus siliculosus virus 1 and Feldmannia spp. virus [11]. PCR ampliﬁcation predictions were done using motif searches in CLC Genomics and the software De-MetaST-BLAST [127]. Viruses 2018, 10, 487 Viruses 2018, 10, 487 14 of 27 A recent clone library of PCR amplicons generated using the two mcp primer sets demonstrates a wide diversity of algal viruses isolated from marine and freshwater environments [124]. This study also used PCR ampliﬁcation to track the occurrence and dynamics of virus groups (deﬁned by sequence clustering as operational taxonomic units, OTUs) over the course of a harmful brown-alga event. Biases aside, the approach used in that study has certainly expanded the known diversity of algal NCLDVs. It has also shown that cultured viral isolates are often distinct from environmental viruses, and that viruses are widely dispersed in the environment [123,124,128–131]. Another recent group of primer sets was developed by Wilson et al. that ampliﬁes a putative algal-Mimiviridae speciﬁc mismatch repair gene (MutS) [132]. Novel groups of algal NCLDVs were detected in all of the samples tested, making this gene/primer set another potentially useful tool for studying virus diversity. RNA virus diversity has been assessed using primer sets targeting RNA dependent RNA polymerase (RdRP), a protein encoded by all RNA viruses [29,133]. This led to the discovery of a highly diverse super group of putative, marine, protist-infecting picorna-like viruses [133] that are consistently represented in metagenomic datasets [134]. Moreover, alignments of conserved regions of RdRP form clades that are congruent with virion structure, host, and epidemiology [29]. While diversity can be addressed with degenerate primer PCR ampliﬁcation, one of the major drawbacks of this approach is that it is generally not suitable for quantitative measurements [135]. Indeed, degeneracies allow for biases in primer-binding and template ampliﬁcation in mixed communities [136]. 3.1. PCR Applications for Estimating Viral Diversity and Dynamics Use of more speciﬁc primer sets and quantitative PCR approaches can avoid this issue [137,138], but at the risk of not detecting closely related viruses. Even when using speciﬁc primer sets, recent duplications of marker genes can result in overestimation of viral abundances. One of the recent developments to overcome this is to spatially separate viruses and subject them to solid-phase, single-molecule PCR polony ampliﬁcation [139]. Family speciﬁc degenerate primers amplify diverse members without the issue of competitive ampliﬁcation, then categorize and quantify the amplicons using probes for virus group speciﬁc genes. Of course, this method is also dependent on prior sequence knowledge on the virus types of interest and has been validated only in cyanophage thus far, but it is certainly an appealing method for the study of eukaryotic alga infecting viruses. Another recently discovered application of PCR is its potential to link viruses and hosts. Microﬂuidics can be used to isolate infected single-cells that can then be subjected to simultaneous PCR detection of viral and host genes [140]. 3.2. Using Omics Approaches to Estimate Virus Diversity and Dynamics Because community scale genomics and transcriptomics are not dependent on target ampliﬁcation, they are better suited for resolving viral diversity and can in some cases allow for the assembly of complete viral genomes. Though this is more readily accomplished in small RNA and DNA viruses [12,109,141], it has also been possible for some large dsDNA viruses and virophage [24,31,32]. This potential is so valuable that a proposal was recently submitted to the International Committee on the Taxonomy of Viruses (ICTV) for the inclusion of metagenomic-assembled viruses into the ofﬁcial classiﬁcation scheme [142]. Not only was this approved, but it initiated a change in the primary approach ICTV uses for virus classiﬁcation from phenotypic characterization based on viral isolates to molecular characterization based on viral DNA sequences. Since this time, metagenome assembled circular Rep-encoding single-stranded (CRESS) DNA viruses have been properly classiﬁed, including the Bacilladnaviridae [109], the putative vertebrate infecting Smacoviridae [143], and many more [112,144]. Some of the initial taxonomic classiﬁcations may also need to be reassessed in light of molecular methods, as classical taxonomy based on phenotype is not always congruent with phylogenetic clustering: The order Nidovirales may in fact belong to the Picornavirales. While becoming more common, sequencing entire viral communities remains challenging and each experimental step must be considered in the context of existing biases and the project objectives. Virus particles have very low nucleic acid contents, necessitating ampliﬁcation, concentration, or enrichment to obtain adequate sequencing depth. Simple approaches to do this involve concentration 15 of 27 Viruses 2018, 10, 487 of environmental samples via ﬁltration [145] or chemical ﬂocculation [146]. Virus enrichment can be done for speciﬁc viral types with some quantitative applications. For example, dsDNA can be quantitatively ampliﬁed using fusion PCR primers, and adaptase will quantitatively amplify both ssDNA and dsDNA viruses [147]. Rolling circle ampliﬁcation can increase detection of circular viruses [109], and recombinant plant proteins that non-speciﬁcally bind dsRNA can select for dsRNA viruses [148]. There are also methods to separate DNA and RNA viruses for separate analyses using hydroxyapatite-mediated techniques [149]. One of the most appealing enrichment strategies recently used involves selection (via binding) of poly-A containing nucleic acid (i.e., mRNA) to focus on the active viral community [12]. 3.2. Using Omics Approaches to Estimate Virus Diversity and Dynamics This is a useful signal to distinguish virus particles from active infection, as the former will not produce an mRNA signal, though this excludes some (+) ssRNA viruses that have polyadenylated genomes independent of infection [150]. Though all of these methods are useful for improving detection, there are biases to be considered before making conclusions about viral abundances. These issues have been elucidated for sampling, extraction, and puriﬁcation methods [151,152], but these studies are not comprehensive. The viral sequences generated from any sequencing approach are subjected to a general analytical workﬂow involving quality ﬁltering, assembly, annotation, and diversity analyses. Many tools are available to perform this bioinformatic workﬂow [153], but few of these are designed to complete the full workﬂow. Moreover, careful understanding of the sequence databases searched in each workﬂow is necessary to know whether biases exist for particular virus types. GenBank and the nt/nr databases are preferred as these are continually updated and contain information for all virus types; however, their large size can slow processing considerably. To overcome this, creating custom workﬂows using marker genes of interest can speed up processing time while maintaining the ability to detect diverse virus types. An example of a bioinformatics workﬂow using a custom marker gene database to interpret NGS sequences (i.e., Illumina™paired-end sequencing) is shown in Figure 5. First, reads must be preprocessed to remove contaminating adapter sequences and trim low-quality reads. The next step involves assembly of reads into larger contigs, followed by contig annotation using a database of known sequences and a homology or alignment search tool (BLAST, HMMER, Bowtie2, etc.). BLAST tools have commonly been used for this purpose in cellular organisms, and even in some virus studies [33], but may be less efﬁcient for identifying novel virus homologs since they often have low pairwise sequence identities [154]. An alternative to using sequence alignments are Hidden Markov models (MMS), which score hits to protein domains. These analyses can be done with the search tool HMMER to create a marker gene database (HMM-build) that can be queried against assembled contigs [155]. Once viral contigs have been identiﬁed, the relevant gene hits can be extracted for post-processing (i.e., phylogenetic analysis). In many cases, especially when using small databases, it is useful to verify viral hits with a second similarity search of the extracted gene. 3.2. Using Omics Approaches to Estimate Virus Diversity and Dynamics Following veriﬁcation, extracted viral hits can be placed unto an existing phylogenetic tree built with homologous reference sequences (e.g., pplacer [156]). Tree topology can be conﬁrmed using a variety of other tree-building software (e.g., FastTree 2.1.7 [157], PhyML [118], RAxML [158], IQ-tree [159]) and methods (e.g., MrBayes for Bayesian tree-building [160]). Viruses 2018, 10, 487 Viruses 2018 10 x 16 of 27 16 f 26 uses 2018, 10, 487 16 of 27 ruses 2018, 10, x 16 of 2 Figure 5. General bioinformatic pipeline using marker gene probing of community sequence data. This framework follows that used by Moniruzzaman et al., 2017 [12], where viral activity was assessed using marker gene detection from environmental mRNA. Though this framework was modeled off the ited tudy it i fle ible e ou h to i o o ate both eta e o i a d etat a i to i Figure 5. General bioinformatic pipeline using marker gene probing of community sequence data. This framework follows that used by Moniruzzaman et al., 2017 [12], where viral activity was assessed using marker gene detection from environmental mRNA. Though this framework was modeled off the cited study, it is ﬂexible enough to incorporate both metagenomic and metatranscriptomic applications. Figure 5. General bioinformatic pipeline using marker gene probing of community sequence data. This framework follows that used by Moniruzzaman et al., 2017 [12], where viral activity was assessed using marker gene detection from environmental mRNA. Though this framework was modeled off Figure 5. General bioinformatic pipeline using marker gene probing of community sequence data. This framework follows that used by Moniruzzaman et al., 2017 [12], where viral activity was assessed using marker gene detection from environmental mRNA. Though this framework was modeled off the cited study, it is ﬂexible enough to incorporate both metagenomic and metatranscriptomic applications. the cited study, it is flexible enough to incorporate both metagenomic and metatranscriptomic applications. Information on virus abundance or activity can be inferred by mapping trimmed metagenomic or metatranscriptomic reads back to viral contigs normalized for between-sample comparisons (e.g., internal standards, library size, length, and reads per kilobase of transcript per million mapped reads [RPKM] values). However, there are some caveats to consider when examining environmental metatranscriptomes. 3.2. Using Omics Approaches to Estimate Virus Diversity and Dynamics Transcript abundance is not directly related to viral abundance for two reasons: First, biases are known to exist for highly transcriptionally active viruses, and second, single host organisms can support high viral loads. Moreover, virus metatranscriptomes can be contaminated with chimeras generated during assembly, remnant viral genes may be expressed from cells [161], and genomic duplications of marker genes could confound expression profiles. Some problems can be avoided with proper sampling and sequencing approaches mentioned previously, but others remain a significant obstacle for quantitative community analyses, though this has been resolved for bacteria-infecting viruses [147,162]. Until these confounding issues can be remedied and benchmarked for all viral types, they must be considered during the analysis of environmental data. Information on virus abundance or activity can be inferred by mapping trimmed metagenomic or metatranscriptomic reads back to viral contigs normalized for between-sample comparisons (e.g., internal standards, library size, length, and reads per kilobase of transcript per million mapped reads [RPKM] values). However, there are some caveats to consider when examining environmental metatranscriptomes. Transcript abundance is not directly related to viral abundance for two reasons: First, biases are known to exist for highly transcriptionally active viruses, and second, single host organisms can support high viral loads. Moreover, virus metatranscriptomes can be contaminated with chimeras generated during assembly, remnant viral genes may be expressed from cells [161], and genomic duplications of marker genes could confound expression proﬁles. Some problems can be avoided with proper sampling and sequencing approaches mentioned previously, but others remain a signiﬁcant obstacle for quantitative community analyses, though this has been resolved for bacteria-infecting viruses [147,162]. Until these confounding issues can be remedied and benchmarked for all viral types, they must be considered during the analysis of environmental data. A recent review by Nooij et al. provided a comprehensive description of workﬂows that have been produced for viromic analyses, including speciﬁc applications, classiﬁcation biases, and open-source availability [153]. 4. Conclusions The opportunities for algal virus ecologists are at an all-time high. Bioinformatic tools are becoming more accessible to a wide variety of scientists through the creation of publicly available genomic databases and graphic interfaces that mediate interactions with traditional command-line software [167]. At the same time, researchers are increasing collaborations with one another by sharing methodologies in an interactive framework on protocols.io (e.g., Viral Ecology Research and Virtual Exchange network, or VERVE Net; https://www.protocols.io/groups/verve-net) and with cross-discipline collaborations fostered at research workshops funded by organizations like the Gordon & Betty Moore Foundation (GBMF) and the Canadian Institute for Advanced Research (CIFAR). The development of long-read sequencing methods, preemptively deemed “third-generation sequencing”, may address many of the issues with short-read assembly and viral quantiﬁcation. DNA barcoding has been suggested as a cheap, reliable method to quickly track virus populations, and has recently been shown to recapitulate general viral community structures using sample volumes no bigger than a cup of water [168]. New virus isolates can be discovered from sequencing of single aquatic viruses sorted by ﬂow cytometry, [169], as closely related, hyper diverse viruses are suggested to be difﬁcult to assemble from metagenomes [170]. Even better, isolation and sequencing of infected single-cells may allow for the identiﬁcation of new virus-host systems. Network analyses of community sequence data predict ecological structures that may lead to the discovery and isolation of several new algal-virus systems, bringing the scientiﬁc community “full-circle” to studying these systems in the lab. In light of that, this exciting frontier cannot be appreciated without recognition of the early work done by some of the ﬁrst aquatic virologists in the ﬁeld. James L. Van Etten, for whom this special issue is in honor of, has spent the last forty years laying the foundation for aquatic virology. Not only did he open doors for other algal virus researchers to join the ﬁeld, but he has set the standard for characterizing the biology and ecology of isolated algal virus systems. Along with the genomic, transcriptomic, and proteomic work done in the Chlorella virus system, the Van Etten lab has also shown that Chlorella viruses are biochemically novel in multiple ways. Virion proteins are glycosylated using a unique viral encoded machinery [171,172], and the viral genomes can be methylated by a range of DNA methyltransferases [173]. A recent review by Nooij et al. provided a comprehe produced for viromic analyses, including specific app 3.3. Other Downstream Applications of Omic Assemblies p y g p pp p availability [153]. 3.3. Other Downstream Applications of Omic Assemblies Another enticing application of community sequence data is the potential to deduce biological interactions using co-occurrence or network analyses. This is a relatively new approach that was developed for microbiome communities but has the potential to identify novel virus-host pairs [163]. Two studies tracking the temporal dynamics of virus communities have been reported thus far [12,33]. From a metagenomics standpoint, these studies were striking because they generated putatively full-length Picornavirales and virophage genomes. Moreover, in the case of Moniruzzaman et al., 2017 [12] the viral genomes were generated from transcripts, indicating these virus genomes were actively expressed and were therefore, produced from infected cells. Beyond these exciting findings, each study used network analyses to link potential virus-host pairs. Clusters created from sequencing data collected over the course of a brown-tide bloom (Aureococcus Another enticing application of community sequence data is the potential to deduce biological interactions using co-occurrence or network analyses. This is a relatively new approach that was developed for microbiome communities but has the potential to identify novel virus-host pairs [163]. Two studies tracking the temporal dynamics of virus communities have been reported thus far [12,33]. From a metagenomics standpoint, these studies were striking because they generated putatively full-length Picornavirales and virophage genomes. Moreover, in the case of Moniruzzaman et al., 2017 [12] the viral genomes were generated from transcripts, indicating these virus genomes were actively expressed and were therefore, produced from infected cells. Beyond these exciting ﬁndings, each study used network analyses to link potential virus-host pairs. Clusters created from sequencing data collected over the course of a brown-tide bloom (Aureococcus anophagefferens) linked the brown alga to its known virus, AaV, demonstrating the ability to extract known relationships with this approach. Several other clusters were generated from the same study, including smaller networks of single virus-host pairs and expected associations between Prasinophyceae and Phycodnaviridae. Roux et al., Viruses 2018, 10, 487 17 of 27 2017 [33] focused on using networks to link virophage with giant NCLDV hosts and found strong speciﬁc associations with Mimiviridae and their extended alga-infecting members to drastically expand the diversity of known virophage hosts. Altogether, predictions stemming from the studies noted above demonstrate how network analyses can generate testable hypotheses for future studies of algal virus-host interactions. A recent review by Nooij et al. provided a comprehe produced for viromic analyses, including specific app 3.3. Other Downstream Applications of Omic Assemblies By deducing sequences of virus-host pairs, one can attempt to conﬁrm probable virus-host interactions. For example, a variation of ﬂuorescent in-situ hybridization, deemed phageFISH, could be used to label virus and host genes in infected cells [164]. Additionally, networks predicting viruses of cultured algae could be followed up with virus tagging experiments [165]. It might even be worthwhile to use more than one network building approach to look at ecosystem structures. Weiss et al. used real and mock in silico data to benchmark eight methods used for bacterial network analyses and found that some methods generate drastically different outputs [166]. This is explained, in part, by differing strengths for detecting particular biological relationships (e.g., mutualism and commensalism) across different network approaches. It was also suggested that p-values of 0.001 should be used for high-precision network detection and rare OTUs should be removed prior to network construction. 4. Conclusions Many of these enzymes are paired with a restriction endonuclease that recognizes the same nucleotide sequence to comprise a viral restriction-modiﬁcation system that recycles host DNA for virus replication [174]. Chlorella viruses have the smallest potassium ion channels that function to depolarize host membranes, concomitantly inhibiting secondary infection and host metabolite transporters [175]. Within seven minutes post-infection, transcriptional activity begins to shift away from the infected host towards producing viral transcripts [20,176]. Along with the extensive biological studies on 18 of 27 Viruses 2018, 10, 487 this system, the Van Etten group has also established many important ﬁndings on their ecology. Chlorella viruses are ubiquitous in freshwaters across the globe, despite their hosts being sequestered as endosymbionts of Paramecium bursaria [6]. This inspired questions about their resistance to degradation as well as how viruses and hosts make contact with one another. Predatory activity on Paramecium bursaria catalyzes this contact by making the endosymbiotic algae available to Chlorella viruses in the environment [177,178]. Another group has shown that Chlorella viruses are more resistant to environmental degradation than other algal viruses, and can even overwinter under ice [179]. Collectively, these questions can be investigated in many types of algal viruses. Although dsDNA viruses certainly have the largest number of genes, even smaller DNA and RNA viruses must deal with many of the same selective pressures. Indeed, there are many lessons algal virus researchers can learn from the body of work produced by James L. Van Etten and his collaborators. Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4915/10/9/487/s1, Supplemental Tables S1 and S2 contain the full information for amino acid sequences used to construct RdRP and polB phylogenetic trees, respectively. Author Contributions: S.R.C. and S.W.W. conceived the paper, and S.R.C., S.W.W., S.M.S., E.R.G. and H.L.P. contributed to the production of ﬁgures, text, and editing. hor Contributions: S.R.C. and S.W.W. conceived the paper, and S.R.C., S.W.W., S.M.S., E.R.G. and H.L.P ributed to the production of ﬁgures, text, and editing. Funding: This research was supported by funds from The Gordon & Betty Moore Foundation #4971, The National Science Foundation IOS-1451528, OCE-1829641, and the Kenneth & Blaire Mossman Endowment to the University of Tennessee (all to S.W.W.). Acknowledgments: The authors would like to thank Mohammad Moniruzzaman, Joshua Stough and Gary LeCleir for insight and advice. 4. Conclusions The authors would further like to thank James Van Etten for providing guidance and support over the last several years, and three anonymous reviewers for their constructive support. Funding for open access to this research was provided by University of Tennessee’s Open Publishing Support Fund. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. References 1. Short, S.M.; Staniewski, M.A.; Chaban, Y.V.; Long, A.M.; Wang, D. Diversity of viruses infecting eukaryotic algae. In Viruses of Microorganisms; Paul, H., Abedon, S.T., Eds.; Caister Academic Press: Poole, UK, 2018. 1. Short, S.M.; Staniewski, M.A.; Chaban, Y.V.; Long, A.M.; Wang, D. Diversity of viruses infecting eukaryotic algae. 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https://openalex.org/W1974938482	https://thescholarship.ecu.edu/bitstream/10342/5541/1/CRIM.HEMATOLOGY2012-194312.PMC3420427.pdf	English	null	Granulocytic Sarcoma of the Male Breast in Acute Myeloblastic Leukemia with Concurrent Deletion of 5q and Trisomy 8	Case reports in hematology	2,012	cc-by	3,476	1. Introduction “granulocytic sarcoma,” recognizing the fact that the green color is not universal [3]. Granulocytic sarcoma is a rare extramedullary tumor con- sisting of primitive myeloid cells. It is also known as chloroma, myeloblastoma, extramedullary leukemia, and myeloid sarcoma. This localized growth of immature myeloid precursors superﬁcially resembles sarcoma. The tumor is a solid collection of immature malignant white blood cells (myoblasts) occurring outside the bone marrow, and is a rare manifestation of acute and chronic leukemias. It can occasionally precede the development of systemic disease by weeks to years. GS was named as chloroma, because of its greenish color. The histology of this unusual tumor was ﬁrst described by Burns in 1811 [1]. The term “chloroma,” literally implying “green tumor”, was suggested by King in 1853 (Greek chloros, meaning green) [2]. The green colora- tion is now known to be due to the presence and oxidation of the myeloperoxidase enzyme. More than a century later, in 1966, Rappaport coined the currently accepted term, These tumors may arise de novo, or could be associated with other hematologic disorders such as acute myelogenous leukemia, myelodysplastic syndrome, or with myeloprolifer- ative disorders such as chronic myelogenous leukemia, poly- cythemia vera, hypereosinophilia, and myeloid metaplasia. GS can develop at any anatomic site and its distribution commonly includes bone, nerve, lymph node, and skin, but may involve a variety of soft tissues. In men, the breast has been reported to be an uncommon site for granulocytic sarcoma [4–6]. This extramedullary neoplasm may repre- sent a diagnostic and therapeutic dilemma for both the hematopathologist and oncologist. Hindawi Publishing Corporation Case Reports in Hematology Volume 2012, Article ID 194312, 5 pages doi:10.1155/2012/194312 Hindawi Publishing Corporation Case Reports in Hematology Volume 2012, Article ID 194312, 5 pages doi:10.1155/2012/194312 Hindawi Publishing Corporation Case Reports in Hematology Volume 2012, Article ID 194312, 5 pages doi:10.1155/2012/194312 Muhammad Rizwan,1 Md. Monirul Islam,2 and Zia ur Rehman3 1Department of Internal Medicine, Vidant Medical Center Greenville, NC 27835, USA 2Division of Pulmonary and Critical Care Medicine, East Carolina University and Vidant Medical Center, Greenville, NC 27835, USA 3Departments of Pulmonary, Critical Care and Sleep Medicine, Brody School of Medicine, 3Departments of Pulmonary, Critical Care and Sleep Medicine, Brody School of Medicine, East Carolina University and Vidant Medical Center, Greenville, NC 27835, USA 3Departments of Pulmonary, Critical Care and Sleep Medicine, Brody School of Medicine, East Carolina University and Vidant Medical Center, Greenville, NC 27835, USA Correspondence should be addressed to Muhammad Rizwan, aawazedost@yahoo.com Correspondence should be addressed to Muhammad Rizwan, aawazedost@yahoo.com Received 13 April 2012; Accepted 22 June 2012 Received 13 April 2012; Accepted 22 June 2012 Academic Editors: C. Imai, Y. Shiozawa, and P. Tsirigotis Academic Editors: C. Imai, Y. Shiozawa, and P. Tsirigotis Copyright © 2012 Muhammad Rizwan et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. We describe a unique case of Granulocytic Sarcoma (GS) in a male, who presented to us with a painless right breast mass without any prior history of Leukemia. GS is an extramedullary tumor of myeloproliferative precursors and may involve multiple sites of the body, but involvement of male breast is extremely rare. In the absence of clinical history or hematological abnormality, GS may be misdiagnosed, depending on the degree of myeloid diﬀerentiation present within the tumor. Often it is misdiagnosed as lymphoma. Diagnosis is made by ﬁnding eosinophilic myelocytes, myeloperoxidase, chloroacetate esterase staining, and lysozyme immunostain. Chemotherapy regimens similar to acute myeloid leukemia are recommended to treat GS. Recognition of this rare entity is important because early, aggressive chemotherapy can induce regression of the tumor and improve patient longevity. 2. A Case Report A 56-year-old Caucasian male presented with right breast enlargement of one month’s duration. It started with swelling 2 Case Reports in Hematology Figure 1: Right breast mass with overlying skin discoloration. Figure 3: Trucut biopsy of the mass. Moderate-to-large size neo- plastic cells with slightly irregular nuclei and conspicuous nucleoli. Figure 3: Trucut biopsy of the mass. Moderate-to-large size neo- plastic cells with slightly irregular nuclei and conspicuous nucleoli. Figure 4: Bone marrow biopsy showing hyperplastic marrow with high M/E ratio. Figure 1: Right breast mass with overlying skin discoloration. Figure 2: CT chest showing eroding mass lesion. Figure 4: Bone marrow biopsy showing hyperplastic marrow with high M/E ratio. Figure 2: CT chest showing eroding mass lesion. and sixth right ribs had been eroded by the mass lesion. An adjacent ﬂuid collection was visible. There was no evidence of lung involvement but a 1.5 cm right adrenal gland nodule was noted. Bone scan performed with gamma camera revealed a suspicious focus of increased uptake in the proxi- mal left femoral shaft and small focus of increased uptake in the superior occipital skull. under the right nipple that had progressed into a large mass. The mass was painless but caused discomfort due to cosmetic reasons. Over the past week the patient had also noticed a change in skin color to orange and then to red, overlying the lower outer quadrant of the right breast. The patient denied any history of trauma, fever, weakness, recent weight loss, or loss of appetite. He complained of mild left hip and upper thigh pains of few days duration. His past medical history was non-contributory. Family history was signiﬁcant for breast cancer in an older sister at age 55 years. under the right nipple that had progressed into a large mass. The mass was painless but caused discomfort due to cosmetic reasons. Over the past week the patient had also noticed a change in skin color to orange and then to red, overlying the lower outer quadrant of the right breast. The patient denied any history of trauma, fever, weakness, recent weight loss, or loss of appetite. He complained of mild left hip and upper thigh pains of few days duration. His past medical history was non-contributory. Family history was signiﬁcant for breast cancer in an older sister at age 55 years. Trucut biopsy of the right breast mass showed myeloid sarcoma (Figure 3). 3. Discussion Isolated granulocytic sarcomas are rare tumors associated with myeloproliferative disorders or leukemias. They have been reported both before and after the hematological diag- nosis [4]. Usually, there is known bone marrow involvement at the time of presentation and a clinical diagnosis is made. Thus, presentation can occur either prior to or in association with the underlying myeloid disorder or upon relapse. GS inﬁltrates in tracts and tissue planes, usually preserv- ing the tissue architecture without extensive tissue destruc- tion and tumor necrosis. Histopathologically, these tumors have been classiﬁed into well diﬀerentiated, poorly diﬀer- entiated, and blastic types depending upon the level of myeloid maturation by hematoxylin-eosin and PAS stained sections [6, 13]. Histologic diagnosis remains diﬃcult. The presence of eosinophilic myelocytes has traditionally been one of the most reliable histologic ﬁndings in making a diagnosis of GS. Tumors are considered well diﬀerentiated if numerous eosinophilic myelocytes can be seen in any section of a given case. Diagnosis can be conﬁrmed with myelo- peroxidase (MPO), chloroacetate esterase staining (leder stain), and lysozyme immunostain(Ly). Histochemical stain- ing to demonstrate the presence of MPO is the most sensitive and speciﬁc antibody test for detection of myeloid diﬀerentiation [14]. Electron microscopy, which reveals azurophilic granules consistent with promyelocytes, helps conﬁrm diagnosis [7] but is rarely performed due to cost and availability. Speciﬁc markers of cluster diﬀerentiation (CD) typical of myeloid lineage can also be demonstrated. For example, Chen and colleagues found that MPO was positive in 97% of cases, Ly in 93%, CD34 in 47%, CD45 in 84%, CD43 in 97%, CD68 in 93%, Bcl-2 in 68%, Tdt in 37%, CD79a in 20%, CD20 in 10%, CD3 in 10%, and c-kit (CD117) reactivity in 87% of the cases [15]. This group of disorders can involve myeloblasts and neu- trophil precursors, monoblasts, or can be a trilineage myeloid tumor of erythroid precursors, megakaryocytic precursors, and granulocytic precursors [5]. It is frequently identiﬁed in patients with chronic myeloid leukemia, myeloproliferative disorders, or myelodysplasia, but most commonly these tumors represent relapse or the initial presentation of acute myeloid leukemia. Incidence is increased in FAB M4/M5 types, CD56 (+) blasts, cytogenetic abnormalities: t (8 : 21), inversion 16, infant leukemia, 11q abnormalities, cellular immune dysfunction, and with new treatments like allo- geneic stem cell transplantation [5]. It has also been reported as an isolated mass without prior history or subsequent development of leukemia [5, 7]. 2. A Case Report Bone marrow biopsy showed hyperplas- tic marrow with high M/E ratio with shift to left (Figure 4). Bone marrow immunohistochemical studies were positive for lysozyme, myeloperoxidase, CD15, CD34, and CD43. These ﬁndings were consistent with a Myeloid Neoplasm, but not speciﬁc for a certain type. The diﬀerential diagnosis proposed was chronic myeloid leukemia (CML), a myelodys- plastic/myeloproliferative disease (e.g., chronic myelomono- cytic leukemia, or CMML), and less likely, a myelodysplastic syndrome. With pending chromosome analysis, patient was started on therapeutic trial of Gleevec 600 mg orally daily and Allopurinol 300 mg orally daily. Arrangements were made for radiation therapy to the mass to improve local symptoms and reduce the size of the tumor. Patient did well on this therapy except for developing anemia for which he was transfused. Physical examination revealed a 13 cm × 15 cm, well- circumscribed, nontender mass, hard in consistency, and ﬁxed with the overlying skin and underlying structures (Figure 1). The skin overlying the mass was pinkish. There was no nipple retraction or discharge. The left breast struc- tures were within normal limits. There was mild conjunctival pallor, but no palpable axillary, inguinal, popliteal, or supra- clavicular lymph nodes. Laboratory studies showed hemoglobin of 9.6 g/dL hematocrit of 28.7%, platelet count of 102 × k/µL and WBC of 22 × k/µL with diﬀerential of 44% neutrophils, 30% bands, 20% lymphocytes, 4% monocytes, and 2% eosino- phils. CT scan of the chest revealed a large, 13 cm × 9 cm mass involving the right anterior and lateral chest walls, eroding into the thoracic cavity (Figure 2). The mass demon- strated areas of low density suggestive of necrosis. The ﬁfth Three days later, the patient sustained a fall resulting in an acute transverse fracture of the proximal left femoral neck. Left hip arthroplasty was performed. Bone reamings from left femur, which were sent for studies, showed diﬀuse inﬁl- tration of neoplastic cells with extensive necrosis. Neoplastic Case Reports in Hematology 3 cells were moderate to large in size and had modest to moderate amount of pink cytoplasm and slightly irregular nuclei with ﬁne chromatin and conspicuous nucleoli. Mitotic activities were frequently present, consistent with myeloid neoplasm. The morphologic ﬁndings were suggestive of the same neoplastic process as the previous bone marrow biopsy and breast biopsy. 2. A Case Report Bone marrow chromosome analysis report came back as well and revealed complex clonal aberrations including deletion of 5q, trisomy 8, and additional material on the short arm of chromosome 17. enough to cause compression symptoms or signs according to their localization. GS is widespread and can involve almost any part of the human body. Common sites of involvement are lymph nodes 15%, skin 14%, head, and spinal cord 13%, small intestine 11%, mediastinum 10%, bone 9%, ovary and uterus 9%, and others 19% [9]. Breast involvement by GS is rare and that of the male is extremely rare. Gynecomastia may be the ﬁrst manifestation of this disease or it may present as an isolated breast mass [6, 10]. In the absence of clinical history or hematological abnormality, granulocytic sarcoma may be misdiagnosed, depending on the degree of myeloid diﬀerentiation present within the tumor. A careful evaluation of the breast mass is important, and GS should be diﬀerentiated from other nonmammary malignancies of the breast; primary and sec- ondary breast lymphoma, primary axillary nodal lymphoma, metastatic acute lymphatic leukemia, metastatic plasma- cytoma, primary angiosarcoma, metastatic rhabdomyosar- coma, hematogenous metastasis from primary lung, ovarian, cervical, thyroid and colonic carcinoma, malignant melano- ma, carcinoma of the nasal cavity, and adenocarcinoma of unknown primary [11]. It was planned to start patient on AML-type chemother- apy upon his recovery, but unfortunately, postoperatively the patient developed respiratory failure and was transferred to intensive care unit on assist control ventilation. He was empirically started on Primaxin and Vancomycin for the pos- sibility of healthcare associated pneumonia. Lovenox dose was changed from prophylactic to therapeutic for possible pulmonary embolism. CT angiogram of chest was obtained which showed bilateral intraluminal ﬁlling defects in second order branches consistent with pulmonary emboli. Multiple attempts to wean him oﬀthe ventilator were unsuccessful. Patient expired ﬁve days after surgery. 3.1. Diagnosis. Mammographically, breast GS are noncalci- ﬁed irregular masses with poorly deﬁned feathery margins [11]. MRI will only show a mass lesion but is helpful in evaluating response to treatment and detect the nonpalpable relapse of the tumor [12]. 3.1. Diagnosis. Mammographically, breast GS are noncalci- ﬁed irregular masses with poorly deﬁned feathery margins [11]. MRI will only show a mass lesion but is helpful in evaluating response to treatment and detect the nonpalpable relapse of the tumor [12]. 3. Discussion Overall, GS has been classiﬁed into four categories: (a) primary GS, (b) GS as a complication of acute myeloblastic leukemia (AML), (c) GS as isolated recurrence of AML par- ticularly during bone marrow remission and not followed by medullary relapse, and (d) GS with concurrent bone marrow relapse of AML [8]. Our patient belongs to category b, GS as a complication of AML as per clinical history and lab results. GS is more common in males. Median age for males and females is 32 and 34, respectively, and most of the patients are in the age range of 20–44 [9]. GS can present as a single lesion or multiple lesions. Sizes can vary greatly and some are large 3.2. Diﬀerential Diagnosis. Diﬀerential diagnosis of GS on ﬁne needle aspiration (FNA) include large cell non-Hodgkin lymphoma, lymphoblastic lymphoma, Hodgkin lymphoma, 4 Case Reports in Hematology suspicion of the diagnosis on examination of routinely stained sections is of paramount importance. extramedullary hematopoiesis, poorly diﬀerentiated carci- noma, infection, inﬂammation, plasmacytoma, and malig- nant melanoma [13, 16]. This case highlights a rare hematological cancer that a clinician should consider when a patient presents with a breast mass. Our objective of presenting this case is to enhance awareness of GS in personnel providing health care. Increased clinical awareness of this entity will facilitate early diagnosis. A high index of suspicion is required and timely recognition of GS is important, because aggressive induction chemotherapy or radiation therapy can aﬀect outcome, minimizing potentially preventable patient morbidity and mortality. In diﬀerential diagnosis, immunohistochemistry is the most powerful tool and is regarded as essential for diagnosing granulocytic myeloid tumor and distinguishing between its various presentations. 3.3. Treatment. Treatment can be planned depending upon the presentation, localization, and size of the tumor. There is a signiﬁcantly lower rate of progression to leukemia and longer survival among patients who received any form of chemotherapy at diagnosis of GS [17]. There is a general con- sensus that all these patients must receive standard systemic induction-intensiﬁcation chemotherapy regimens similar to those given in acute myeloid leukemia [5, 7, 18]. Surgical excision and radiotherapy (tumor is highly radiosensitive) can be curative, but mostly are performed to reduce the bulk the of tumor and to relieve the compressive symptoms. High dose methylprednisolone treatment has been reported to markedly reduce the size of the tumor. References [1] A. Burns, Observations of Surgical Anatomy, Head and Neck, Thomas Royce, Edinburgh, UK, 1811. [2] A. King, “A case of chloroma,” Monthly Journal of the Medical Society, vol. 17, p. 97, 1853. [3] H. Rappaport, “Tumors of the hematopoietic system,” in Atlas of Tumor Pathology, Section III, Fascicle 8, Armed Forces Instit- ute of Pathology, Washington, DC, USA, 1966. [4] W. McClintock Todd, “Acute myeloid leukemia and related conditions,” Hematology/Oncology Clinics of North America, vol. 16, no. 2, pp. 301–319, 2002. [5] S. Paydas, S. Zorludemir, and M. Ergin, “Granulocytic sar- coma: 32 cases and review of the literature,” Leukemia and Lymphoma, vol. 47, no. 12, pp. 2527–2541, 2006. Reports have emphasized the use of combined local and systemic radical options, including chemotherapy, radiation therapy, surgical intervention, and bone marrow transplan- tation. Because of information on a limited number of patients, there is no agreed upon optimal therapy. However, systemic chemotherapy similar to that given for acute myeloid leukemia with or without local radiotherapy may result in long remissions [18, 19]. [6] J. R. Valbuena, J. H. Admirand, G. Gualco, and L. J. Medeiros, “Myeloid sarcoma involving the breast,” Archives of Pathology and Laboratory Medicine, vol. 129, no. 1, pp. 32–38, 2005. [7] K. Yamauchi and M. Yasuda, “Comparison in treatments of nonleukemic granulocytic sarcoma: report of two cases and a review of 72 cases in the literature,” Cancer, vol. 94, no. 6, pp. 1739–1746, 2002. [8] J. C. Byrd, W. J. Edenﬁeld, D. J. Shields, and N. A. Dawson, “Extramedullary myeloid cell tumors in acute nonlympho- cytic leukemia: a clinical review,” Journal of Clinical Oncology, vol. 13, no. 7, pp. 1800–1816, 1995. 3.4. Prognosis. Generally, GS is associated with decreased overall survival. Blastic types, age more than 50 years, and extramedullary relapse following allogeneic bone marrow transplant are signiﬁcant adverse prognostic factors [5, 12, 19]. The median overall survival is about 20–22 months [12, 13]. The median survival of patients with chromosome 8 abnormalities is 12 months [13]. In our patient, chro- mosome analysis revealed complex clonal aberrations with deletion of 5q. Deletion of 5q occurs in MDS/AML. In the International Scoring System, MDS patients with this karyotype pattern were placed in a poor prognostic category [20]. [9] A. F. List, G. Gonzalez-Osete, T. Kummet, and D. C. Doll, “Granulocytic sarcoma in myelodysplastic syndromes: clinical marker of disease acceleration,” American Journal of Medicine, vol. 90, no. 2, pp. 3. Discussion Other adjuvant treat- ment modalities that have been applied successfully include low dose alpha-interferon and disodium pamidronate, low dose methotrexate, allogeneic bone marrow transplant, and autologous stem cell transplantation [5, 18]. References 274–276, 1991. [10] W. T. Yang, M. Muttarak, and L. W. C. Ho, “Nonmammary malignancies of the breast: ultrasound, CT, and MRI,” Semi- nars in Ultrasound CT and MRI, vol. 21, no. 5, pp. 375–394, 2000. [11] T. J. Barloon, D. C. Young, and S. H. Bass, “Multicentric gran- ulocytic sarcoma (chloroma) of the breast: mammographic ﬁndings,” American Journal of Roentgenology, vol. 161, no. 5, pp. 963–964, 1993. 4. Conclusion [12] T. Kinoshita, M. Yokokawa, and N. Yashiro, “Multicentric granulocytic sarcoma of the breast: mammographic, sono- graphic, and MR ﬁndings,” Clinical Imaging, vol. 30, no. 4, pp. 271–274, 2006. GS is diﬃcult to recognize and may be easily overlooked or misdiagnosed. GS may precede the diagnosis of a chronic myeloproliferative disorder or acute myeloid leukemia, or may present parallel to a hematologic diagnosis. An accurate diagnosis of GS is of great clinical importance in the ongoing management of hematologic malignancies. Precise diagnosis is essential because all GS should be treated as acute myeloid leukemias. Immunohistochemical and enzyme histochemi- cal staining are useful in establishing the diagnosis, although [13] Y. K. Suh and H. J. C. Shin, “Fine-needle aspiration biopsy of granulocytic sarcoma: a clinicopathologic study of 27 cases,” Cancer, vol. 90, no. 6, pp. 364–372, 2000. [14] L. P. Menasce, S. S. Banerjee, E. Beckett, and M. Harris, “Extra- medullary myeloid tumour (Granulocytic sarcoma) is often misdiagnosed: a study of 26 cases,” Histopathology, vol. 34, no. 5, pp. 391–398, 1999. 5 Case Reports in Hematology 5 [15] J. Chen, R. R. Yanuck, S. L. Abbondanzo, W. S. Chu, and N. S. I. Aguilera, “c-Kit (CD117) reactivity in extramedullary myeloid tumor/granulocytic sarcoma,” Archives of Pathology and Labo- ratory Medicine, vol. 125, no. 11, pp. 1448–1452, 2001. [16] I. W. Y. Ngu, E. C. Sinclair, S. Greenaway, and M. L. Green- berg, “Unusual presentation of granulocytic sarcoma in the breast: a case report and review of the literature,” Diagnostic Cytopathology, vol. 24, no. 1, pp. 53–57, 2001. [17] K. R. Imrie, M. J. Kovacs, D. Selby et al., “Isolated chloroma: the eﬀect of early antileukemic therapy,” Annals of Internal Medicine, vol. 123, no. 5, pp. 351–353, 1995. [18] A. M. Tsimberidou, H. M. Kantarjian, E. Estey et al., “Out- come in patients with nonleukemic granulocytic sarcoma treated with chemotherapy with or without radiotherapy,” Leukemia, vol. 17, no. 6, pp. 1100–1103, 2003. [19] K. Yener, K. B. Miller, D. P. Schenkein, P. Daoust, K. Sprague, and E. Berkman, “Extramedullary tumors of myeloid blasts in adults as a pattern of relapse following allogeneic bone marrow transplantation,” Cancer, vol. 85, no. 3, pp. 608–615, 1999. [20] P. Greenberg, C. Cox, M. M. LeBeau et al., “International scoring system for evaluating prognosis in myelodysplastic syndromes,” Blood, vol. 89, no. 6, pp. 2079–2088, 1997.
https://openalex.org/W2073408820	https://hrmars.com/papers_submitted/602/does-foreign-direct-investment-really-affect-ghanas-economic-growth.pdf	English	null	Does Foreign Direct Investment really affect Ghanaâs Economic Growth?	International journal of academic research in economics and management sciences	2,014	cc-by	5,345	Abstract In this paper, we investigate the linkage between FDI and economic growth using macro econometric model in the Ghanaian context. Structural shocks in an SVAR model were used to identify the contemporaneous and short run relationships effects of these variables. The AB model restriction approach was used for the Identification and was compared to the Cholesky decomposition. We showed that, there exit a contemporaneous short run positive effects of FDI inflows on GDP growth but as the time horizon expands these effects tend to converge to the equilibrium, however FDI’s deteriorate domestic investment. Keywords: Foreign Direct Investment, Gross Domestic Product, Impulses Responses, Structural Var Does Foreign Direct Investment really affect Ghana’s Economic Growth? Gabriel Obed Fosu1, Eric Amoo Bondzie2, Gabriel Asare Okyere3 Department of Mathematics, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana1,3 ,DEFAP, Università Cattolica del Sacro Cuore, Italy 2 Emails: gabrielobedpeters@gmail.com1, EricAmoo.Bondzie@unicatt.it2, goasare@yahoo.co.uk3 Okyere Department of Mathematics, Kwame Nkrumah University of Science and Technology Kumasi, Ghana1,3 ,DEFAP, Università Cattolica del Sacro Cuore, Italy 2 Emails: gabrielobedpeters@gmail.com1, EricAmoo.Bondzie@unicatt.it2, goasare@yahoo.co.uk3 To Link this Article: http://dx.doi.org/10.6007/IJAREMS/v3-i1/602 DOI:10.6007/IJAREMS/v3-i1/602 Published Online: 02 January, 2014 To Link this Article: http://dx.doi.org/10.6007/IJAREMS/v3-i1/602 DOI:10.6007/IJAREMS/v3-i1/602 Published Online: 02 January, 2014 http://dx.doi.org/10.6007/IJAREMS/v3-i1/602 DOI:10.6007/IJAREMS/v3-i1/602 Vol 3, Issue 1, (2014) E-ISSN: 2226-3624 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN ECONOMICS AND MANAGEMENT SCIENCES Vol. 3, No. 1, 2014, E-ISSN: 2226-3624 © 2014 TERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN ECONOMICS AND MANAGEMENT SCIENCES l. 3, No. 1, 2014, E-ISSN: 2226-3624 © 2014 the development of local financial markets (Alfaro 2009). Alfaro et al. (2004) provide evidence that only countries with well-developed financial markets gain significantly from FDI in terms of their growth rates. the development of local financial markets (Alfaro 2009). Alfaro et al. (2004) provide evidence that only countries with well-developed financial markets gain significantly from FDI in terms of their growth rates. In this paper we ascertain the nexus between FDI and economic growth in the Ghanaian context within the broader increasingly competitive world market for FDI based on macro econometric modeling. We identify the contemporaneous and short run relationships effects of these variables using Structural shocks in an SVAR model. We further find out the impact of FDI on other economic variables like Inflation, Gross Fixed Capital Formation as well as Government Expenditure. There is a vast body of empirical literature: Azmat (1999), Andrea Marino (2000), Balasundram (2000), Kishor (2000), Chakrabarti (2001), Gordon (2001), on whether foreign direct investment is beneficial to host country’s growth or not. Trade theorist believes, market size, trade policy regime followed by host countries development policies influences significantly both the amount of inward FDI received by recipient countries and the impact of foreign direct investment on growth. With respect to the Ghanaian economy, Okyere, Fosu and Boakye (2014) examined the causality of macroeconomic variables using multivariate vector autoregressive model. Antwi and Zhao (2013) applied the cointegration method to determine how FDI, GDP and Gross National Income (GNI) are related. The study established a long-run equilibrium and causal relationship between these variables. But in the short-run, the effects of GDP and GNI volatility on FDI are nearly imaginary. Baba Insah (2013) also investigated the relationship between economic growth and FDI inflows using Dynamic Ordinary Least Squares (DOLS) technique. He indicated that, the elasticity of economic growth with respect to FDI had a positive sign. However, the effect of a three year lag of FDI on economic growth had a negative sign. Methodology This study used the Vector Autoregressive (VAR) model. Under the VAR model methodologies, the relationships of the variables were determined with their optimal lag length effects. The Causality was determined based on one-way causality or either direction techniques suggested by Engle and Granger (1987). These techniques were accompanied with the impulse response functions and the variance decomposition functions. The standard procedure of using both techniques to measure the change in one of the variable and keeping all other variables constant and finding the covariance matrix of the reduce form residuals was to orthogonalize the innovations. The technique gave us the forecasting capability of each of the variables defining to the other variables. The necessary model checking and identification procedure was applied for the suitability of the model, optimal lag lengths based on criterion used by the FPE (Final Prediction Error), AIC (Akakai’s information Criterion). Structural shocks in a SVAR model was identified by placing some restrictions on contemporaneous and short run relationships. With this, the AB model of Amisano and Giannini (1997) restriction approach was used for the Identification and later compared to the Cholesky decomposition. The unit roots and order of the integration of the variables using Augmented Dickey Fuller (ADF) and Phillips-Person tests were applied. Introduction The economic progress of countries depends to a large extent on the opportunity of making profitable investments and accumulating capital. Having access to foreign capital and investments allows a country to invest in both human and physical capital and to exploit opportunities that otherwise could not be used. Beginning from a general mistrust of foreign direct investment (FDI) in the 1960s and early 1970s, developing country governments have now come to embrace it warmly within the last two decades. The growing interest in FDI is not only a result of globalization but also a consequence of the steady decline in official development assistance. Developing country share of FDI has increased from a paltry 5% in 1980 to 36% in 2004 (UNCTAD, 2008). Foreign direct investment is now viewed as a source of capital and a major tool in the fight against poverty. It is also viewed as a catalyst for technology transfer from the developed to developing countries. It is known from economic theory that, international capital inflows, inter alia, promote efficient allocation of resources, which in turn enhances economic growth (Asafu-Adjaye 2005). There is a widespread belief that foreign direct investment (FDI) enhances the productivity of host countries and promotes economic development. FDI may not only provide direct capital financing but also create positive externalities via the adoption of foreign technology and know-how. A country's capacity to take advantage of FDI externalities might be limited by local conditions, such as 137 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN ECONOMICS AND MANAGEMENT SCIENCES Vol. 3, No. 1, 2014, E-ISSN: 2226-3624 © 2014 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN ECONOMICS AND MANAGEMENT SCIENCES Vol. 3, No. 1, 2014, E-ISSN: 2226-3624 © 2014 where GDP is a function of policy distortions (FDI) and v control variables that can impact GDP growth overtime. The model was linearized for estimation as: Where GE is Government Expenditure, GFCF is Gross Fixed Capital Formation and CPI is Consumer Price Index as a measure of inflation. The linear specification of the model might be questioned, however, Chakrabarti (2001), has confirmed that in country-specific analysis, modeling FDI determinants in semi-log form can improve the overall fit and the significance of the coefficients. To ensure that the predictive power of the model is unquestionable a battery of tests for the normality of residuals, homoscedasticity of errors, serial correlation and structural stability were run to support the empirical results. where GDP is a function of policy distortions (FDI) and v control variables that can impact GDP growth overtime. The model was linearized for estimation as: Where GE is Government Expenditure, GFCF is Gross Fixed Capital Formation and CPI is Consumer Price Index as a measure of inflation. The linear specification of the model might be questioned, however, Chakrabarti (2001), has confirmed that in country-specific analysis, modeling FDI determinants in semi-log form can improve the overall fit and the significance of the coefficients. To ensure that the predictive power of the model is unquestionable a battery of tests for the normality of residuals, homoscedasticity of errors, serial correlation and structural stability were run to support the empirical results. where GDP is a function of policy distortions (FDI) and v control variables that can impact GDP growth overtime. The model was linearized for estimation as: The Model To capture the relationship between GDP and FDI inflows, a simple model by Matthias Busse (2003) in his analysis of democracy and FDI was adopted and modified to suit the peculiarities of Ghana as: 138 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN ECONOMICS AND MANAGEMENT SCIENCES Vol. 3, No. 1, 2014, E-ISSN: 2226-3624 © 2014 where denotes the Lag operator and is a dimensional identity matrix. The AB model by Amisano and Giannini is obtained by multiplying (7) by and assuming that Thus Given that is a triangular matrix, the Cholesky decomposition is calculated as Given that is a triangular matrix, the Cholesky decomposition is calculated as Given that is a triangular matrix, the Cholesky decomposition is calculated as Structural VAR The basic form of a vector autoregressive model of order is described by (Lutkepohl, 1993 & 2003): where is a vector of endogenous variable. is a k-dimensional process with and time invariant positive covariance matrix (white noise). The impulse responses functions are calculated from the moving average representation of the VAR p Th t l b The contemporaneous relationships between the variables can be included into the model by transforming the VAR model (1) into the structural vector autoregressive (SVAR) model (Hamilton, 1994): The contemporaneous relationships between the variables can be included into the model by transforming the VAR model (1) into the structural vector autoregressive (SVAR) model (Hamilton, 1994): h h l where the structural errors , are white noise and are the structural coefficient matrices. The reduced form of the SVAR is given as where the structural errors , are white noise and are the structural coefficient matrices. The reduced form of the SVAR is given as The recursive form is equation one is given as: The recursive form is equation one is given as: 139 Discussion of Results Annual time series data covering the period 1975-2010 were obtained from World Bank’s World Development Indicators 2012. These were transformed to quarterly data with 144 observations by EViews software packages. This study looked beyond the traditional regression problems of autocorrelation, multicollinearity and simultaneity and considered the dynamic specification of the series. Unit root tests suggest that almost all of the variables included in the model are non-stationary at levels. Johansen cointergration test was carried out, and the results are indicated in Table 4 of the Appendix. The Johansen test indicates the presence of one co-integrating vector at lag 2 but statistical checking proved that the co-integrating vector is not statistically significant. To capture best impulse response and variance decomposition results; a lag structure of 2, as suggested by Akaike, Schwarz and Hannan-Quinn information criterion was specified for the explanatory variables and gradually reduced to the parsimonious model. For numerical illustration see Table 5. The AB model of Amisano and Giannini suggests that the restrictions are to be placed on the matrix A and matrix B should be a diagonal matrix. Theoretical evidence to support this restriction is inadequate therefore these restrictions were done based on empirical findings. The series of matrix restrictions and changes being made in the lag order the matrix equation below represents the restrictions being imposed for proper impulse response and variance decomposition. Table 1: Matrix A restriction of the AB model: GDP shock GE shock GFCF shock CPI shock FDI shock GDP 1 0 0 N/A N/A GE N/A 1 0 0 0 GFCF N/A N/A 1 N/A 0 CPI 0 N/A 0 1 N/A FDI 0 N/A N/A 0 1 The above restrictions were imposed on the matrix A of the AB model and the results are shown in Table 2. These restrictions give statistically significant co-efficients and the proper impulse response and variance decomposition functions. The N/A coefficients in the matrix equation indicate that the shocks in column variable affect its corresponding row variable. The zero coefficients indicate that those entries in the matrix are constrained to be zero. The above restrictions were imposed on the matrix A of the AB model and the results are shown in Table 2. These restrictions give statistically significant co-efficients and the proper impulse response and variance decomposition functions. The N/A coefficients in the matrix equation indicate that the shocks in column variable affect its corresponding row variable. Discussion of Results The zero coefficients indicate that those entries in the matrix are constrained to be zero. The above restrictions were imposed on the matrix A of the AB model and the results are shown in Table 2. These restrictions give statistically significant co-efficients and the proper impulse response and variance decomposition functions. The N/A coefficients in the matrix equation indicate that the shocks in column variable affect its corresponding row variable. The zero coefficients indicate that those entries in the matrix are constrained to be zero. The results of ADF unit root tests on the GDP growth and FDI in their log-levels and log- differenced forms indicate that, real GDP growth and FDI ratio are non-stationary in their respective levels. Then again, after first differencing the variables, the null hypothesis of a 140 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN ECONOMICS AND MANAGEMENT SCIENCES Vol. 3, No. 1, 2014, E-ISSN: 2226-3624 © 2014 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN ECONOMICS AND MANAGEMENT SCIENCES Vol. 3, No. 1, 2014, E-ISSN: 2226-3624 © 2014 Figure 5 represents the impulse responses as a result of a shock in FDI. It shows that a shock or an inward flow of FDI corresponds to contemporaneous increase in Gross Domestic Product, Government Expenditure and lowers the Gross Fixed Capital Formation. This result follows the various empirical literatures presented on FDI and economic growth as well as no positive improvements in GFCF. The figure also depicts that foreign investors are sensitive to INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN ECONOMICS AND MANAGEMENT SCIENCES Vol. 3, No. 1, 2014, E-ISSN: 2226-3624 © 2014 1, 2014, E-ISSN: 2226-3624 © 2014 the levels of inflation. This is because as FDI increases within the first-five quarter the inflation levels are at its minimal and as inflation rises FDI reduces. Because we seek to find the relationship among GDP growth and FDI, a shock in GDP was imposed to see its effects. There is no contemporaneous effect of growth in GDP on FDI. But rather as time goes on this shock in GDP turns to increase the inflow of FDI. Figure 1 indicates that inflation is a sensitive phenomenon which needs to be addressed since it influences the decision of both foreign and domestic investment. decision of both foreign and domestic investment. Figure 1: Responses to FDI Shocks Therefore the Ghanaian economy needs to tackle the issue of inflation to attract a sizable FDI. The variance decomposition shows the variation explained by the other variables to the policy variables. There is a huge variation in both FDI and GDP shocks; this is due to fact that the Ghanaian economy is linked strongly to FDI. This is illustrated in Figure 3.We further compared the Cholesky decomposition and the AB model. As shown in Figure 4, the Cholesky Decomposition gives an impulse model which deviates from the various theoretical and empirical evidences of GDP growth and FDI inflows relation. This is because the Cholesky Decomposition indicates that the relation between these two variables is negative whiles most literatures suggest otherwise. This argument concludes that the AB Model of Amisano and Giannini will be the best model to use to describe the Ghanaian economy. -.012 -.008 -.004 .000 .004 .008 .012 5 10 15 20 25 30 Response of GDP to Shock5 -.020 -.015 -.010 -.005 .000 .005 .010 5 10 15 20 25 30 Response of GE to Shock5 -.020 -.016 -.012 -.008 -.004 .000 .004 .008 5 10 15 20 25 30 Response of GFCF to Shock5 -.06 -.04 -.02 .00 .02 .04 5 10 15 20 25 30 Response of CPI to Shock5 -.04 -.02 .00 .02 .04 .06 .08 .10 5 10 15 20 25 30 Response of FDI to Shock5 Response to Structural One S.D. Innovations ± 2 S.E. Response to Structural One S.D. Innovations ± 2 S.E. Response to Structural One S.D. Innovations ± 2 S.E. INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN ECONOMICS AND MANAGEMENT SCIENCES Vol. 3, No. 1, 2014, E-ISSN: 2226-3624 © 2014 unit root in the ADF tests were rejected at the 5% significance level for all the series. Thus the series are integrated of order one, I (1). Moreover AIC, SBC and Likelihood Ratio (LR) information criteria established the optimum lag length of the VAR. Table 5 presents the output of the choice criteria for selecting the order of the VAR model. The Adjusted LR test statistics adjusted for the samples rejects the zero lag. On the basis of the results, the LR, FPE and AIC selects 6 lags and the SBC selects 2 lag. The minimized SBC’s two (2) lag order for the VAR model is selected because it captures best impulse response and variance decomposition results. Table 2: Results of Matrix A Restriction of The AB Model ab e : Results of Matrix A Restriction of The AB Model Results of Matrix A Restriction of The AB Model A = 1 0 0 C(7) C(9) C(1) 1 0 0 0 C(2) C(3) 1 C(8) 0 0 C(4) 0 1 C(10) 0 C(5) C(6) 0 1 B = C(11) 0 0 0 0 0 C(12) 0 0 0 0 0 C(13) 0 0 0 0 0 C(14) 0 0 0 0 0 C(15) Coefficient Std. Error z-Statistic Prob. C(1) -0.770768 0.068416 -11.26586 0.0000 C(2) -0.520218 0.226069 -2.301146 0.0214 C(3) -0.352901 0.177454 -1.988691 0.0467 C(4) -0.280162 0.501029 -0.559174 0.5760 C(5) 1.161884 0.493474 2.354499 0.0185 C(6) -1.464532 0.377356 -3.881037 0.0001 C(7) -0.000264 0.026160 -0.010083 0.9920 C(8) -0.159559 0.049376 -3.231509 0.0012 C(9) -0.018290 0.033566 -0.544890 0.5858 C(10) 0.747677 0.148895 5.021512 0.0000 C(11) 0.010438 0.000627 16.65447 0.0000 C(12) 0.008345 0.000495 16.85141 0.0000 C(13) 0.017352 0.001300 13.35232 0.0000 C(14) 0.049318 0.003278 15.04332 0.0000 C(15) 0.044390 0.003090 14.36337 0.0000 Coefficient Std. Error z-Statistic Prob. Figure 5 represents the impulse responses as a result of a shock in FDI. It shows that a shock or an inward flow of FDI corresponds to contemporaneous increase in Gross Domestic Product, Government Expenditure and lowers the Gross Fixed Capital Formation. This result follows the various empirical literatures presented on FDI and economic growth as well as no positive improvements in GFCF. The figure also depicts that foreign investors are sensitive to 141 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN ECONOMICS AND MANAGEMENT SCIENCES Vol. 3, No. 1, 2014, E-ISSN: 2226-3624 © 2014 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN ECONOMICS AND MANAGEMENT SCIENCES Vol. 3, No. INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN ECONOMICS AND MANAGEMENT SCIENCES Vol. 3, No. 1, 2014, E-ISSN: 2226-3624 © 2014 -.020 -.015 -.010 -.005 .000 .005 .010 5 10 15 20 25 30 Response of GE to Shock5 -.012 -.008 -.004 .000 .004 .008 .012 5 10 15 20 25 30 Response of GDP to Shock5 p Response of GDP to Shock5 -.06 -.04 -.02 .00 .02 .04 5 10 15 20 25 30 Response of CPI to Shock5 Response of CPI to Shock5 Response of GFCF to Shock5 -.020 -.016 -.012 -.008 -.004 .000 .004 .008 5 10 15 20 25 30 Response of GFCF to Shock5 Response of FDI to Shock5 -.04 -.02 .00 .02 .04 .06 .08 .10 5 10 15 20 25 30 Response of FDI to Shock5 Figure 1: Responses to FDI Shocks Therefore the Ghanaian economy needs to tackle the issue of inflation to attract a sizable FDI. The variance decomposition shows the variation explained by the other variables to the policy variables. There is a huge variation in both FDI and GDP shocks; this is due to fact that the Ghanaian economy is linked strongly to FDI. This is illustrated in Figure 3.We further compared the Cholesky decomposition and the AB model. As shown in Figure 4, the Cholesky Decomposition gives an impulse model which deviates from the various theoretical and empirical evidences of GDP growth and FDI inflows relation. This is because the Cholesky Decomposition indicates that the relation between these two variables is negative whiles most literatures suggest otherwise. This argument concludes that the AB Model of Amisano and Giannini will be the best model to use to describe the Ghanaian economy. 142 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN ECONOMICS AND MANAGEMENT SCIENCES Vol. 3, No. 1, 2014, E-ISSN: 2226-3624 © 2014 Figure 3: Combined Variance Decomposition: 0 20 40 60 80 100 5 10 15 20 25 30 35 40 45 50 Shock1 Shock2 Shock3 Shock4 Shock5 Variance Decomposition of GDP 0 10 20 30 40 50 60 70 5 0 10 20 30 40 50 60 70 5 10 15 20 25 30 35 40 45 50 Shock1 Shock2 Shock3 Shock4 Shock5 Variance Decomposition of GFCF 0 10 20 30 40 50 60 70 5 0 10 20 30 40 50 60 70 80 5 10 15 20 25 30 35 40 45 50 Shock1 Shock2 Shock3 Shock4 Shock5 Variance Decomposition of FDI 0 10 20 30 40 50 60 70 5 10 15 20 25 30 35 40 45 50 Shock1 Shock2 Shock3 Shock4 Shock5 Variance Decomposition of GE 0 10 20 30 40 50 60 70 5 10 15 20 25 30 35 40 45 50 Shock1 Shock2 Shock3 Shock4 Shock5 Variance Decomposition of CPI Figure 3: Combined Variance Decomposition: 0 20 40 60 80 100 5 10 15 20 25 30 35 40 45 50 Shock1 Shock2 Shock3 Shock4 Shock5 Variance Decomposition of GDP 0 10 20 30 40 50 60 70 5 10 15 20 25 30 35 40 45 50 Shock1 Shock2 Shock3 Shock4 Shock5 Variance Decomposition of GE 0 10 20 30 40 50 60 70 5 10 15 20 25 30 35 40 45 50 Shock1 Shock2 Shock3 Shock4 Shock5 Variance Decomposition of GFCF 0 10 20 30 40 50 60 70 5 10 15 20 25 30 35 40 45 50 Shock1 Shock2 Shock3 Shock4 Shock5 Variance Decomposition of CPI 0 10 20 30 40 50 60 70 80 5 10 15 20 25 30 35 40 45 50 Shock1 Shock2 Shock3 Shock4 Shock5 Variance Decomposition of FDI Variance Decomposition of GE Figure 3: Combined Variance Decomposition: Conclusion In this paper we have demonstrated that foreign direct investment correlates with economic growth in Ghana. Using Amisaso amd Giannini restrictions, the model suggests that there exit contemporaneous short run positive effects of FDI inflows on GDP growth. However, as the time horizon expands, these effects tend to converge to the equilibrium. This research also indicates that inflation (CPI) influences the inflow of FDI into the country and therefore needs to be given a closer attention. Andrea Marino (2000). The impact of FDI on developing countries growth: Trade policy matters. ISTAT (National Institute of Statistics), Italy. CEMAFI, Université de Nice-Sophia Antipolis, France. INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN ECONOMICS AND MANAGEMENT SCIENCES Vol. 3, No. 1, 2014, E-ISSN: 2226-3624 © 2014 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN ECONOMICS AND MANAGEMENT SCIENCES Vol. 3, No. 1, 2014, E-ISSN: 2226-3624 © 2014 143 Figure 2: Responses to CPI Shocks: -.012 -.008 -.004 .000 .004 .008 5 10 15 20 25 30 Response of GDP to Shock4 -.016 -.012 -.008 -.004 .000 .004 .008 .012 5 10 15 20 25 30 Response of GE to Shock4 -.010 -.005 .000 .005 .010 .015 .020 5 10 15 20 25 30 Response of GFCF to Shock4 -.04 -.02 .00 .02 .04 .06 .08 5 10 15 20 25 30 Response of CPI to Shock4 -.03 -.02 -.01 .00 .01 .02 .03 .04 .05 5 10 15 20 25 30 Response of FDI to Shock4 Response to Structural One S.D. Innovations ± 2 S.E. Figure 2: Responses to CPI Shocks: -.012 -.008 -.004 .000 .004 .008 5 10 15 20 25 30 Response of GDP to Shock4 -.016 -.012 -.008 -.004 .000 .004 .008 .012 5 10 15 20 25 30 Response of GE to Shock4 -.010 -.005 .000 .005 .010 .015 .020 5 10 15 20 25 30 Response of GFCF to Shock4 -.04 -.02 .00 .02 .04 .06 .08 5 10 15 20 25 30 Response of CPI to Shock4 -.03 -.02 -.01 .00 .01 .02 .03 .04 .05 5 10 15 20 25 30 Response of FDI to Shock4 Response to Structural One S.D. Innovations ± 2 S.E. Response to Structural One S.D. Innovations ± 2 S.E. -.016 -.012 -.008 -.004 .000 .004 .008 .012 5 10 15 20 25 30 Response of GE to Shock4 ne S.D. Innovations ± 2 S.E. -.012 -.008 -.004 .000 .004 .008 5 10 15 20 25 30 Response of GDP to Shock4 Response of GE to Shock4 Response of GE to Shock4 Response of GDP to Shock4 Response of GDP to Shock4 -.04 -.02 .00 .02 .04 .06 .08 5 10 15 20 25 30 Response of CPI to Shock4 -.010 -.005 .000 .005 .010 .015 .020 5 10 15 20 25 30 Response of GFCF to Shock4 Response of GFCF to Shock4 Response of CPI to Shock4 -.03 -.02 -.01 .00 .01 .02 .03 .04 .05 5 10 15 20 25 30 Response of FDI to Shock4 Response of FDI to Shock4 Figure 2: Responses to CPI Shocks: 143 Alfaro, L., Chanda, A., Kalemli Ozcan, S., and Sayek, S. (2004). FDI and Economic Growth, The Role of Local Financial Markets. Journal of International Economics 64, 113-134. Amisano, G. and Giannini, C. (1997). Topics in Structural VAR Econometrics, Second edition. Springer, Berlin. Alfaro, L. and Rodriguez-Clare, A. (2004). Multinationals and Linkages: Evidence from Latin America. Economia 4, 113-170. Alfaro, L., Chanda, A., Kalemli-Ozcan, S., and Sayek, S. (2004). FDI and Economic Growth, The Role of Local Financial Markets. Journal of International Economics 64, 113-134. Amisano, G. and Giannini, C. (1997). Topics in Structural VAR Econometrics, Second edition. Springer, Berlin. Andrea Marino (2000). The impact of FDI on developing countries growth: Trade policy matters. ISTAT (National Institute of Statistics), Italy. CEMAFI, Université de Nice-Sophia Antipolis, France. Alfaro, L. and Rodriguez-Clare, A. (2004). Multinationals and Linkages: Evidence from Latin America. Economia 4, 113-170. References Alfaro, L. and Rodriguez-Clare, A. (2004). Multinationals and Linkages: Evidence from Latin America. Economia 4, 113-170. Alfaro, L., Chanda, A., Kalemli-Ozcan, S., and Sayek, S. (2004). FDI and Economic Growth, The Role of Local Financial Markets. Journal of International Economics 64, 113-134. Amisano, G. and Giannini, C. (1997). Topics in Structural VAR Econometrics, Second edition. Springer, Berlin. A d M i (2000) Th i f FDI d l i i h T d li Andrea Marino (2000). The impact of FDI on developing countries growth: Trade policy matters. ISTAT (National Institute of Statistics), Italy. CEMAFI, Université de Nice-Sophia Antipolis, France. Andrea Marino (2000). The impact of FDI on developing countries growth: Trade policy matters. ISTAT (National Institute of Statistics), Italy. CEMAFI, Université de Nice-Sophia Antipolis, France. 144 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN ECONOMICS AND MANAGEMENT SCIENCES Vol. 3, No. 1, 2014, E-ISSN: 2226-3624 © 2014 Asafu-Adjaye, J. (2005). What has been the Impact of Foreign Direct Investment in Ghana? The Institute of Economic Affairs Publications Vol 1. Azmat Ghani (1999). Foreign Direct Investment in Fiji’.Pacific economic bulletin, volume 14, number 1, June 1999 Asia Pacific Press. Baba Insah (2013). Foreign Direct Investment Inflows and Economic Growth in Ghan International Journal of Economic Practices and Theories, Vol. 3, No. 2, 2013. Balasundram Maniam (2000). U.S. FDI in Latin America: A new perspective. Sam Houston State University Proceedings of the Academy for Economics and Economic Education, 3(2) Chakrabarti, A. (2001). The Determinants of Foreign Direct Investment: Sensitivity Analyses of Cross-Country Regressions, Kyklos, 54(1):89-113 Engle, F. and Granger, C. (1987). Cointegration and Error Correction: Representation, Estimation and Testing. Econometrica 55: 251-76 Gordon H. Hanson (2001). Should Countries Promote Foreign Direct Investment? United Nations Conference on Trade and Development- Center for International Development Harvard University. Research papers for the Intergovernmental Group of Twenty-Four on International Monetary Affairs. Johanson S. (1995). Likelihood-Based Inference in Cointegrated Vector Autoregressive Models. New York: Oxford University Press. Hamilton, J. D. (1994). Time series analysis. Princeton: Princeton University Press. Kishor Sharma (2000). Export Growth In India: Has FDI Played A Role’ Center Discussion Paper No. 816 Charles Stuart University Australia. Kishor Sharma (2000). Export Growth In India: Has No. 816 Charles Stuart University Australia. Lutkepohl, H. (1993). Introduction to multiple times series analysis. Second edition , Berlin: Springer. Lutkepohl, H. (2001). Vector autoregressions. In Baltagi, B. (Ed.) Companion to theoretical econometrics, Oxford: Blackwell, 678–699. Matthias, B. (2003). Democracy and FDI, Hwwa Discussion Paper 220 Hamburgisches Welt- Wirtschafts Archives. Okyere, G. A., Fosu, G. O. and Boakye R. O. (2014). Granger Causality Analysis of Some Macroeconomic Variables in Ghana. Journal of Research in Business and Management, Volume 2, Issue 1, 09-17 Antwi, S. and Zhao, X. (2013). Impact of Foreign Direct Investment and Economic Growth in Ghana: A Cointegration Analysis. International Journal of Business and Social Research, Volume -3, No.-1. UNCTAD (2008), Foreign Direct Investment Database (online), Internet Posting: http://www.unctad.org/Templates/Page.asp?intItemID=1923&lang=1. 145 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN ECONOMICS AND MANAGEMENT SCIENCES Vol. 3, No. 1, 2014, E-ISSN: 2226-3624 © 2014 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN ECONOMICS AND MANAGEMENT SCIENCES Vol. 3, No. 1, 2014, E-ISSN: 2226-3624 © 2014 Appendix Table 3 Correlation Matrix: GDP GE GFCF CPI FDI GDP 1 GE 0.95907 1 GFCF 0.89044 0.93406 1 CPI -0.60903 -0.61201 -0.67233 1 FDI 0.77892 0.83918 0.84939 -0.50934 1 Table 4: Johansen Co-Integration Test Data Trend: None None Linear Linear Quadratic Test Type No Intercept Intercept Intercept Intercept Intercept No Trend No Trend No Trend Trend Trend Trace 1 1 1 1 2 Max-Eig 1 1 1 1 1 Critical values based on MacKinnon-Haug-Michelis (1999) Critical values based on MacKinnon-Haug-Michelis (1999) Table 5: Lag Length Selection Lag LogL LR FPE AIC SC HQ 0 703.0791 NA 2.58e-11 -10.19234 -9.978174 -10.10531 1 1602.429 1706.120 6.72e-17 -23.05043 -22.30085 -22.74582 2 1731.696 235.7217 1.45e-17 -24.58376 -23.29877* -24.06157 3 1745.938 24.92371 1.71e-17 -24.42556 -22.60515 -23.68579 4 1751.492 9.310753 2.29e-17 -24.13959 -21.78376 -23.18224 5 1850.566 158.8100 7.81e-18 -25.22891 -22.33767 -24.05398 6 1915.103 98.70404* 4.44e-18* -25.81034* -22.38369 -24.41784* 7 1923.577 12.33727 5.81e-18 -25.56731 -21.60525 -23.95723 8 1930.523 9.601525 7.84e-18 -25.30181 -20.80433 -23.47415 * indicates lag order selected by the criterion LR: sequential modified LR test statistic (each test at 5% level) FPE: Final prediction error AIC: Akaike information criterion SC: Schwarz information criterion HQ: Hannan-Quinn information criterion Table 5: 146 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN ECONOMICS AND MANAGEMENT SCIENCES Vol. 3, No. 1, 2014, E-ISSN: 2226-3624 © 2014 Figure 4: Impulse Response to Cholesky Decomposition -.012 -.008 -.004 .000 .004 .008 5 10 15 20 25 30 35 40 45 50 Response of GDP to FDI -.016 -.012 -.008 -.004 .000 .004 .008 5 10 15 20 25 30 35 40 45 50 Response of GE to FDI -.016 -.012 -.008 -.004 .000 .004 .008 5 10 15 20 25 30 35 40 45 50 Response of GFCF to FDI -.03 -.02 -.01 .00 .01 .02 .03 5 10 15 20 25 30 35 40 45 50 Response of CPI to FDI -.04 -.02 .00 .02 .04 .06 .08 5 10 15 20 25 30 35 40 45 50 Response of FDI to FDI Response to Cholesky One S.D. Innovations ± 2 S.E. Response to Cholesky One S.D. Innovations ± 2 S.E. -.016 -.012 -.008 -.004 .000 .004 .008 5 10 15 20 25 30 35 40 45 50 Response of GE to FDI ne S.D. Innovations ± 2 S.E. -.012 -.008 -.004 .000 .004 .008 5 10 15 20 25 30 35 40 45 50 Response of GDP to FDI p y Response of GE to FDI -.016 -.012 -.008 -.004 .000 .004 .008 5 10 15 20 25 30 35 40 45 50 Response of GFCF to FDI -.03 -.02 -.01 .00 .01 .02 .03 5 10 15 20 25 30 35 40 45 50 Response of CPI to FDI Response of GFCF to FDI Response of CPI to FDI -.04 -.02 .00 .02 .04 .06 .08 5 10 15 20 25 30 35 40 45 50 Response of FDI to FDI Response of FDI to FDI Figure 4: Impulse Response to Cholesky Decomposition 147
https://openalex.org/W338236958	https://revistas.lasallep.edu.mx/index.php/xihmai/article/download/117/104, https://dialnet.unirioja.es/descarga/articulo/4953773.pdf	es		Origen Y Fundamento De La Educación Basada En Competencias	Xihmai	2,012	cc-by	3,657	ORIGEN Y FUNDAMENTO DE LA EDUCACIÓN BASADA EN COMPETENCIAS Alejandro López Ibarra* Resumen El presente ensayo trata de establecer cuáles son las premisas históricas y pedagógicas en las que se basa el Modelo Educativo Basado en Competencias. En primer término, se hace una reflexión sobre la situación de la escuela en la Era del Conocimiento y cómo una enseñanza que gire en torno a la promoción de competencias es la que puede dar respuestas a las exigencias del mundo actual. Sin embargo, se hace una distinción entre lo que es una perspectiva estrecha y una perspectiva amplia en la aplicación de este modelo educativo y cómo esta última se presenta como una respuesta plausible para formar a los ciudadanos que requiere esta época tan convulsionada. Abstract The present essay tries to establish which are the historical and pedagogical premises based on the Educative Model Based on Competitions. In first term, a reflection is made on the situation of the school in the Era of Knowledge and how an education that turns around the promotion of competitions is the one that can give answers to the exigencies of the present world. Nevertheless, a distinction is made between a narrow perspective and a wide one in the application of this educative model and how this last one appears like a reasonable answer to train the citizens whom this so convulsed time requires. PALABRAS CLAVE: Competencias, evaluación del conocimiento, ciudadanía, modelo educativo *Comunicólogo de profesión, tiene una Especialidad en Estrategias Psicopedagógicas por parte de la Universidad La Salle y está por concluir, en el Tecnológico de Monterrey, sus estudios de la Maestría en Educación con Acentuación en Procesos de Enseñanza y Aprendizaje. Su carrera docente la inició desde 1997 y ha impartido clases en diversas facultades, pero siempre en asignaturas relacionadas con el Área de las Ciencias Sociales. Ha sido asesor de tesis y sinodal en varios exámenes profesionales. Actualmente se desempeña como Director de Comunicación, Difusión y Promoción de la Universidad La Salle Cancún donde, a la par de ser el responsable de toda la comunicación institucional, está al frente de la Licenciatura en Ciencias de la Comunicación. comunicacion@lasallecancun.edu.mx La Nueva Educación en la Era del Conocimiento. Sin lugar a dudas, el mundo se ha transformado a un ritmo desenfrenado en los últimos años. Ya desde el advenimiento de la Revolución Industrial se perfilaban una serie de cambios que impactarían profundamente en el tejido social. Pero lo que hemos experimentado en la llamada Era del Conocimiento es algo para lo que, en muchos sentidos, no estábamos preparados. Un editorial del Periódico “El Comercio” de Miami (2006, p.1) señalaba sobre la Era del Conocimiento: Estamos viviendo una transformación radical. Por primera vez, el factor de producción más importante está en manos de los trabajadores y éste es el conocimiento… Las reglas de juego han cambiado. Hemos pasado de la Era Industrial a la Era del Conocimiento. En la sociedad del conocimiento, el trabajador del conocimiento gana acceso al trabajo y posición social a través de la educación. Por lo tanto, la adquisición y distribución de conocimiento formal, tiene la misma importancia que la que ha tenido la adquisición y distribución de la propiedad e ingresos en los últimos siglos. Por lo tanto, en el pasado, el ser dueño de propiedades y tener un gran capital eran elementos fundamentales para descollar en el terreno profesional. En cambio, en la actualidad, si bien es cierto el tema económico sigue siendo importante, nos encontramos con que el conocimiento es considerado, en sí mismo, un producto valioso y objeto de transacciones comerciales. En este contexto no es de extrañarnos que muchos de los profesionistas más destacados de nuestra época se dediquen a la consultoría. En otras palabras, su conocimiento es el bien que intercambian con otros. Por ello, al hablar de la Era del Conocimiento, autores como Cardona (2002, p. 2) nos indican que “la educación debe replantear sus objetivos, sus metas, sus pedagogías y sus didácticas si quiere cumplir con su misión en el siglo XXI”. Sin embargo, en el entorno nacional e internacional, podemos ver cómo muchas escuelas o universidades no han sabido enfrentarse a las nuevas demandas sociales de nuestra época. Ante esta, llamada por algunos, crisis educativa, no comparto la visión de quienes profetizan el cierre de las escuelas y el surgimiento de un mundo de autodidactas conectados a la Red (Katz, 1999). Empero, estoy plenamente convencido que la escuela del futuro (y del presente) necesita cambios estructurales significativos. Pero, ¿por qué debemos cambiar a las escuelas? ¿Qué no han hecho bien su trabajo? Es decir, muchas personas podrían argumentar que el gran avance tecnológico y científico que hemos tenido ha sido promovido por individuos que pasaron años preparándose en instituciones educativas con estructuras y métodos tradicionales. Por lo tanto, si los resultados son tan positivos, ¿dónde está el problema? Podríamos analizar si estos científicos y profesionistas han destacado en sus ámbitos de desarrollo, a pesar de las escuelas en las que estudiaron y si su éxito se debe, en gran medida, al autodidactismo. Pero más bien quisiera centrarme en las razones históricas del surgimiento de las universidades y cómo las condiciones imperantes en dicha época han sido rebasadas y, por ende, las instituciones educativas que siguen trabajando bajo el modelo de antaño no están preparadas para responder a las necesidades de un mundo como el actual. Quisiera enfatizar que este análisis histórico, aunque esté centrado en las universidades, impacta la realidad de todos los niveles educativos; ya que como veremos más adelante, la Educación Basada en Competencias se empezó a implementar en las instituciones de Educación Superior, para luego influir el currículum de escuelas que van desde el preescolar hasta el bachillerato. Ginés (2004), en su artículo “La necesidad del cambio educativo para la sociedad del conocimiento” comenta que, aunque la universidad como tal surgió en la Edad Media, no fue sino hasta el siglo XIX, en plena Era Moderna, que se fueron configurando los tres modelos universitarios que imperan en muchas de las instituciones de Educación Superior actuales: a) El Modelo Alemán, cuyo objetivo era que los estudiantes adquirieran una amplia cultura sobre diversos temas, que tuvieran una investigación científica que los sustentara, independientemente de si éstos tenían un uso en el ámbito laboral. La premisa de este modelo era que las personas con un amplio dominio de conocimientos científicos, los aportaran al desarrollo del país en diversas facetas. b) El Modelo Francés, el cual surgió para responder a las demandas del Imperio Napoleónico cuyo crecimiento demandaba profesionistas en muchos rubros. Por lo tanto, la universidad estaba al servicio de las necesidades del Estado y preparaba a los burócratas que el gobierno necesitaba. c) El Modelo Anglosajón combinaba algunas de las características de los dos anteriores, pero dándoles ciertos elementos distintivos. Al igual que el Modelo Alemán, pretendía dotar a los estudiantes de un conocimiento amplio sobre diversos temas, pero a diferencia de éste, no promovía la investigación. Asimismo, los estudiantes que concluyeran sus estudios podían terminar trabajando para el Estado, al igual que en el Modelo Francés. Pero lo anterior, no significaba que también había una gran posibilidad de que los jóvenes trabajaran para la iniciativa privada. Además, a diferencia de los otros dos modelos, el Modelo Anglosajón fue desarrollado por instituciones privadas y su administración no estaba en manos del Estado. Poco a poco, estos modelos se fueron combinando; en la actualidad, podemos encontrar en nuestros sistemas universitarios rastros de cada uno. Sin embargo, a pesar de sus diferencias, un rasgo característico de estos tres modelos de universidad era la premisa de que los estudiantes se preparaban para ejercer profesiones que no sufrían grandes cambios con el paso del tiempo. Es decir, era un pensamiento común el hecho de que un egresado de una carrera obtenía una licencia (de ahí, la palabra licenciado) para ejercer una profesión de por vida. Si nos ubicamos en el siglo XIX y en la importancia que las élites (como las universitarias) le daban a la ciencia exacta, podemos entender que se pretendía transmitir a las nuevas generaciones los principios inamovibles que el genio humano había logrado “arrancarle” a la naturaleza. La enseñanza de leyes era importante, pero no sólo en las Ciencias Naturales. Esa tendencia, también era un anhelo de los estudiosos del mundo social. Una vez que se ha comprendido el origen de nuestras universidades actuales, nos percatamos cómo sus estructuras y fundamentos han sido rebasados. ¿Por qué basarnos en las necesidades y realidades del Modernismo del siglo XIX cuando nos encontramos ante el influjo del Posmodernismo del siglo XX? ¿Transmitir a unos cuantos, cual tesoro o “iluminación”, un conocimiento “eterno” cuando hoy sabemos que hasta ciertos principios que considerábamos inamovibles están sujetos a revisión? El mundo ha cambiado y el conocimiento que adquiere un individuo, cambia cada cinco años (Argudín, 2005). Lejos ha quedado la época en que los egresados de las universidades concluían sus estudios y estaban “preparados para la vida”. La Era del Conocimiento nos demanda mucho más que eso y una de las respuestas que se ha generado a esta problemática es la llamada Educación Basada en Competencias. El movimiento en pro del desarrollo de competencias en los estudiantes universitarios surgió a finales de la década de los 60 y principios de los 70. En esa época, un profesor de Psicología de Harvard, David McClelland, se percató que los exámenes o pruebas que se aplicaban en las universidades no podían predecir el futuro éxito o fracaso profesional del sustentante (Adams, 1996, citado por Brundrett, 2000). McClelland se empezó a preguntar el porqué ocurría esto y trató de encontrar las variables que le permitirían predecir el futuro profesional de los jóvenes universitarios. Fue en ese momento, que el psicólogo estadounidense fundó la firma consultora “McBer”, con el objetivo de encontrar lo que hacía competente a un trabajador. En otras palabras, intentaba encontrar los factores o competencias que podían ser determinantes en la adecuada ejecución de una labor, para lo cual elaboró la llamada “Evaluación de Competencia Laboral”. Brundrett (2000) nos narra que en 1981, Richard Boyatzis, un consultor de la empresa “McBer”, intentó definir un “Modelo Genérico de Competencia Gerencial”. Para ello aplicó la “Evaluación de Competencia Laboral” desarrollada por su jefe a más de 2,000 personas que tenían puestos gerenciales en 12 compañías distintas. La intención de Boyatzis era encontrar las características de un desempeño laboral sobresaliente. Su trabajo derivó en una lista de 19 competencias básicas que todo gerente debía poseer si pretendía realizar su trabajo de forma sobresaliente. Ahora bien, ¿por qué los resultados del trabajo realizado en la firma “McBer” generaron tantas implicaciones en el terreno educativo? Porque estas investigaciones produjeron una “lista de oro” sobre lo que debía poseer una persona para ser considerada competente en su trabajo. Y aunque el estudio de Boyatzis estaba enfocado a la Administración de Empresas y, particularmente, a la mejora gerencial; las preguntas que empezaban a rondar en las mentes de los educadores de distintas disciplinas eran: ¿será posible generar listas similares de competencias para otras profesiones? Y si esto es posible, ¿por qué no enseñar a las personas esas competencias? Tratando de poner en contexto la revolución que significó el trabajo de McClelland y Boyatzis, pensemos cómo un maestro “tradicional” enseñaba (o sigue enseñando) las “virtudes del buen gerente” a los alumnos de Administración. Lo más probable es que el maestro les diera textos teóricos sobre el liderazgo que un gerente debe tener sobre su equipo, sobre la importancia de la motivación en el logro de las metas, etc. Es más, quizá podrían hacer discusiones sobre los tópicos y los docentes más “abiertos”, incluso, les pedirían hacer simulaciones de los ambientes de trabajo. Pero ¿esas actividades harían que los alumnos, en el momento que egresaran, fueran gerentes competentes? Tristemente, la respuesta es no. Ahora bien, ¿por qué no? Porque la lista de Boyatzis incluía competencias que rara vez son enseñadas en el aula; como el autocontrol o el pensamiento analítico (Carriel, Ruiz, Ruiz y Suazo, 2004). Entonces podemos empezar a vislumbrar el cambio de rumbo que significa la Educación Basada en Competencias. Si nos apegamos al caso que hemos venido explicando, un maestro de Administración que quisiera promover buenos gerentes en “potencia” tendría que diseñar actividades que sometieran al alumno a niveles de stress similares a los que se viven en el ámbito laboral, con el objeto de que el estudiante pudiera ir moldeando su autocontrol. El docente también debería pensar cómo desarrollar en los alumnos la capacidad cognitiva de hacer análisis certeros, pues esta competencia es fundamental para que no se pierda el rumbo en la obtención de las metas. Pero detengámonos un momento a reflexionar sobre lo que tendría que hacer nuestro hipotético maestro. En el párrafo anterior se mencionó que un buen gerente debe tener autocontrol pero, parafraseando nuestro mismo ejemplo, ¿cómo enfrentarse al diseño de actividades que sometan al alumno a niveles de stress similares a los que se viven en el ámbito laboral, con el objeto de que el estudiante pueda ir moldeando su autocontrol? Es más, ¿se puede hacer eso en el aula? Muchos autores piensan que no y que la única manera es llevando la universidad a la empresa. Es decir, el campus deja de estar confinado a las cuatro paredes del salón y los alumnos, vía prácticas profesionales, cátedras empresariales o estancias por proyecto, entran en contacto con la realidad profesional. Sin embargo, algunas personas pensamos que, si bien es cierto que es muy importante el llevar la universidad a la empresa, también se pueden generar competencias trayendo la empresa a la escuela. El dar conferencias, pláticas o el generar proyectos escolares de manera conjunta con instituciones privadas o públicas es otro tipo de acercamiento válido y necesario para promover el desarrollo de alumnos competentes. Además, esta última vertiente no queda circunscrita a la Educación Superior y es una de las formas en que los profesores de otros niveles educativos pueden vincular sus materias con la realidad que sus alumnos enfrentan en su vida cotidiana. Ahora bien, ¿cómo se definen las competencias? Pensamos que el recorrido que, hasta el momento, hemos tenido sobre el origen y fundamento de este modelo educativo, permite entender algunas de las definiciones que se presentan a continuación. Sin embargo, quisiera hacer hincapié en que éstas son tan variadas como los autores que escriben sobre el tema. Es decir, la Educación Basada en Competencias es un tópico tan nuevo que todavía nos encontramos en un proceso de consenso. Boyatzis (1982, citado por Brundrett, 2000) definía a las competencias como las características que marcan la diferencia entre una actuación sobresaliente y un desempeño promedio o abajo del promedio. Contrario a lo que los autores estadounidenses como McClelland y Boyatzis sugerían, en el Reino Unido se considera que una persona ha adquirido una competencia cuando puede desempeñar adecuadamente una labor, sin que necesariamente tenga una actuación sobresaliente (Brundett, 2000). Independientemente de esta diferencia, lo que se puede apreciar es que ambas posturas tienen una fuerte tendencia a relacionar sus definiciones con el ámbito empresarial, y pareciera que se confunde el término “actuación en el trabajo” con el de competencia. Este hecho es una de las principales críticas a la Educación Basada en Competencias. Muchas voces se alzan en contra de esta dependencia educativa a las necesidades de la empresa y en esa visión tan estrecha que constriñe la capacidad personal con la capacidad laboral. Hacket (2001) en su artículo “Educando para la Competencia y la Práctica Reflexiva” comenta que, dentro de la Educación Basada en Competencias, se puede considerar que hay dos perspectivas antagónicas entre sí. La perspectiva estrecha, la cual sostiene que el entrenamiento estandarizado produce resultados que pueden ser alcanzados, en un nivel aceptable, por todos los educandos. Los pasos de ese entrenamiento estandarizado surgen de la observación de un trabajador que ha adquirido cierta competencia. En cambio, la perspectiva amplia toma en cuenta las facetas sociales, intelectuales, emocionales y de proceso de las diversas circunstancias educativas en la que la Educación Basada en Competencias es practicada. No confunde el término “actuación en el trabajo” con el de competencia y enfatiza los aspectos humanos en las descripciones sobre competencia. Considero que el antagonismo entre la perspectiva estrecha y la visión amplia es el punto central de la discusión en torno a la definición de lo que es una competencia; y es en este sentido que, para los fines de este ensayo, se tomará una postura ante esta disyuntiva. Pienso que la perspectiva estrecha tiene su razón de ser, tanto práctica como histórica. Es decir, los empresarios se percataron que las universidades no estaban “haciendo su trabajo” y los egresados de las mismas llegaban al ámbito laboral sin todos los elementos necesarios para su buen desempeño. Por lo tanto, decidieron generar procesos de capacitación específicos para sus empleados en las áreas en las que consideraban que requerían apoyo. Obviamente, todos estos esfuerzos estaban (y están) encaminados a mejorar la productividad de la empresa y no toman mucho en cuenta a la persona como tal. Sin embargo, el hecho de que exista una justificación del surgimiento de la perspectiva estrecha, no significa que esa deba ser la visión con la que la Educación Basada en Competencias sea llevada al ámbito de las escuelas. Estoy firmemente convencido que la perspectiva amplia, la cual toma en cuenta el desarrollo integral del ser humano, es la que debe prevalecer en los diseños curriculares de las escuelas de todos los niveles. Esta visión más holística no hace a un lado las preocupaciones “pragmáticas y empresariales” de la postura estrecha. Más bien, le añade elementos que son muy valiosos. Incluso, la UNESCO (Organización de las Naciones Unidas para la Educación, la Ciencia y la Cultura, por sus siglas en inglés) ha adoptado la perspectiva amplia en su propia definición de lo que es un competencia, la cual es: “conjunto de comportamientos socio afectivos y habilidades cognoscitivas, psicológicas, sensoriales y motoras que permiten llevar a cabo adecuadamente un desempeño, una función, una actividad o una tarea” (Argudín, 2005, p. 12). Podemos concluir que dependiendo de la perspectiva con la que uno asuma la Educación Basada en Competencias, se podrán determinar los alcances de la aplicación de este modelo educativo en los alumnos. ¿Qué es lo que queremos? ¿Sólo alumnos que sepan cómo hacer sus trabajos y que adquieran competencias procedimentales? Entonces, hagamos uso de la perspectiva estrecha. En cambio, ¿queremos alumnos que sepan hacer su trabajo pero que, al mismo tiempo, tengan una serie de actitudes y comportamientos que los hagan mejores personas y no sólo trabajadores? Si la respuesta es afirmativa, la perspectiva amplia de la Educación Basada en Competencias es lo que estamos buscando. Además, ésta tiene la gran ventaja de adaptarse a todos los niveles educativos y no constreñirse al ámbito universitario. De todos los textos que abogan por la Educación Basada en Competencias, el escrito por Argudín (2005) parece el más adecuado para expresar los puntos a favor de este modelo educativo. Retomando los puntos más importantes de los primeros tres capítulos del libro de esta autora, podemos señalar que la Educación Basada en Competencias tiene las siguientes ventajas: a) En primer término, la Educación Basada en Competencias hace frente a una sociedad donde el conocimiento cambia de forma muy rápida. Es decir, si en esta época de transformaciones constantes, el único “tesoro” del estudiante es la información que tiene, la valía de lo que conoce se irá perdiendo con el paso de los años y de una forma acelerada. De lo anterior se deriva que, si el conocimiento se renueva tan rápido, las escuelas tienen que enseñar a los alumnos a aprender a aprender. De esa manera, no importa que la información cambie o sea mucha, el estudiante tendrá la competencia de indagar, sintetizar y valorar los nuevos datos que surjan en su ámbito profesional y personal. b) También la Educación Basada en Competencias le permite al estudiante identificar el procedimiento utilizado para llevar a cabo las cosas. De esa manera, el alumno podrá tener un autocontrol sobre los pasos que lleva a cabo para lograr las metas que se proponga. c) La Educación Basada en Competencias evita la desvinculación de los contenidos escolares y las demandas laborales de la sociedad posmoderna. d) ¿Cuál es la función de la educación? Que el individuo se integre a la sociedad y que sea transformado bajo las pautas culturales aceptadas. Sin embargo, en el modelo de competencias también se espera que el aprendiz llegue a ocupar un lugar en el sector productivo. Es decir, en una perspectiva amplia de una Educación Basada en Competencias, se pretende generar personas con una buena formación (normas, valores, actitudes, código ético, etc.) y una buena capacitación (conceptos y procedimientos para desempeñar correctamente una función). FuentesdeConsulta ARGUDÍN, Y., (2005), Educación basada en competencias. Distrito Federal, México: Trillas. BRUNDRETT, M. (2000). The question of competence: the origins, strengths and inadequacies of a leadership training paradigm, School Leadership & Management Abingdon. CARDONA, G. (2002). Tendencias educativas para el siglo XXI: educación virtual, on line y @learning. Elementos para la discusión, Revista Electrónica de Tecnología Educativa, CARRIEL, J., Ruiz, S., Ruiz, N. y Suazo, E. (2004). Diseño de un sistema de evaluación de las competencias a desarrollar por los usuarios de las TIC. (Disertación para obtener el grado de licenciado, Universidad de Concepción en Chile, 2004). GINÉS Mora, J. (2004). La necesidad del cambio educativo para la sociedad del conocimiento. Revista Iberoamericana de Educación, 35. Disponible en: http://www.campus-oei.org/revista/rie35a01.htm HACKET, S. (2001), Educating for competency and reflective practice: fostering a conjoint approach in education and training, Journal of Workplace Learning, 13 (3/4). KATZ, R. (1999), Information Technology and the New Competition in Higher Education. San Francisco, California, USA: Jossey-Bass Higher and Adult Education Series. Opinión-Hemos pasado de la era industrial a la era del conocimiento; [Fuente: El Comercio]. (28 de Junio). Noticias Financieras, 1. Recuperado en Octubre 22, 2007, de Latin American Newsstand Database. (Document ID: 1068319921).
https://openalex.org/W2194890348	https://tspace.library.utoronto.ca/bitstream/1807/87170/1/40635_2015_Article_569.pdf	English	null	High flow nasal cannula oxygen therapy in immunocompromised patients with acute hypoxemic respiratory failure	Intensive care medicine experimental	2,015	cc-by	1,089	High flow nasal cannula oxygen therapy in immunocompromised patients with acute hypoxemic respiratory failure From ESICM LIVES 2015 Berlin, Germany. 3-7 October 2015 in this study, knowing that patients with profound neutro- penia were excluded. Objectives To compare intubation and mortality rates in the subset of immunocompromised patients admitted to ICU for ARF. Methods We performed a subgroup analysis of the FLORALI study. This study included all patients with non-hypercapnic (PaCO2 ≤45 mm Hg) ARF excluding patients with cardio- genic pulmonary edema and those with underlying chronic lung disease. Patients were assigned to three groups according to treatment: High-Flow Oxygen, O2 or NIV. The primary outcome was the intubation rate and secondary outcome included 90-day mortality. We focused on the subset of immunocompromised patients included Introduction In the early 2000’s, two randomized controlled trials have shown that non-invasive ventilation (NIV) could decrease mortality of immunocompromised patients admitted to ICU for acute respiratory failure (ARF) as compared to standard oxygen therapy (O2) [1,2]. However, the benefits of NIV in immunocompetent patients with ARF failure are debated. High flow nasal cannula oxygen therapy (High-Flow Oxygen) may offer an alternative in hypoxe- mic patients. We recently found in a randomized controlled trial including 310 patients with ARF that High-Flow Oxygen decreased mortality as compared to NIV [3]. Immunocompromised patients could be also included in this study, except those with profound neutro- penia. Therefore, we assessed the benefits of High-Flow Oxygen or NIV in this subgroup of patients. © 2015 Frat et al.; This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http:// creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Conclusions In immunocompromised patients admitted to ICU for acute hypoxemic respiratory failure, High-Flow Oxygen was associated with lower intubation and mortality rates, and a reduced duration of invasive mechanical ventilation as compared to O2 or NIV. Results Among the 310 patients with ARF, 82 (26%) were immu- nocompromised including 26 patients in the High-Flow Oxygen group, 30 in the O2 group, and 26 in the NIV group. Intubation rates were 31%, 43% and 55% in the High-Flow Oxygen, O2 and NIV groups, respectively (p = 0.04). The 90-day mortality rates were 15%, 27% and 46% in the High-Flow Oxygen, O2 and NIV groups (p = 0.046). Ventilator-free days at day 28 were 26 ± 6, 23 ± 10 and 14 ± 13 days in the High-Flow Oxygen, O2 and NIV groups, respectively (p < 0.0001). Frat et al. Intensive Care Medicine Experimental 2015, 3(Suppl 1):A425 http://www.icm-experimental.com/content/3/S1/A425 Authors’ details 1 1CHU Poitiers, Service de Réanimation Médicale, Poitiers, France. 2Université Poitiers, INSERM CIC 1402 (Équipe 5 ALIVE), Poitiers, France. 3Université Poitiers, INSERM CIC 1402 Biostatistics, Poitiers, France. 4CHU Rouen, Service de Réanimation Médicale, Rouen, France. 5Université Rouen, UPRES EA 3830- IRIB, Institute for Biomedical Research and Innovation, Rouen, France. 6CHU Clermont Ferrand, Pôle de Médecine Périopératoire, Clermont-Ferrand, France. 7Université d’Auvergne, R2D2, EA-7281, Clermont-Ferrand, France. 8CHU de la Cavale Blanche, Service de Réanimation Médicale, Brest, France. 9CHR Orléans, Réanimation Médico-Chirurgicale, Orléans, France. 10CHU Angers, Service de Réanimation Médicale et Médecine Hyperbare, Angers, France. 11St Michael’s Hospital, Keenan Research Centre and Critical Care Department, Toronto, Canada. 12University of Toronto, Interdepartmental Division of Critical Care Medicine, Toronto, Canada. 13Université Créteil, INSERM UMR 955 Eq13, Créteil, France. 1CHU Poitiers, Service de Réanimation Médicale, Poitiers, France Full list of author information is available at the end of the article Authors details 1CHU Poitiers, Service de Réanimation Médicale, Poitiers, France. 2Université Poitiers, INSERM CIC 1402 (Équipe 5 ALIVE), Poitiers, France. 3Université Poitiers, INSERM CIC 1402 Biostatistics, Poitiers, France. 4CHU Rouen, Service de Réanimation Médicale, Rouen, France. 5Université Rouen, UPRES EA 3830- IRIB, Institute for Biomedical Research and Innovation, Rouen, France. 6CHU Clermont Ferrand, Pôle de Médecine Périopératoire, Clermont-Ferrand, France. 7Université d’Auvergne, R2D2, EA-7281, Clermont-Ferrand, France. 8CHU de la Cavale Blanche, Service de Réanimation Médicale, Brest, France. 9CHR Orléans, Réanimation Médico-Chirurgicale, Orléans, France. 10CHU Angers, Service de Réanimation Médicale et Médecine Hyperbare, Angers, France. 11St Michael’s Hospital, Keenan Research Centre and Critical Care Department, Toronto, Canada. 12University of Toronto, Interdepartmental Division of Critical Care Medicine, Toronto, Canada. 13Université Créteil, INSERM UMR 955 Eq13, Créteil, France. POSTER PRESENTATION Open Access 1CHU Poitiers, Service de Réanimation Médicale, Poitiers, France Full list of author information is available at the end of the article Page 2 of 2 Page 2 of 2 Frat et al. Intensive Care Medicine Experimental 2015, 3(Suppl 1):A425 http://www.icm-experimental.com/content/3/S1/A425 Published: 1 October 2015 References 1. Antonelli M, Conti G, Bufi M, Costa MG, Lappa A, Rocco M, et al: Noninvasive ventilation for treatment of acute respiratory failure in patients undergoing solid organ transplantation: a randomized trial. JAMA 2000, 283(2):235-241. 2. Hilbert G, Gruson D, Vargas F, Valentino R, Gbikpi-Benissan G, Dupon M, et al: Noninvasive ventilation in immunosuppressed patients with pulmonary infiltrates, fever, and acute respiratory failure. N Engl J Med 2001, 344(7):481-487. 3. Frat JP, Thille AW, Mercat A, Girault C, Ragot S, Perbet S, et al: High-Flow Nasal Cannulae Oxygen Therapy in Acute Hypoxemic Respiratory Failure. N Engl J Med 2015, 372:2185-2196. doi:10.1186/2197-425X-3-S1-A425 Cite this article as: Frat et al.: High flow nasal cannula oxygen therapy in immunocompromised patients with acute hypoxemic respiratory failure. Intensive Care Medicine Experimental 2015 3(Suppl 1):A425. References 1. Antonelli M, Conti G, Bufi M, Costa MG, Lappa A, Rocco M, et al: Noninvasive ventilation for treatment of acute respiratory failure in patients undergoing solid organ transplantation: a randomized trial. JAMA 2000, 283(2):235-241. 2. Hilbert G, Gruson D, Vargas F, Valentino R, Gbikpi-Benissan G, Dupon M, et al: Noninvasive ventilation in immunosuppressed patients with pulmonary infiltrates, fever, and acute respiratory failure. N Engl J Med 2001, 344(7):481-487. 3. Frat JP, Thille AW, Mercat A, Girault C, Ragot S, Perbet S, et al: High-Flow Nasal Cannulae Oxygen Therapy in Acute Hypoxemic Respiratory Failure. N Engl J Med 2015, 372:2185-2196. doi:10.1186/2197-425X-3-S1-A425 Cite this article as: Frat et al.: High flow nasal cannula oxygen therapy in immunocompromised patients with acute hypoxemic respiratory failure. Intensive Care Medicine Experimental 2015 3(Suppl 1):A425. References doi:10.1186/2197-425X-3-S1-A425 Cite this article as: Frat et al.: High flow nasal cannula oxygen therapy in immunocompromised patients with acute hypoxemic respiratory failure. Intensive Care Medicine Experimental 2015 3(Suppl 1):A425. Submit your manuscript to a journal and beneﬁ t from: 7 Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the ﬁ eld 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.com
https://openalex.org/W4313650035	https://discovery.ucl.ac.uk/id/eprint/10163786/1/s10509-022-04157-z.pdf	English	null	Effects of noise on the accuracy of plasma bulk parameters derived from velocity moments of in-situ observations	Astrophysics and space science	2,023	cc-by	6,983	G. Nicolaou g.nicolaou@ucl.ac.uk 1 Department of Space and Climate Physics, Mullard Space Science Laboratory, University College London, Dorking, Surrey, RH5 6NT, UK RESEARCH RESEARCH RESEARCH Abstract We expose and quantify the inaccuracies of plasma bulk parameters derived from the calculation of velocity moments of noisy in-situ plasma observations. First, we simulate typical solar wind proton plasma observations, obtained by a typical top-hat electrostatic analyzer instrument. We add background noise to the simulated observations and analyze them by applying standard methods to derive the plasma density, speed, and temperature. We then compare the analysis results with the parameters we use to simulate the observations in the ﬁrst place, in order to quantify the inaccuracies in the calculated plasma parameters as functions of the noise level in the observations. We ﬁnd that even noise levels that are smaller than 1% of the signal peak, lead to signiﬁcant inaccuracies in some plasma parameters. The plasma temperature suffers the biggest inaccuracies and the plasma speed the smallest. Our results highlight the importance of removing noise from observations when calculating the moments of the constructed plasma distributions. We ﬁnally, evaluate one simple method to remove uniform background noise automatically from measurements, which is useful for future on-board analyses. Keywords Plasmas · Instrumentation: miscellaneous · Methods: data analysis Eﬀects of noise on the accuracy of plasma bulk parameters derived from velocity moments of in-situ observations Georgios Nicolaou1 Received: 2 August 2022 / Accepted: 10 December 2022 © The Author(s) 2023 Astrophysics and Space Science (2023) 368:3 https://doi.org/10.1007/s10509-022-04157-z Astrophysics and Space Science (2023) 368:3 https://doi.org/10.1007/s10509-022-04157-z Astrophysics and Space Science (2023) 368:3 https://doi.org/10.1007/s10509-022-04157-z 1 Introduction Another popular method for estimating plasma bulk pa- rameters from in-situ observations, is the calculation of the VDF’s velocity moments. This method estimates the aver- age (bulk) parameters of the plasma via numerical integra- tion of the constructed VDFs, weighted by velocity dyadic products. For instance, the zeroth order moment calculates the plasma density, while the ﬁrst and second order moments calculate the plasma bulk velocity and pressure (or tempera- ture), respectively. Compared to the ﬁtting method, the cal- culation of moments is faster, demanding much less com- putational time. This is because, unlike the ﬁtting method, the calculation of moments does not require the evaluation of an analytical function. Some studies have calculated the velocity moments of VDFs, constructed in several environ- ments, such as the Saturnian magnetotail (Dialynas et al. 2018), Jovian magnetosphere (Mauk et al. 2004), and the solar wind (e.g., Marsch et al. 1982; Kasper 2003; Nicolaou et al. 2021). The velocity distribution functions (VDFs) of space plasma particles are typically constructed from in-situ plasma ob- servations. A proper analysis of the constructed VDFs de- termines the bulk parameters of the plasma. One popular analysis method is the ﬁtting of analytical functions, param- eterized by the bulk parameters, to the constructed VDFs or the raw observations. In this method, the bulk parameters are determined by the best “ﬁt” of the model function to the measurement. Several studies applied ﬁtting algorithms to in-situ observations in order to estimate bulk parameters in several space regions, such as the Jovian magnetotail (e.g., Nicolaou et al. 2014, 2015), Saturnian magnetosphere (Livi et al. 2014; Wilson et al. 2017), cometary plasma electrons (Broiles et al. 2016), and the solar wind (e.g., Kasper 2003; Ebert et al. 2009; Elliott et al. 2016; Nicolaou et al. 2021; Abraham et al. 2022). The fact that the calculation of moments demands very low computational time, it makes it suitable for on-board data processing. This is extremely beneﬁcial in missions requiring high time resolution measurements, or when the plasma parameters must be calculated and downlinked very fast (e.g., on space weather monitoring missions, Page 2 of 9 G. Nicolaou 3 Nicolaou et al. 2020a). However, if the original measure- ments are not downlinked, we cannot construct the plasma VDFs on-ground and evaluate the accuracy of the derived plasma parameters. Therefore, it is important to evaluate our processing methods, using models of realistic plasma and measurement conditions. 1 Introduction Background noise is one factor that can result in erroneous calculations of plasma param- eters, depending on the signal-to-noise ratio level. A com- plete evaluation of the derived moments should quantify the effects of noise, which are often overlooked. The expected number of counts in each instrument pixel s is Cexp (E,Θ,Φ) = 2 m2 Gf f (E,Θ,Φ)E2τ, (1) (1) where Gf is the instrument’s geometric factor, which we consider constant for all E, Θ and Φ. In practice, the ge- ometric factor is determined form laboratory calibrations (e.g., Owen et al. 2020). τ is the acquisition time for each measurement (obtained at each E, Θ, Φ) and f (E,Θ,Φ) is the VDF of the plasma particles, with the particle velocity vector expressed in E, Θ, Φ. The formula in Eq. (1) as- sumes that the VDF does not vary over the E, Θ, Φ pixel acceptance bandwidth (e.g., Nicolaou et al. 2019, 2020a,b, 2021). Furthermore, the VDF is assumed to be invariant with time over the full E, Θ scan of the instrument. Here, we examine the inaccuracies of calculated mo- ments using simulations of in-situ plasma observations of solar wind proton particles, with a uniform background noise. The comparison between the simulated observations analysis results and the plasma model input, allows us to expose and quantify the effects of the background noise to the ﬁnal product of the analysis. In Sect. 2, we explain the method we follow to simulate proton plasma observations with background noise, obtained by a typical electrostatic analyzer design. Further, we explain how we calculate the plasma bulk parameters via the velocity moments of the ve- locity distribution functions constructed from the modeled measurements. In Sect. 3, we show the comparison between the output moments and the input parameters for different noise levels. This comparison quantiﬁes the accuracy of the derived bulk parameters. In Sect. 4, we discuss our results and we evaluate a novel method to remove the uniform, background noise form the measurements. Finally, Sect. 5 summarizes our study and lists our conclusions. 1 Introduction For our simulations, we consider solar wind plasma protons with their velocities −→ U (or energies) following a Maxwellian distribution function: f (E,Θ,Φ) = N m 2πkBT 3 2 e−E+E0−2EE0 cos[ω(Θ,Φ)] kB T , (2) (2) where N is the number density of the simulated protons with mass m ∼1.67 × 10−27 kg, T is their temperature, E0 their bulk energy, ω (Θ,Φ) the angle between the par- ticle velocity ⃗U = 2E m ⃗U ⃗U and the bulk velocity vector ⃗ ⃗U0 = 2E0 m ⃗U0 −→ U 0 , and kBis the Boltzmann constant. We ⃗U0 = 2E0 m ⃗U0 −→ U 0 , and kBis the Boltzmann constant. We ⃗U0 = 2E0 m ⃗U0 −→ U 0 , and kBis the Boltzmann constant. We simulate plasma protons with input bulk parameters N = Nin = 35 cm−3, ⃗U0 = ⃗U0,in ∼440 kms−1 (E0 = 1 keV) to- wards Θ = 0 and Φ = 9, and T = Tin = 20 eV, respec- tively. This set of input parameters models typical solar wind protons at ∼1 au (e.g., Barouch 1977). simulate plasma protons with input bulk parameters N = Nin = 35 cm−3, ⃗U0 = ⃗U0,in ∼440 kms−1 (E0 = 1 keV) to- wards Θ = 0 and Φ = 9, and T = Tin = 20 eV, respec- tively. This set of input parameters models typical solar wind protons at ∼1 au (e.g., Barouch 1977). Eﬀects of noise on the accuracy of plasma bulk parameters derived from velocity moments... For the spe- ciﬁc plasma parameters, the simulated counts peak at ∼1000 counts. Top panels correspond to noise level nl = 0 counts, middle panels to nl = 1 counts, and bottom panels to nl = 10 counts. (9) × 2E m sinΘ fout (E,Θ,Φ)E cosθθϕ, (10) (10) respectively, and we calculate the scalar temperature as: kBTout = 1 3Nout E Θ Φ 2E m kBTout = 1 3Nout E Θ Φ 2E m Typically, we construct the observed particle VDF fout, using the inverse formula in Eq. (1), such that × w2 x + w2 y + w2 z fout (E,Θ,Φ)E cosθθϕ, (11) (11) fout (E,Θ,Φ) = m2C (E,Θ,Φ) 2Gf E2τ . (6) (6) where where wx = 2E m cosΘ cosΦ −Ux,out, wy = 2E m cosΘ sinΦ −Uy,out and wz = 2E m sinΘ −Uz,out. (12) wx = 2E m cosΘ cosΦ −Ux,out, wy = 2E m cosΘ sinΦ −Uy,out and wz = 2E m sinΘ −Uz,out. (12) We then calculate the statistical moments of fout from which we get the plasma bulk parameters (see also Nicolaou et al. 2020a). We calculate the density as: Nout = E Θ Φ 2E m3 fout (E,Θ,Φ)E cosθθϕ, (12) (7) In the above formulas, E, Θ, and Φ are the differences between consecutive E, Θ, and Φ pixels, respectively. while the velocity components are Finally, we quantify the accuracy of the derived moments by investigating the ratios Nout/Nin, Uout/Uin, and Tout/Tin for different input noise levels. In the next section we show how these ratios deviate from unity for increasing noise lev- els, revealing potential misestimations of the plasma bulk parameters when those are estimated from the moments of the constructed plasma VDFs. Ux,out = 1 Nout E Θ Φ 2E m3 × 2E m cosΘ cosΦ fout (E,Θ,Φ)E cosθθϕ, Ux,out = 1 Nout E Θ Φ 2E m3 × 2E m cosΘ cosΦ fout (E,Θ,Φ)E cosθθϕ, (8 U 1 2E (8) Eﬀects of noise on the accuracy of plasma bulk parameters derived from velocity moments... Page 3 of 9 3 Page 3 of 9 3 Fig. 1 Schematic of our concept instrument’s ﬁeld-of-view. (a) Elevation direction bins and (b) azimuth sectors In Fig. 2, we show three examples of simulated counts C (E,Θ,Φ), considering the same distribution function for the plasma protons, but different background noise levels. Left panels show the logarithm of the number of counts log10(C) as a function of the logarithm of the sampled energy log10(E) and elevation angle Θ, obtained at the azimuth sector centered at Φ = 9◦. Right panels show log10(C) as a function of log10(E) and azimuth angle Φ, obtained at elevation bins centered at Θ = 0◦. For the spe- ciﬁc plasma parameters, the simulated counts peak at ∼1000 counts. Top panels correspond to noise level nl = 0 counts, × 2E m cosΘ sinΦ fout (E,Θ,Φ)E cosθθϕ, (9) and Uz,out = 1 Nout E Θ Φ 2E m3 × 2E m sinΘ fout (E,Θ,Φ)E cosθθϕ, (10) Fig. 1 Schematic of our concept instrument’s ﬁeld-of-view. (a) Elevation direction bins and (b) azimuth sectors Fig. 1 Schematic of our concept instrument’s ﬁeld-of-view. (a) Elevation direction bins and (b) azimuth sectors In Fig. 2, we show three examples of simulated counts C (E,Θ,Φ), considering the same distribution function for the plasma protons, but different background noise levels. Left panels show the logarithm of the number of counts log10(C) as a function of the logarithm of the sampled energy log10(E) and elevation angle Θ, obtained at the azimuth sector centered at Φ = 9◦. Right panels show log10(C) as a function of log10(E) and azimuth angle Φ, obtained at elevation bins centered at Θ = 0◦. For the spe- ciﬁc plasma parameters, the simulated counts peak at ∼1000 counts. Top panels correspond to noise level nl = 0 counts, middle panels to nl = 1 counts, and bottom panels to nl = 10 counts. In Fig. 2, we show three examples of simulated counts C (E,Θ,Φ), considering the same distribution function for the plasma protons, but different background noise levels. Left panels show the logarithm of the number of counts log10(C) as a function of the logarithm of the sampled energy log10(E) and elevation angle Θ, obtained at the azimuth sector centered at Φ = 9◦. Right panels show log10(C) as a function of log10(E) and azimuth angle Φ, obtained at elevation bins centered at Θ = 0◦. 2 Methodology First, we consider a typical top-hat electrostatic analyzer instrument, obtaining measurements of space plasma parti- cles. Our concept instrument is similar to the design of So- lar Wind Analyser’s Proton Alpha Sensor (SWA-PAS, Owen et al. 2020) on board Solar Orbiter and measures number of particles C in speciﬁc energy steps E, elevation directions Θ, and azimuth directions Φ. As we show in Fig. 1, the elevation direction Θ is deﬁned by the angle between the particle velocity vector and the top-hat plane (x-y plane) of the instrument, while the azimuth direction Φ is deﬁned by the angle between the particle velocity vector projection on the top-hat plane and the x-axis. The ﬁeld-of-view (FOV) of the instrument covers elevation directions from −22.5° to +22.5° in 9 Θ steps, while the azimuth direction is re- solved in 11 azimuth sectors, covering the range from −24° to +42° (see Fig. 1). Finally, our concept instrument mea- sures particles with energies between 200 eV and 20 keV. This energy range is covered in 96, exponentially spread steps E (see also Owen et al. 2020 and the instrument mod- els by Nicolaou et al. 2018, 2019). To account for the statistical measurement error, we model the signal counts in each E, Θ, Φ pixel of the instru- ment, assuming that each measurement Csignal appears with a probability that follows the Poisson distribution function, such that P Csignal = C Csignal exp e−Cexp Csignal! . (3) (3) Then, we add a uniform background noise of a certain level nl to each measurement. The noise also follows the Poisson distribution Then, we add a uniform background noise of a certain level nl to each measurement. The noise also follows the Poisson distribution P (Cnoise) = nCnoise l e−nl Cnoise! , (4) (4) so that the ﬁnal measurement in each pixel is (5) C (E,Θ,Φ) = Csignal (E,Θ,Φ) + Cnoise (E,Θ,Φ). (5) Eﬀects of noise on the accuracy of plasma bulk parameters derived from velocity moments... Eﬀects of noise on the accuracy of plasma bulk parameters derived from velocity moments... 3 Results Uy,out = 1 Nout E Θ Φ 2E m3 In the top panel of Fig. 3, we show histograms of Nout, Uout, and Tout for 1000 simulated measurements for the in- Page 4 of 9 G. Nicolaou Fig. 2 Modeled measurements of plasma protons with Nin = 35 cm−3, ⃗Uin ∼440 kms−1 along Θ = 0P ◦and Φ = 9◦, and Tin = 20 eV, for three different levels of uniform background noise. (a) C(E,Θ,Φ = 9◦) and (b) C(E,Θ = 0◦,Φ) for nl = 0 counts. (c) C(E,Θ,Φ = 9◦) and (d) C(E,Θ = 0◦,Φ) for nl = 1 counts. (e) C(E,Θ,Φ = 9◦) and (f) C(E,Θ = 0◦,Φ) for nl = 10 counts put parameters we deﬁne in Sect. 2 and noise level nl = 0.1 counts. The bottom panels of Fig. 3, show the histograms of Nout/Nin, Uout/Uin, and Tout/Tin for the same simulated samples. According to this result, even when the noise level is considerably smaller than the peak of the signal (∼1000 counts), the calculation of the VDF moments misestimates signiﬁcantly the plasma temperature (by a factor of ∼1.37). In Fig. 4, we plot the ratios Nout/Nin, Uout/Uin, and T /T f diff t i l l i th i t −→ U in ∼440 kms−1 along Θ = 0◦and Φ = 9◦, and Tin = 20 eV). The main plots show the noise level in logarithmic scale. Inside each panel however, we show the same plot, using a linear scale. First, our results show that for non-zero noise, all the plasma parameters are overestimated. This is expected, considering that the E steps of our instrument in- crease exponentially and the uniform background noise will be misinterpreted as part of the VDF, increasing the overall VDF i ll i th hi h S d ﬁd Fig. 2 Modeled measurements of plasma protons with Nin = 35 cm−3, ⃗Uin ∼440 kms−1 along Θ = 0P ◦and Φ = 9◦, and Tin = 20 eV, for three different levels of uniform background noise. (a) C(E,Θ,Φ = 9◦) and (b) C(E,Θ = 0◦,Φ) for nl = 0 counts. (c) C(E,Θ,Φ = 9◦) and (d) C(E,Θ = 0◦,Φ) for nl = 1 counts. (e) C(E,Θ,Φ = 9◦) and (f) C(E,Θ = 0◦,Φ) for nl = 10 counts Fig. 3 Results 2 Modeled measurements of plasma protons with Nin = 35 cm−3, ⃗Uin ∼440 kms−1 along Θ = 0P ◦and Φ = 9◦, and Tin = 20 eV, for three different levels of uniform background noise. (a) C(E,Θ,Φ = 9◦) and (b) C(E,Θ = 0◦,Φ) for nl = 0 counts. (c) C(E,Θ,Φ = 9◦) and (d) C(E,Θ = 0◦,Φ) for nl = 1 counts. (e) C(E,Θ,Φ = 9◦) and (f) C(E,Θ = 0◦,Φ) for nl = 10 counts put parameters we deﬁne in Sect. 2 and noise level nl = 0.1 counts. The bottom panels of Fig. 3, show the histograms of Nout/Nin, Uout/Uin, and Tout/Tin for the same simulated samples. According to this result, even when the noise level is considerably smaller than the peak of the signal (∼1000 counts), the calculation of the VDF moments misestimates signiﬁcantly the plasma temperature (by a factor of ∼1.37). In Fig. 4, we plot the ratios Nout/Nin, Uout/Uin, and Tout/Tin for different noise levels, using the same input plasma conditions in our simulations (Nin = 35 cm−3, −→ U in ∼440 kms−1 along Θ = 0◦and Φ = 9◦, and Tin = 20 eV). The main plots show the noise level in logarithmic scale. Inside each panel however, we show the same plot, using a linear scale. First, our results show that for non-zero noise, all the plasma parameters are overestimated. This is expected, considering that the E steps of our instrument in- crease exponentially and the uniform background noise will be misinterpreted as part of the VDF, increasing the overall VDF, especially in the high energy range. Second, we ﬁnd that among the three bulk parameters we examine here, the put parameters we deﬁne in Sect. 2 and noise level nl = 0.1 counts. The bottom panels of Fig. 3, show the histograms of Nout/Nin, Uout/Uin, and Tout/Tin for the same simulated samples. According to this result, even when the noise level is considerably smaller than the peak of the signal (∼1000 counts), the calculation of the VDF moments misestimates signiﬁcantly the plasma temperature (by a factor of ∼1.37). In Fig. 4, we plot the ratios Nout/Nin, Uout/Uin, and Tout/Tin for different noise levels, using the same input plasma conditions in our simulations (Nin = 35 cm−3, In Fig. 3 Results 4, we plot the ratios Nout/Nin, Uout/Uin, and Tout/Tin for different noise levels, using the same input plasma conditions in our simulations (Nin = 35 cm−3, Eﬀects of noise on the accuracy of plasma bulk parameters derived from velocity moments... Page 5 of 9 3 Fig. 3 Histograms of (top) Nout, Uout, and Tout, and (bottom) Nout/Nin, Uout/Uin, and Tout/Tin, derived from the analysis of 1000 simulated measurement samples considering plasma with Nin = 35 cm−3, ⃗Uin ∼440 kms−1 along Θ = 0◦and Φ = 9◦, and Tin = 20 eV and noise level nl = 0.1 counts 35 cm−3, ⃗Uin ∼440 kms−1 along Θ = 0◦and Φ = 9◦, and Tin = 20 eV and noise level nl = 0.1 counts Fig. 3 Histograms of (top) Nout, Uout, and Tout, and (bottom) Nout/Nin, Uout/Uin, and Tout/Tin, derived from the analysis of 1000 simulated measurement samples considering plasma with Nin = Nout as a function of the measured counts: Nout as a function of the measured counts: plasma temperature suffers the largest inaccuracies, while bulk speed is affected the least. More speciﬁcally, the tem- perature is overestimated by a factor of ∼18 when the noise level is ∼10 counts (1% of the signal peak). For the same nl, the calculated density is overestimated by a factor of ∼2.3 and the bulk speed by a factor of ∼1.03. Interestingly, even a typical noise level, such as 0.1% of the peak (nl = 1 count in our simulations), has a signiﬁcant impact on the calculations of the bulk parameters of the plasma we model here. Nout = E Θ Φ √m 2E3 C(E,Θ,Φ) Gf τ E cosθθϕ, (13) (13) which is the same equation as in Nicolaou and Livadi- otis 2020, expressed in terms of particle energy instead of particle speed. Since C (E,Θ,Φ) = Csignal (E,Θ,Φ) + Cnoise(E,Θ,Φ), we split in two terms: The misestimation of the bulk parameters depends on the input plasma parameters as well, in a rather complicated way (se also Nicolaou et al. 2018, 2019, 2020a). Here, we do not provide a detailed quantiﬁcation of the accuracy of the cal- culated moments for a wide range of plasmas. Instead, we want to expose potential inaccuracies that can result from noise, even when the noise level seems insigniﬁcant com- pared to the signal. 3 Results In addition, we demonstrate method- ologies suitable to quantify the noise effects for any input plasma VDF, observed by any other instrument operating similarly to our concept instrument. In the next section, we discuss our results further and we evaluate a method that re- duces signiﬁcantly potential inaccuracies. Nout = E Θ Φ √m 2E3 Csignal(E,Θ,Φ) Gf τ E cosθθϕ + E Θ Φ √m 2E3 Cnoise(E,Θ,Φ) Gf τ E cosθθϕ. E Θ Φ (14) (14) The ﬁrst term on the right-hand side is the actual plasma density (Nin), while the second term is the contribution from noise. We can then express Nout/Nin as: Nout/Nin Nout/Nin = 1 + E Θ Φ √m 2E3 Cnoise(E,Θ,Φ) Gf τ E cosθθϕ E Θ Φ √m 2E3 Csignal(E,Θ,Φ) Gf τ E cosθθϕ . (15) 4 Discussion Our results indicate that the calculation of the statistical moments requires a proper treatment of the observations in order to minimize the effects of noise, even when the noise level seems insigniﬁcant. Although on-ground analyses can investigate the 3D data properly and come-up with sophisti- cated noise removal tools, some missions require on-board moments calculations to enable high-time resolution science within the available telemetry, or to provide early solar wind estimations for space weather forecasting. One idea is to measure the noise level on-board, using a background anode (e g McComas et al 2017) which al- Page 6 of 9 Page 6 of 9 G. Nicolaou 3 Fig. 4 Ratios of output over input plasma parameters as functions of the noise level in the simulated plasma observations. Our simulations consider plasma with Nin=35 cm−3, ⃗Uin ∼440 kms−1 along Θ = 0◦and Φ = 9◦, and Tin = 20 eV. Each data point is the average ratio over 1000 samples and the error bars are the associated standard deviations. Within each panel we show the same plot using linear scale for the x-axis Fig. 5 Ratio of output over input plasma density as a function of noise level as predicted in our simulations (black circles), along with the an- alytical expression (dashed red line) Fig. 5 Ratio of output over input plasma density as a function of noise level as predicted in our simulations (black circles) along with the an- value. This misestimation can be reduced by using beam- tracking methods (e.g., De Keyser et al. 2018), or any sim- ilar method which excludes from the analysis pixels away from the VDF peak. The successful use of these techniques in data-analyses depends on the ability to select the optimal energy and angular range of the observations. Nevertheless, even with the proper selection of pixels to analyze, we do not solve the problem entirely, as the analyzed parts of the VDFs are still polluted by noise. Our results indicate that the calculation of the statistical moments requires a proper treatment of the observations in order to minimize the effects of noise, even when the noise level seems insigniﬁcant. Although on-ground analyses can investigate the 3D data properly and come-up with sophisti- cated noise removal tools, some missions require on-board moments calculations to enable high-time resolution science within the available telemetry, or to provide early solar wind estimations for space weather forecasting. Fig. 4 Discussion (15) As it is expected, background noise in in-situ measurements of plasma particles, leads to erroneous calculations of the velocity moments of the constructed plasma particle VDFs. We quantify the errors of the calculations as functions of the noise level, using simulated plasma observations of a typi- cal solar wind plasma. Figure 4 shows that there is a linear relationship between the estimated plasma density and the background noise. By combining Eqs. (6) and (7), we get For the cases we examine here, Cnoise is a function of a uniform nl, as shown in Eq. (4). Then, for the same input plasma parameters, we expect a linear relationship between Nout/Nin, on average. In Fig. 5, we show our model results for Nout/Nin, along with the analytical function in Eq. (15). There is a perfect agreement between the model and ex- pected result. If someone follows a similar algebra for the higher order moments, will ﬁnd that the rest of the plasma 3 Page 6 of 9 G. Nicolaou Fig. 4 Ratios of output over input plasma parameters as functions of the noise level in the simulated plasma observations. Our simulations consider plasma with Nin=35 cm−3, ⃗Uin ∼440 kms−1 along Θ = 0◦and Φ = 9◦, and Tin = 20 eV. Each data point is the average ratio over 1000 samples and the error bars are the associated standard deviations. Within each panel we show the same plot using linear scale for the x-axis Fig. 5 Ratio of output over input plasma density as a function of noise level as predicted in our simulations (black circles), along with the an- alytical expression (dashed red line) parameters do not have a linear relationship with the noise level exactly as shown in Fig 4 value. This misestimation can be reduced by using beam- tracking methods (e.g., De Keyser et al. 2018), or any sim- ilar method which excludes from the analysis pixels away from the VDF peak. The successful use of these techniques in data-analyses depends on the ability to select the optimal energy and angular range of the observations. Nevertheless, even with the proper selection of pixels to analyze, we do not solve the problem entirely, as the analyzed parts of the VDFs are still polluted by noise. Eﬀects of noise on the accuracy of plasma bulk parameters derived from velocity moments... (Right) From top to bottom Nout/Nin, Uout/Uin, and Tout/Tin as functions of the input nl, calculated after removing the estimated noise level from the observations The moments are then calculated exactly as in Eqs. (7)–(12), using fout,corr. The right panels of Fig. 6, show the ratios Nout/Nin, Uout/Uin, and Tout/Tin as functions of the in- put nl, calculated after subtracting the estimated noise level from the measured (simulated) counts. The data-points cor- respond to the mean values over 1000 samples, while the error bars are the associated standard deviations. With this analysis, the mean values of the estimated bulk parameters do not deviate signiﬁcantly from the input, which is great improvement compared to the analysis of the raw counts. However, there is a statistical uncertainty (error bars) in- creasing with nl. Our methodology can estimate this uncer- tainty for any similar application in space and judge if it is a threat for a speciﬁc mission concept and goals. Such eval- uations are crucial for the success of missions employing in-situ plasma observations. the signal is spread over a large portion of the instrument’s energy and angular range, the median value will be affected by the signal and will be larger than the actual background counts. Here, we propose to estimate the noise level from the average number of counts obtained in an energy range, in which we don’t expect to measure any particles (e.g., largest E step). Typically, the energy tables of plasma instruments include E steps beyond the expected energy range of the measured particles. If the noise is quasi-uniform over the entire energy and angular range of the instrument, the noise level estimated from the background counts in a limited en- ergy range is a representative for the noise level in every E, Θ, Φ pixel. We validate such a method by estimating the noise level as the average number of counts obtained at the highest energy sampled by our concept instrument (96th en- ergy step E96 = 20 keV). In this case This study uses the concept of the uniform background noise. In real life however, the background noise may de- pend on energy and/or particle direction. For instance, ob- servations by the electron sensor of the Jovian Auroral Dis- tributions Experiment (McComas et al. 2017) on board Juno, reveal background noise in energies above ∼1 keV, caused by penetrating radiation and the detector’s internal noise. Eﬀects of noise on the accuracy of plasma bulk parameters derived from velocity moments... Page 7 of 9 3 Fig. 6 (Left) The noise level estimated from the average number of counts obtained in the highest E bin of the instrument. The red dashed line is the identity function y=x. (Right) From top to bottom Nout/Nin, Uout/Uin, and Tout/Tin as functions of the input nl, calculated after removing the estimated noise level from the observations the signal is spread over a large portion of the instrument’s energy and angular range, the median value will be affected by the signal and will be larger than the actual background counts. Here, we propose to estimate the noise level from the average number of counts obtained in an energy range, in which we don’t expect to measure any particles (e.g., largest E step). Typically, the energy tables of plasma instruments include E steps beyond the expected energy range of the measured particles. If the noise is quasi-uniform over the entire energy and angular range of the instrument, the noise level estimated from the background counts in a limited en- ergy range is a representative for the noise level in every E, Θ, Φ pixel. We validate such a method by estimating the noise level as the average number of counts obtained at the highest energy sampled by our concept instrument (96th en- The moments are then calculated exactly as in Eqs. (7)–(12), using fout,corr. The right panels of Fig. 6, show the ratios Nout/Nin, Uout/Uin, and Tout/Tin as functions of the in- put nl, calculated after subtracting the estimated noise level from the measured (simulated) counts. The data-points cor- respond to the mean values over 1000 samples, while the error bars are the associated standard deviations. With this analysis, the mean values of the estimated bulk parameters do not deviate signiﬁcantly from the input, which is great improvement compared to the analysis of the raw counts. However, there is a statistical uncertainty (error bars) in- creasing with nl. Our methodology can estimate this uncer- tainty for any similar application in space and judge if it is a threat for a speciﬁc mission concept and goals. Such eval- uations are crucial for the success of missions employing in-situ plasma observations. Fig. 6 (Left) The noise level estimated from the average number of counts obtained in the highest E bin of the instrument. The red dashed line is the identity function y=x. Fig. 6 (Left) The noise level estimated from the average number of counts obtained in the highest E bin of the instrument. The red dashed line is the identity function y=x. (Right) From top to bottom Nout/Nin, Uout/Uin, and Tout/Tin as functions of the input nl, calculated after removing the estimated noise level from the observations 4 Discussion 5 Ratio of output over input plasma density as a function of noise level as predicted in our simulations (black circles), along with the an- alytical expression (dashed red line) parameters do not have a linear relationship with the noise level, exactly as shown in Fig. 4. One idea is to measure the noise level on-board, using a background anode (e.g., McComas et al. 2017), which al- though operates regularly, it has a blocked FOV. Addition- ally, we could perform on-board estimations similar to previ- ous on-ground analyses, which have estimated background noise and ultra-violet contamination by calculating the me- dian number of counts over the measurement samples (e.g., Nilsson et al. 2012; Rojas-Castillo et al. 2018). This robust approach is reasonable when the useful signal is not spread over the majority of the instrument’s pixels. In cases where We ﬁnd that the plasma temperature is signiﬁcantly over- estimated even for noise levels that are by >1000 times smaller than the peak of the measured counts. This is reason- able, considering that the temperature moment is the mean kinetic energy of the particles in the plasma rest frame and background noise in energy and angular pixels away from the VDF peak is misinterpreted as measurement of parti- cles with high kinetic energies, increasing the mean energy ﬀects of noise on the accuracy of plasma bulk parameters derived from velocity moments... Eﬀects of noise on the accuracy of plasma bulk parameters derived from velocity moments... 5 Summary and conclusions Jeong, S-Y., Berˇciˇc, L.: Astrophys. J. 931, 118 (2022) We model observations of typical solar wind protons, ob- tained by a typical electrostatic analyzer. We consider dif- ferent levels of background noise in the observations and we use a standard analysis method to construct the underline proton velocity distribution functions. We then calculate the velocity moments of the constructed VDFs to determine the proton density, bulk speed, and temperature. Our analysis concludes that: Barabash, S., Lundin, R., Andersson, H., Brinkfeldt, K., Grigoriev, A., Gunell, H., Holmström, M., Yamauchi, M., Asamura, K., Bochsler, P., et al.: Space Sci. Rev. 126, 113 (2006) A., Gunell, H., Holmström, M., Yamauchi, M., Asamura, K., Bochsler, P., et al.: Space Sci. Rev. 126, 113 (2006) Barouch, E.: J. Geophys. Res. 82(10), 1493 (1977) Broiles, T.W., Livadiotis, G., Burch, J.L., Chae, K., Clark, G., Cravens, T.E., Davidson, R., Frahm, R.A., Fuselier, S.A., Goldstein, R., Henri, P., Madanian, H., Mandt, K., Mokashi, P., Pollock, C., Rah- mati, A., Samara, M., Schwartz, S.J.: J. Geophys. Res. 121, 7407 (2016) De Keyser, J., Lavraud, B., Pˇrech, L., Neefs, E., Berkenbosch, S., Beeckman, B., Fedorov, A., Marcucci, F.M., De Marco, R., Brienza, D.: Ann. Geophys. 36, 1285 (2018) • The presence of noise in typical in-situ measurements of 3D velocity distribution functions of plasma particles, leads to misestimations of the statistical moments. Even small noise levels (<1% of the signal peak) cause signif- icant inaccuracies. Therefore, derivations of plasma mo- ments must pay extra caution in reducing the effects of noise; Dialynas, K., Roussos, E., Regoli, L., Paranicas, C.P., Krimigis, S.M., Kane, M., Mitchell, D.G., Hamilton, D.C., Krupp, N., Carbary, J.F.: J. Geophys. Res. 123, 8066 (2018) Ebert, R.W., McComas, D.J., Elliott, H.A., Forsyth, R.J., Gosling, J.T.: J. Geophys. Res. 114, A01109 (2009) Elliott, H.A., McComas, D.J., Valek, P., Nicolaou, G., Weidner, S., Li- vadiotis, G.: Astron. Astrophys. Suppl. Ser. 223(2), 19 (2016) Kasper, J.C.: Solar Wind Plasma: Kinetic Properties and MicroInsta- bilities. PhD thesis, M (2003) • From the bulk parameters we examine here, plasma tem- perature is affected the most by noise, while bulk speed is affected the least; Livi, R., Goldstein, J., Burch, J.L., Crary, F., Rymer, A.M., Mitchell, D.G., Persoon, A.M.: J. Geophys. Res. 119, 3683 (2014) Marsch, E., Mühlhäuser, K.-H., Schwenn, R., Rosenbauer, H., Pilipp, W., Neubauer, F.M.: J. Geophys. Res. References Abraham, J.B., Owen, C.J., Verscharen, D., Bakrania, M., Stansby, D., Wicks, R.T., Nicolaou, G., Whittlesey, P.L., Agudelo Rueda, J.A., Jeong, S-Y., Berˇciˇc, L.: Astrophys. J. 931, 118 (2022) Eﬀects of noise on the accuracy of plasma bulk parameters derived from velocity moments... The noise level peaks at 2 Hz. In another example, Ion Mass Analyzer on-board Mars Express (Barabash et al. 2006) shows two types of noise, an independent of energy and elevation direction, and an energy dependent one. The Ion Composition Analyser (ICA, Nilsson et al. 2007) has simi- lar noise proﬁles and the noise background increases as the spacecraft gets closer to the Sun. In such cases, if the noise proﬁle is not characterized, or cannot be characterized by laboratory calibrations, someone may be able to construct it using ﬂight observations obtained in range of energies, an- gles, and environment conditions. Finally, it is important to nl,estimated = 1 99 9 i=1 11 j=1 C E96,Θi,Φj . (16) (16) In the left panel of Fig. 6, we show the estimated noise level as a function of the input noise level for simulations consid- ering Nin = 35 cm−3, −→ U in = 440 kms−1 along Θ = 0° and Φ = 9°, and Tin = 20 eV. Each data-point is the mean value over 1000 samples with the same input nl, while the error bar is the associated standard deviation. All the data-points are along the y=x line (red dashed), conﬁrming that the cal- culated noise level is accurate. Therefore, we can construct a corrected VDF using Eq. (6), after we subtract the estimated noise level from the raw counts, such that fout,corr. (E,Θ,Φ) = m2 C (E,Θ,Φ) −nl,estimated 2Gf E2τ . (17) (17) Page 8 of 9 G. Nicolaou 3 note that noise does not have impact only in the calculation of statistical moments. Nicolaou et al. 2022 use simulations and laboratory experiments to demonstrate and quantify the effects of background noise in the accuracy of plasma pa- rameters, derived by ﬁtting noisy observations with model distributions. 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/. 5 Summary and conclusions 87, 52 (1982) • In the case of uniform background noise, we can fairly es- timate the noise level from the average number of counts obtained in an energy where no signal is expected. Then, the signiﬁcant inaccuracies we demonstrate in this study can be reduced by subtracting the estimated noise level from the raw measurements. Mauk, B.H., Mitchell, D.G., McEntire, R.W., Paranicas, C.P., Roelof, E.C., Williams, D.J., Krimigis, S.M., Lagg, A.: J. Geophys. Res. 109, A09S12 (2004) McComas, D.J., Alexander, N., Allegrini, F., Bagenal, F., Beebe, C., Clark, G., et al.: Space Sci. Rev. 213, 547 (2017) Nicolaou, G., McComas, D.J., Bagenal, F., Elliott, H.A.: J. Geophys. Res. 119, 3463 (2014) Acknowledgements G.N. thanks Diana Rojas-Castillo and Hans Nils- son for useful discussions related to the topic of this study. Nicolaou, G., McComas, D.J., Bagenal, F., Elliott, H.A., Ebert, R.W.: Planet. Space Sci. 111, 116 (2015) Nicolaou, G., Livadiotis, G., Owen, C.J., Verscharen, D., Wicks, R.T.: Astrophys. J. 864, 3 (2018) Author contributions This is a single author paper. G.N. is responsible for the entire study. Author contributions This is a single author paper. G.N. is responsible for the entire study. Nicolaou, G., Verscharen, D., Wicks, R.T., Owen, C.J.: Astrophys. J. 886, 101 (2019) Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Nicolaou, G., Wicks, R.T., Rae, I.J., Kataria, D.O.: Space Weather 18, e2020SW002559 (2020a) Nicolaou, G., Wicks, R.T., Livadiotis, G., Verscharen, D., Owen, C., Kataria, D.: Entropy 22, 103 (2020b) Owen, C.J., Bruno, R., Livi, S., Louarn, P., Al Janabi, K., Allegrini, F., Amoros, C., Baruah, R., Barthe, A., Berthomier, M., et al.: Astron. Astrophys. 642, A16 (2020) Rojas-Castillo, D., Nilsson, H., Stenberg Wieser, G.: J. Geophys. Res. 123, 8806 (2018) Wilson, R.J., Bagenal, F., Persoon, A.M.: J. Geophys. Res. 122, 7256 (2017) Eﬀects of noise on the accuracy of plasma bulk parameters derived from velocity moments... Owen, C.J., Bruno, R., Livi, S., Louarn, P., Al Janabi, K., Allegrini, F., Amoros, C., Baruah, R., Barthe, A., Berthomier, M., et al.: Astron. Astrophys. 642, A16 (2020) Wilson, R.J., Bagenal, F., Persoon, A.M.: J. Geophys. Res. 122, 7256 (2017) p y Rojas-Castillo, D., Nilsson, H., Stenberg Wieser, G.: J. Geophys. Res. 123, 8806 (2018) Declarations Nicolaou, G., Wicks, R.T., Owen, C.J., Kataria, D.O., Chandrasekhar, A., Lewis, G.R., Verscharen, D., Fortunato, V., Mele, G., De- Marco, R., Bruno, R.: Astron. Astrophys. 656, A10 (2021) Competing interests The authors declare no competing interests. Competing interests The authors declare no competing interests. Nicolaou, G., Livadiotis, G.: Entropy 22, 541 (2020) 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, pro- vide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included Nicolaou, G., Allegrini, F., Livadiotis, G., Ebert, R.W.: Rev. Sci. In- strum. 93, 103305 (2022) Nilsson, H., Stenberg, G., Futaana, Y., Holmström, M., Barabash, S., Lundin, R., Edberg, N.J.T., Fedorov, A.: Earth Planets Space 64, 9 (2012) Nilsson, H., Lundin, R., Lundin, K., Barabash, S., Borg, H., Norberg, O., Fedorov, A., Sauvaud, J.-A., Koskinen, H., Kallio, E., et al.: Space Sci. Rev. 128, 671 (2007) Page 9 of 9 3 Publisher’s Note Springer Nature remains neutral with regard to juris- dictional claims in published maps and institutional afﬁliations. Rojas-Castillo, D., Nilsson, H., Stenberg Wieser, G.: J. Geophys. Res. 123, 8806 (2018) Wilson, R.J., Bagenal, F., Persoon, A.M.: J. Geophys. Res. 122, 7256 (2017)
https://openalex.org/W2866577345	https://www.nature.com/articles/s41598-018-29030-4.pdf	English	null	O2-inducible H2O2-forming NADPH oxidase is responsible for the hyper O2 sensitivity of Bifidobacterium longum subsp. infantis	Scientific reports	2,018	cc-by	9,131	O2-inducible H2O2-forming NADPH oxidase is responsible for the hyper O2 sensitivity of Bifidobacterium longum subsp. infantis Received: 6 February 2018 Accepted: 4 July 2018 Published: xx xx xxxx Bifidobacteria are beneficial anaerobes, and their O2 sensitivity levels differ among species as a function of unknown molecular mechanisms. Bifidobacterium longum subspecies infantis (B. infantis), a predominant colonizer of the gastrointestinal tract of infants, showed a hyper O2-sensitive growth profile with accompanying a production of H2O2. In this study, we characterized an NADPH oxidase as a key enzyme responsible for this microbe’s hyper O2 sensitivity. A dominant active elution peak of H2O2-forming NADPH oxidase activity was detected in the first step of column chromatography, and the purified NADPH oxidase (NPOX) was identified as a homolog of nitroreductase family proteins. The introduction of the gene encoding B. infantis NPOX (npoxA) into O2-tolerant Bifidobacterium minimum made the strain O2 sensitive and allowed it to produce H2O2. Knockout of the npoxA gene in B. infantis decreased the production of H2O2 and mitigated its B. infantis hyper O2 sensitivity. A transcript of B. infantis npoxA is induced by O2, suggesting that the aerobic production of toxic H2O2 is functionally conserved in B. infantis. Oxygen (O2) has a negative effect on the growth of anaerobes1,2; however, the habitats of anaerobes, such as the gastrointestinal tract, are frequently contaminated with O2 that has been dissolved in food and beverages. Therefore, anaerobes must possess systems to adapt to aerated environments for survival in nature3,4. Recent studies have identified an O2-inducible O2- and ROS-reducing enzyme complex in obligate anaerobes such as sulfate-reducing bacteria5,6, Clostridium7,8, and Bacteroides9. These findings indicate that these obligate anaer- obes possess excellent systems to avoid oxidative damage for maintaining their obligate anaerobiosis in aerated environments.i Several gut anaerobes such as O2-sensitive bifidobacteria and lactic acid bacteria are known to produce H2O2, a toxic reactive oxygen species (ROS), after exposure to O2, which inhibits the cell growth10–12. These anaerobes do not harbor the above-mentioned genes encoding the ROS-reducing enzyme complex in their genomes. NAD(P)H peroxidase or alkylhydroperoxide (Ahp) reductase systems (AhpF-AhpC or thioredoxin reductase–AhpC sys- tem), which detoxify H2O2 to H2O using NAD(P)H in aerobes and facultatively anaerobes13–16, have been identi- fied in these anaerobes17–19; however, exogenous H2O2 production has been detected in these anaerobes20,21. These results suggest that H2O2 production and decomposition are controlled in vivo and play a role in the biological functions of these bacteria in aerated environments. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports O2-inducible H2O2-forming NADPH oxidase is responsible for the hyper O2 sensitivity of Bifidobacterium longum subsp. infantis H2O2 is highly toxic for a bacterium’s own cells; thus, the reason for this H2O2 production remains unclear22,23.i p Members of the genus Bifidobacterium are gram-positive, catalase-negative anaerobes that are known to be beneficial to human health24–26. The degree of O2 sensitivity differs among species and strains in the genus11,27–35. de Vries and Stouthamer (1969) proposed that O2-sensitive Bifidobacterium species produce H2O2 under 1Department of Molecular Microbiology, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo, 156-8502, Japan. 2Department of Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo, 156-8502, Japan. 3Department of Molecular Genetics, Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto, 607-8414, Japan. 4Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan. 5United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan. Correspondence and requests for materials should be addressed to S.K. (email: kawashin@ nodai.ac.jp) Scientific REPOrTS \| (2018) 8:10750 \| DOI:10.1038/s41598-018-29030-4 Scientific REPOrTS \| (2018) 8:10750 \| DOI:10.1038/s41598-018-29030-4 1 www.nature.com/scientificreports/ Figure 1. Growth of Bifidobacterium infantis JCM 1222T in liquid shake culture at various O2 concentrations. Circles, 100% N2 cultures; squares, 5% O2/95% N2 cultures; triangles, 10% O2/90% N2 cultures; diamonds, 20% O2/80% N2 cultures. Data represent the average ± SD of three independent cultures. Figure 1. Growth of Bifidobacterium infantis JCM 1222T in liquid shake culture at various O2 concentrations. Circles, 100% N2 cultures; squares, 5% O2/95% N2 cultures; triangles, 10% O2/90% N2 cultures; diamonds, 20% O2/80% N2 cultures. Data represent the average ± SD of three independent cultures. aerobic growth conditions through a reaction catalyzed by NADH oxidase activity, which can be detected in cell extracts11. According to our studies using liquid shaking cultures at different O2 concentrations, bifidobacterial strains can be classified into four groups: O2 hypersensitive (5% O2-sensitive), O2-sensitive (10% O2-sensitive), O2-tolerant (21% O2-tolerant), and O2-hypertolerant (microaerophilic)33,36,37. H2O2 production has been detected in strains belonging to the O2-hypersensitive and O2-sensitive groups when cell growth was inhibited by O2 33. The majority of O2-tolerant and hyper O2-tolerant strains have been isolated from non-human sources such as a honeybee hindgut38 and bovine rumen39. Bifidobacterium asteroides (B. asteroides) was isolated from a honeybee hindgut and possesses a heme-catalase, which is a rare characteristic among bifidobacteria24,38,40,41. O2-inducible H2O2-forming NADPH oxidase is responsible for the hyper O2 sensitivity of Bifidobacterium longum subsp. infantis B. infantis has been reported as a champion colonizer in the gut microbiota of human infants45 and is reported to confer benefits to premature infants in terms of promoting anti-inflammatory activity and decreasing the risk of necrotizing enterocolitis46,47. Use of B. infantis as a bioactive probiotic for infants is highly anticipated; therefore we set out to identify the mechanisms underlying its hyper O sensitivity Here we identi i g p q In the present study, Bifidobacterium longum subspecies infantis (B. infantis) showed a hyper O2-sensitive growth profiles. B. infantis has been reported as a champion colonizer in the gut microbiota of human infants45 and is reported to confer benefits to premature infants in terms of promoting anti-inflammatory activity and decreasing the risk of necrotizing enterocolitis46,47. Use of B. infantis as a bioactive probiotic for infants is highly anticipated; therefore, we set out to identify the mechanisms underlying its hyper-O2 sensitivity. Here, we identi- fied an enzyme that produces H2O2 in B. infantis. The role of this protein and the mechanisms and evolutionary significance of H2O2 production in this bacterium are discussed. O2-inducible H2O2-forming NADPH oxidase is responsible for the hyper O2 sensitivity of Bifidobacterium longum subsp. infantis Several reports have revealed that the addition of catalase to the culture medium11,33 or the introduction of ROS-detoxifying enzymes into O2-sensitive Bifidobacterium strains42,43 improved their aerobic growth, indicating that H2O2 pro- duction strongly correlates with the inhibition of aerobic growth. With respect to the enzymes that produce H2O2 in bifidobacteria, an H2O2-forming NADH oxidase was identified in an O2-sensitive strain of Bifidobacterium bifi- dum (B. bifidum), a type species in the genus Bifidobacterium, and the purified enzyme was identified as a b-type dihydroorotate dehydrogenase (DHOD), which is the enzyme that catalyzes oxidation of dihydroorotate to oro- tate in pyrimidine biosynthesis44. This enzyme is suggested to be involved in H2O2 production in B. bifidum, but its function has not been confirmed in vivo because of the lack of a gene disruption technique for this bacterium.i aerobic growth conditions through a reaction catalyzed by NADH oxidase activity, which can be detected in cell extracts11. According to our studies using liquid shaking cultures at different O2 concentrations, bifidobacterial strains can be classified into four groups: O2 hypersensitive (5% O2-sensitive), O2-sensitive (10% O2-sensitive), O2-tolerant (21% O2-tolerant), and O2-hypertolerant (microaerophilic)33,36,37. H2O2 production has been detected in strains belonging to the O2-hypersensitive and O2-sensitive groups when cell growth was inhibited by O2 33. The majority of O2-tolerant and hyper O2-tolerant strains have been isolated from non-human sources such as a honeybee hindgut38 and bovine rumen39. Bifidobacterium asteroides (B. asteroides) was isolated from a honeybee hindgut and possesses a heme-catalase, which is a rare characteristic among bifidobacteria24,38,40,41. Several reports have revealed that the addition of catalase to the culture medium11,33 or the introduction of ROS-detoxifying enzymes into O2-sensitive Bifidobacterium strains42,43 improved their aerobic growth, indicating that H2O2 pro- duction strongly correlates with the inhibition of aerobic growth. With respect to the enzymes that produce H2O2 in bifidobacteria, an H2O2-forming NADH oxidase was identified in an O2-sensitive strain of Bifidobacterium bifi- dum (B. bifidum), a type species in the genus Bifidobacterium, and the purified enzyme was identified as a b-type dihydroorotate dehydrogenase (DHOD), which is the enzyme that catalyzes oxidation of dihydroorotate to oro- tate in pyrimidine biosynthesis44. This enzyme is suggested to be involved in H2O2 production in B. bifidum, but its function has not been confirmed in vivo because of the lack of a gene disruption technique for this bacterium. In the present study, Bifidobacterium longum subspecies infantis (B. infantis) showed a hyper O2-sensitive growth profiles. Results Data represent the average ± SD of three independent cultures. Table 1. H2O2 accumulation of B. infantis and B. minimum under 0% to 20% O2 conditions. aH2O2 accumulation in the medium (24 h). bVector pKKT427 was transformed into B. minimum. cThe B. infantis npoxA gene was cloned in pKKT427 and transformed into B. minimum. dND, not detected (less than 3 µM). eData in parentheses are close to the reliable detection limit. Data represent the average ± SD of three independent cultures. Table 1. H2O2 accumulation of B. infantis and B. minimum under 0% to 20% O2 conditions. aH2O2 accumulation in the medium (24 h). bVector pKKT427 was transformed into B. minimum. cThe B. infantis npoxA gene was cloned in pKKT427 and transformed into B. minimum. dND, not detected (less than 3 µM). eData in parentheses are close to the reliable detection limit. Data represent the average ± SD of three independent cultures. Table 1. H2O2 accumulation of B. infantis and B. minimum under 0% to 20% O2 conditions. aH2O2 accumulation in the medium (24 h). bVector pKKT427 was transformed into B. minimum. cThe B. infantis npoxA gene was cloned in pKKT427 and transformed into B. minimum. dND, not detected (less than 3 µM). eData in parentheses are close to the reliable detection limit. Data represent the average ± SD of three independent cultures. Table 1. H2O2 accumulation of B. infantis and B. minimum under 0% to 20% O2 conditions. aH2O2 bh Table 1. H2O2 accumulation of B. infantis and B. minimum under 0% to 20% O2 conditions. aH2O2 bh accumulation in the medium (24 h). bVector pKKT427 was transformed into B. minimum. cThe B. infantis npoxA gene was cloned in pKKT427 and transformed into B. minimum. dND, not detected (less than 3 µM). eData in parentheses are close to the reliable detection limit. Data represent the average ± SD of three independent cultures. eData in parentheses are close to the reliable detection limit. Data represent the average ± SD of three independent cultures. Figure 2. (A) Chromatographic elution profiles of B. infantis NADH and NADPH oxidases. Cell extracts, after treatment with streptomycin sulfate and ammonium sulfate, were applied to a Butyl-TOYOPEARL column equilibrated with 1.2 M ammonium sulfate in 50 mM potassium phosphate buffer (pH 7.0). Results B. infantis showed a hyper O2-sensitive growth profile. The degree of O2 sensitivity of B. infantis JCM1222T (=ATCC15697 = DSM20088) was tested at several O2 concentrations in liquid shaking cultures. B. infantis grew well under anoxic conditions, and the optical density at 660 nm (OD660) reached 8 to 10 in the final growth stage. This level of growth was one of the most productive observed in MRS medium among the tested Bifidobacterium species36,37. Although several O2-sensitive bifidobacterial strains, including B. bifidum and Bifidobacterium longum (B. longum), grew well in liquid shaking cultures at 5% O2 33, the growth of B. infantis was found to be significantly inhibited at 5% O2 (Fig. 1). Therefore, we classified B. infantis as O2-hypersensitive. H2O2 production in the culture medium was detected at 10% and 20% O2 (Table 1). Elution profiles of NAD(P)H oxidase activity in the first step of column chromatography. We previously identified the enzyme fractions that are involved in H2O2 production in an O2-sensitive B. bifidum strain using a Butyl-TOYOPEARL column44. In this study, we performed the same experiments for B. infantis. Proteins in the cell free extracts from B. infantis were fractionated according to their hydrophobic properties using a Butyl-TOYOPEARL column (Fig. 2A). One dominant elution peak of NADPH-dependent oxidase activ- ity was detected, whereas NADH-dependent oxidase activity was detected as a minor elution peak. These major and minor peak fractions showed an H2O2-forming oxidase reaction in which stoichiometric production of H2O2 was detected by means of the reduction of O2. No significant NAD(P)H oxidase activity was detected in either the unbound fraction or the washed fractions in the first step of column chromatography. Scientific REPOrTS \| (2018) 8:10750 \| DOI:10.1038/s41598-018-29030-4 2 www.nature.com/scientificreports/ O2 (%) H2O2 (µM)a B. infantis wild type B. infantis ∆npoxA B. minimum pkkt427b B. minimum + npoxA in pKKT427c 0 NDd ND ND ND 5 ND ND ND ND 10 29 ± 10 (3 ± 1)e ND 172 ± 13 20 31 ± 6 14 ± 10 ND 169 ± 18 Table 1. H2O2 accumulation of B. infantis and B. minimum under 0% to 20% O2 conditions. aH2O2 accumulation in the medium (24 h). bVector pKKT427 was transformed into B. minimum. cThe B. infantis npoxA gene was cloned in pKKT427 and transformed into B. minimum. dND, not detected (less than 3 µM). eData in parentheses are close to the reliable detection limit. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Electron acceptora Relative activity (%)b O2 100 DCIP 358 FAD 229 FMN 74 Nitrobenzene 2090 Nitrofurazone 725 Table 2. Identification of electron acceptors of B. infantis NPOX. aRelative activity (%) was calculated in comparison with the activity of NADPH oxidase. Data are the average of two independent measurements that varied by less than 5%. Electron acceptora Relative activity (%)b O2 100 DCIP 358 FAD 229 FMN 74 Nitrobenzene 2090 Nitrofurazone 725 Table 2. Identification of electron acceptors of B. infantis NPOX. aRelative activity (%) was calculated in comparison with the activity of NADPH oxidase. Data are the average of two independent measurements that varied by less than 5%. Table 2. Identification of electron acceptors of B. infantis NPOX. aRelative activity (%) was calculated in comparison with the activity of NADPH oxidase. Data are the average of two independent measurements that varied by less than 5%. The gene encoding NPOX (designated as npoxA) was cloned from the B. infantis genome, and the amino acid sequence identity (% amino acid identity calculated from ClustalW alignment) showed 100% to that derived from BLON_RS12650 (Blon_2447). The translated product of B. infantis npoxA showed homology to proteins of the bacterial nitroreductase family and encodes a protein of 254 amino acids that shows ~35% identity to Escherichia coli oxygen-insensitive NADPH nitroreductase NfsA (accession number P17117)48, Bacillus subti- lis NADPH-dependent nitroreductase NfrA1 (32% identity, accession number P39605)49, and Vibrio harveyi NADPH-flavin oxidoreductase FRP1 (35% identity, accession number Q56691)50. B. subtilis NfrA1 is reported to catalyze H2O2-forming NADH oxidase activity51, and V. harveyi FRP1 is reported to catalyze an FMN reduc- tase reaction that is involved in a luciferase reaction that generates light in luminescent bacteria52. These NPOX homologs utilize NADH or NADPH or both as an electron donor to reduce substrates. The protein homologs are widely distributed in the genus Bifidobacterium. Homologous proteins were found in several B. longum strains (99% to 100%), Bifidobacterium breve (85% identity), B. bifidum (70% identity), Bifidobacterium minimum (B. minimum) (58% identity), and B. asteroides (52% identity). None of these proteins have been characterized in terms of function. Enzymatic properties. The purified enzyme showed high affinity for NADPH, rather than NADH, as the electron donor. The Km values for NADPH and NADH when O2 was used as an electron acceptor were 1.38 ± 0.12 and 113.3 ± 7.3 µM, respectively. The purified B. www.nature.com/scientificreports/ infantis NPOX showed a typical flavoprotein spectrum with absorbance maxima at 280, 373, and 444 nm (Fig. S2). The bound flavin was identified as FMN by HPLC analysis. In the NADPH oxidase reaction, stoichiometric production of H2O2 was detected following the reaction with O2; e.g., 1 µg enzyme consumed 28.3 nmol O2/5 min/mL, and 13.8 nmol O2/mL was produced after the addition of catalase. This result indicated that the purified enzyme reduces O2 by two reducing equivalents to H2O2. The puri- fied enzyme used flavins and nitro compounds as electron acceptors under anoxic conditions (Table 2). The pH optimum for the NADPH oxidase reaction was found to be 5.5–6.0. The temperature optimum was approximately 30–40 °C, and activity significantly decreased at temperatures above 40 °C. The specific activity of this protein under air-saturated conditions at 37 °C was 13.8 ± 4.3 U/mg protein. Transformation of the B. infantis npoxA into an O2-tolerant B. minimum strain. B. minimum DSM 20102T, which is an isolate from sewage53, showed an O2-tolerant profile32,37, and liquid shaking growth was not significantly inhibited in the presence of 20% O2 (Fig. 3A). Therefore, we classified B. minimum as a member of the O2-tolerant group and used this microbe for further study. The E. coli-Bifidobacterium shuttle vector pKKT427, originally developed for the transformation of B. longum strain 105-A54,55, was successfully transformed into B. minimum in this study; therefore, we used this vector to express B. infantis NPOX in B. min- imum. B. infantis npoxA was cloned into pKKT427, and then the growth of transformants was tested at different O2 concentrations. The production of H2O2 was not detected in a B. minimum wild-type strain carrying pKKT427 in the presence of O2 (Table 1). However, B. minimum carrying npoxA in pKKT427 showed an O2-sensitive growth profile in the presence of 10% and 20% O2 with accompanying the production of H2O2 (Fig. 3B, Table 1). These results indicated that B. infantis NPOX is responsible for the O2 sensitivity of and H2O2 production by B. minimum. The growth of a B. infantis npoxA gene knockout strain. Single-gene transformation of the O2-tolerant B. minimum strain significantly increased H2O2 production and O2 sensitivity. To determine the function of the npoxA product in B. infantis, we constructed an npoxA deletion mutant (∆npoxA) (Fig. S1), and then the growth of this mutant was compared with that of a wild-type strain of B. infantis. B. Results After sample loading, the bound proteins were eluted with a linear gradient of ammonium sulfate, from 1.2 to 0 M, dissolved in the same buffer. Black circles, NADH oxidase activity; white circles, NADPH oxidase activity; dashed line, ammonium sulfate concentration (conc.). (B) SDS-PAGE of the purified B. infantis NPOX protein. After electrophoresis, the gel was stained with Coomassie brilliant blue. The protein standards (lane 1) and purified protein (lane 2) are indicated, along with the corresponding molecular masses (indicated on the left in kDa). Figure 2. (A) Chromatographic elution profiles of B. infantis NADH and NADPH oxidases. Cell extracts, after treatment with streptomycin sulfate and ammonium sulfate, were applied to a Butyl-TOYOPEARL column equilibrated with 1.2 M ammonium sulfate in 50 mM potassium phosphate buffer (pH 7.0). After sample loading, the bound proteins were eluted with a linear gradient of ammonium sulfate, from 1.2 to 0 M, dissolved in the same buffer. Black circles, NADH oxidase activity; white circles, NADPH oxidase activity; dashed line, ammonium sulfate concentration (conc.). (B) SDS-PAGE of the purified B. infantis NPOX protein. After electrophoresis, the gel was stained with Coomassie brilliant blue. The protein standards (lane 1) and purified protein (lane 2) are indicated, along with the corresponding molecular masses (indicated on the left in kDa). Purification and characterization of the NADPH oxidase in B. infantis. The fractions that showed NADPH oxidase activity were collected and purified by additional column chromatography steps. At each purification step, only one predominant active elution peak was detected. After the final step of affinity chromatography, the purified fractions appeared as a single band (29 kDa) by SDS-PAGE (Fig. 2B). The N-terminal sequence of the purified NADPH oxidase (NPOX) was sequenced, and the amino acid sequence (MVTNATIEALLGRRSIRKFK) showed 100% identity with the N-terminal sequence of the Blon_2447 gene product of B. infantis ATCC15697 in the genome database. Scientific REPOrTS \| (2018) 8:10750 \| DOI:10.1038/s41598-018-29030-4 3 www.nature.com/scientificreports/ infantis ∆npoxA grew well at 5% O2, and H2O2 production was significantly decreased at 10% and 20% O2 (Fig. 4, Table 1). These results indicated that the npoxA product contributes to both aerobic growth inhibition and H2O2 production in B. infantis. Candidates for the production of residual H2O2 were the minor active fractions of the NADH-dependent H2O2-forming oxidase (Fig. 2A). Expression profiles of genes encoding B. infantis NADPH oxidase and alkylhydroperoxide reductase. To determine the effect of O2 on the expression of the gene encoding B. infantis NADPH oxi- dase (npoxA), we performed a Northern blot analysis. The result showed that B. infantis npoxA was significantly upregulated within 30 min after the start of 5% O2 aeration (Fig. 5B). We also determined the expression of genes encoding alkylhydroperoxide reductase (Ahp), which is an enzyme complex composed of AhpF and AhpC that detoxifies ROS. TrxR is an AhpF homolog in B. infantis, and ahpC-trxR genes are tandemly located in the genome Scientific REPOrTS \| (2018) 8:10750 \| DOI:10.1038/s41598-018-29030-4 4 www.nature.com/scientificreports/ Figure 3. Growth of Bifidobacterium minimum DSM 20102T in liquid shake culture at various O2 concentrations with (B) or without (A) the B. infantis npoxA gene in the vector pKKT427. Circles, 100% N2 cultures; squares, 5% O2/95% N2 cultures; triangles, 10% O2/90% N2 cultures; diamonds, 20% O2/80% N2 cultures. Data represent the average ± SD of three independent cultures. Figure 3. Growth of Bifidobacterium minimum DSM 20102T in liquid shake culture at various O2 concentrations with (B) or without (A) the B. infantis npoxA gene in the vector pKKT427. Circles, 100% N2 cultures; squares, 5% O2/95% N2 cultures; triangles, 10% O2/90% N2 cultures; diamonds, 20% O2/80% N2 cultures. Data represent the average ± SD of three independent cultures. Figure 4. Growth of the ∆npoxA mutant of B. infantis in liquid shake culture at various O2 concentrations. Circles, 100% N2 cultures; squares, 5% O2/95% N2 cultures; triangles, 10% O2/90% N2 cultures; diamonds, 20% O2/80% N2 cultures. Data represent the average ± SD of four independent experiments. Figure 4. Growth of the ∆npoxA mutant of B. infantis in liquid shake culture at various O2 concentrations. Circles, 100% N2 cultures; squares, 5% O2/95% N2 cultures; triangles, 10% O2/90% N2 cultures; diamonds, 20% O2/80% N2 cultures. Data represent the average ± SD of four independent experiments. (Fig. 5A). Northern analyses indicated that B. www.nature.com/scientificreports/ infantis ahpC was strongly upregulated within 10 minutes after the start of aeration (Fig. 5B). These results indicate that B. infantis express the genes for ROS production and for ROS detoxification in response to O2 stress. (Fig. 5A). Northern analyses indicated that B. infantis ahpC was strongly upregulated within 10 minutes after the start of aeration (Fig. 5B). These results indicate that B. infantis express the genes for ROS production and for ROS detoxification in response to O2 stress. Discussionh The growth of bifidobacterial strains is inhibited by O2, and this inhibition has been shown to occur in conjunc- tion with H2O2 production. To date, b-type DHOD from an O2-sensitive strain of B. bifidum, has been identi- fied as the only purified H2O2-forming NADH oxidase in the genus Bifidobacterium. In this study, we detected a dominant H2O2-forming NADPH oxidase activity peak in an O2-hypersensitive strain of B. infantis. This NADPH-specific peak was not detected in B. bifidum using the same hydrophobic column chromatography44, thus suggesting that the mechanisms of O2 sensitivity in these O2-sensitive strains differ from each other.i gg g 2 y 2f Purified B. infantis H2O2-forming NPOX showed high similarity (~30% identity) with proteins of the bac- terial nitroreductase family. B. infantis NPOX exhibited nitroreductase- and flavin reductase-activity under Scientific REPOrTS \| (2018) 8:10750 \| DOI:10.1038/s41598-018-29030-4 Scientific REPOrTS \| (2018) 8:10750 \| DOI:10.1038/s41598-018-29030-4 5 www.nature.com/scientificreports/ p / Figure 5. Genome structures and gene expression profiles of B. infantis npoxA and ahpC. (A) Genome structure of the npoxA (upper) and the ahpC (lower). (B) Northern blot analyses of 5 µg of B. infantis total RNA probed with npoxA or ahpC. 0, immediately before the start of aeration with 5% O2 in the mid-exponential phase; 10, after 10 min; 30, after 30 min. The estimated sizes of the observed transcripts are indicated on the right. Ethidium bromide staining of the ribosomal RNA (rRNA) to confirm equal RNA loading is shown below the autoradiogram. The membranes were reused for reprobing different probes. Figure 5. Genome structures and gene expression profiles of B. infantis npoxA and ahpC. (A) Genome structure of the npoxA (upper) and the ahpC (lower). (B) Northern blot analyses of 5 µg of B. infantis total RNA probed with npoxA or ahpC. 0, immediately before the start of aeration with 5% O2 in the mid-exponential phase; 10, after 10 min; 30, after 30 min. The estimated sizes of the observed transcripts are indicated on the right. Ethidium bromide staining of the ribosomal RNA (rRNA) to confirm equal RNA loading is shown below the autoradiogram. The membranes were reused for reprobing different probes. anoxic conditions. The role of B. infantis H2O2-forming NPOX under anaerobic conditions is unclear; however, based on the O2-inducible gene expression profile, NPOX is expected to be functionally involved in the aer- obic life of B. infantis. Scientific REPOrTS \| (2018) 8:10750 \| DOI:10.1038/s41598-018-29030-4 Discussionh Similar expression of a transcript, from a gene that encodes an NPOX ortholog (Gene ID: BL0139), was observed by Oberg et al. in microarray data from the H2O2-sensitive strain B. longum D2975 and the H2O2-tolerant strain B. longum NCC270556. The NPOX homolog transcript was induced only in the H2O2-sensitive D2975 strain under oxidative stress. To clarify the expression of NPOX and its correlation with bifidobacterial O2 sensitivity, it would be desirable to compare the expression profiles and kinetics of NPOX homologs in other O2-tolerant and O2-sensitive strains37.i g 2 2 H2O2 is highly toxic to cells, and therefore, Bifidobacterium strains need to conserve systems to reduce the amount of H2O2 to avoid cell death. Alkylhydroperoxide reductase, a component of the AhpF-AhpC system that is known to decompose H2O2 efficiently, is widely conserved in bifidobacterial genomes. Although the function of this enzyme complex has not been characterized in the genus Bifidobacterium, the upregulation of ahpC (or the protein AhpC) in response to O2 or H2O2 stress has been reported in strains such as Bifidobacterium animalis subsp. lactis35 and B. longum56. These data suggested that ahpC is involved in the oxidative stress protection of bifidobacterial strains. In the present study, B. infantis induced ahpC within 10 minutes after exposure to O2. Interestingly, the induction of ahpC was more rapid than that of B. infantis npoxA (Fig. 5). This gene expression program is considered to be important for the strain to rapidly prepare the defense system against the generation of H2O2. 2 2 In this study, knockout of npoxA in B. infantis enabled the strain to grow under microaerobic conditions, with a decrease in H2O2 production under aerobic conditions. Improvement of bacterial O2 sensitivity by single-gene knockout has been demonstrated in the obligate anaerobe Bacteroides fragilis, with deletion of oxe enabling the strain to grow under micro-oxic conditions (as high as 2% O2)57. Although the function of the oxe gene product has not yet been identified, a mutation in oxe enhanced the H2O2-scavenging activity of the mutant relative to that of the wild type. The reason for the production of a toxic H2O2 in anaerobes has been unknown; however, it is obvious that both B. fragilis and B. infantis control the production and degradation of H2O2 according to their own genetic programs. g p g We conclude here that B. Methods Bacterial strain and growth conditions. B. infantis (Bifidobacterium longum subsp. infantis) JCM 1222T (=ATCC 15697T, DSM 20088T) was used in this study. B. infantis was grown anaerobically at 37 °C in modified MRS medium (pH 6.7). The culturing conditions under several O2 concentrations were as described previously33. Chemicals. All chemicals were of analytical grade. ß-NADPH, ß-NADH, FAD, FMN, riboflavin, nitroben- zene, nitrofurazone, and 2,6-dichlorobenzenoneindophenol (DCIP) were from Sigma. Water was prepared with an Ellix-10 Milli-Q ultra-pure water system (Millipore, Tokyo, Japan). Enzyme purification. Microaerobically grown B. infantis cells (cultured statically but stirred with a mag- netic stirrer in 3 liters of medium in a 5-liter flask with a cotton plug) were harvested for enzyme purification. NAD(P)H oxidase activity was assayed spectrophotometrically in 1 ml of air saturated 50 mM potassium phos- phate buffer (pH 6.5) at 37 °C. The reaction was started by the addition of enzyme solution, and the decrease in absorbance at 340 nm (ε340 = 6,220 M−1·cm−1) was monitored with a spectrophotometer (HITACHI U-3300, Hitachi, Japan). One unit of activity was defined as the amount of enzyme that catalyzes the oxidation of 1 µmol NAD(P)H per minute. ( ) p Microaerobically grown B. infantis cells (80 g) were suspended in 240 ml of 50 mM potassium phosphate buffer, pH 6.5, containing 0.1 mM DTT, 0.2 mM PMSF, and 5 mM EDTA, and then the cells were disrupted by treatment with a French pressure cell at 140 MPa. All purification procedures were carried out at 4 °C or on ice. Cell free extracts (CFE) were obtained by removing cell debris by centrifugation at 39,000 g for 15 min. The cyto- plasmic solution obtained by ultracentrifugation at 100,000 g for 2 h was treated with 10% streptomycin sulfate (dissolved in 10 mM potassium phosphate buffer, pH 7.0) to remove nucleic acids. After centrifugation at 39,000 g for 15 min, the supernatant was fractionated by the stepwise addition of solid ammonium sulfate (20%). The supernatant obtained after centrifugation at 30,000 g was dissolved in 50 mM potassium phosphate buffer, pH 7.0, containing 1 M ammonium sulfate, 5 mM EDTA, 0.2 mM phenylmethanesulfonyl fluoride (PMSF), and 0.1 mM dithiothreitol (DTT). After centrifugation at 39.000 g for 15 min, the supernatant was applied on a hydrophobic interaction chromatography using Butyl-TOYOPEARL 650 s (TOSOH, Tokyo, Japan) column chromatography. Discussionh infantis induces the expression of NPOX in response to an increase in O2, le ing to the production of H2O2 to maintain anaerobiosis. B. infantis ∆npoxA still produced H2O2 at elevated 6 www.nature.com/scientificreports/ concentrations, indicating the presence of another H2O2-forming oxidase in vivo. A b-type DHOD, that is con- served in the genome of B. infantis, is a likely candidate. Further characterization of the residual H2O2 production in B. infantis, as well as analysis of the enzyme kinetics of the O2-tolerant B. minimum NPOX homolog, will be needed to describe the mechanisms of anaerobiosis in these strains. concentrations, indicating the presence of another H2O2-forming oxidase in vivo. A b-type DHOD, that is con- served in the genome of B. infantis, is a likely candidate. Further characterization of the residual H2O2 production in B. infantis, as well as analysis of the enzyme kinetics of the O2-tolerant B. minimum NPOX homolog, will be needed to describe the mechanisms of anaerobiosis in these strains. Methods The enzyme was eluted with a linear gradient from 1.2 M to 0 M ammonium sulfate dissolved in the same buffer. After the Butyl-TOYOPEARL chromatography, active fractions of NADPH oxidase activity were obtained. The solution after Butyl-TOYOPEARL chromatography was dialyzed against 50 mM potassium phosphate buffer, pH 6.5, containing 0.5 mM EDTA, 0.2 mM PMSF, and 0.1 mM DTT for 6 h, and this procedure was repeated 3 times using freshly prepared buffer each time. The enzyme solution was then applied to DEAE Sephacel (GE Healthcare, USA) column chromatography. The column was sequentially washed with the same buffer containing 0 mM and 150 mM NaCl, and eluted with buffer containing 250 mM NaCl. The active fractions were pooled and dialyzed for 12 h against 10 mM potassium phosphate buffer, pH 6.5, containing 0.5 mM EDTA and 0.1 mM DTT and subjected to hydroxyapatite (WAKO, Japan) column chromatography. After applying the enzyme solution to the hydroxyapatite column, the protein was sequentially washed with 10 mM and 50 mM potassium phosphate buffer, pH 6.5, and eluted with 100 mM potassium phosphate buffer, pH 6.5. The active fractions were collected and concentrated using Amicon Ultra centrifugal filter units (30,000 Da cutoff; MILLIPORE, Cork, Ireland). The concentrated enzyme was applied to POLOS HQ/H (PerSeptive Biosystems, Framingham, USA) column chro- matography. The column was sequentially washed with 50 mM potassium phosphate buffer (pH 6.5), then eluted with a linear gradient of 0 mM–250 mM NaCl. The active fraction was collected and concentrated using Amicon Ultra. After this chromatography, the enzyme purity in the active fractions was checked by SDS-PAGE with Coomassie Brilliant Blue G-250 staining or silver staining. By using this enzyme concentrator, the basal buffer of the enzyme solution was changed to 50 mM potassium phosphate buffer, pH 6.5, by diluting the concentrated enzyme solution 100-fold with the new buffer, concentrating to the original volume, and then repeating the dilu- tion and concentration steps; the second concentrated enzyme solution was subjected to enzyme assay. Protein concentration was determined by the dye-binding assay58.hil y y g y The purified enzyme was subjected to SDS-PAGE and electroblotted onto polyvinylidene difluoride mem- branes (NIPPON GENETICS, Tokyo, Japan). The N-terminal amino acid sequence was determined by the Edman degradation method using the peptide sequencer described previously7. The UV-Vis absorption spectrum was recorded on a Hitachi U3300 spectrophotometer (Hitachi, Tokyo, Japan) using a 1 cm path length quartz cuvette. www.nature.com/scientificreports/ conversion of 1 mol H2O2 to 1 mol H2O and 1/2 mol O2. The H2O2-forming type NAD(P)H oxidase produces 50% O2 after the addition of catalase to the total amount of O2 consumed by the NAD(P)H oxidase reaction.hii conversion of 1 mol H2O2 to 1 mol H2O and 1/2 mol O2. The H2O2-forming type NAD(P)H oxidase produces 50% O2 after the addition of catalase to the total amount of O2 consumed by the NAD(P)H oxidase reaction.hii 2t 2 y ( ) The substrate specificity of the purified enzyme was assayed spectrophotometrically at 37 °C using 50 mM potassium phosphate buffer, pH 6.5. Electron acceptors for a purified enzyme were investigated by adding various substrates (final concentration, 50 µM) to the anaerobic cuvette together with the enzyme solution. For all accep- tors except O2, the reactions were carried out under anoxic conditions by purging the anaerobic glass cuvette containing the reaction buffer with O2-free argon gas. NAD(P)H:O2 oxidoreductase activity was assayed under air saturated conditioin. NAD(P)H (final 150 µM) was used as an electron donor for the enzyme. For the reactions of NAD(P)H:acceptor oxidoreductase, one unit of activity was defined as the amount of enzyme that catalyzes the reduction of 1 µmol of NAD(P)H (ε340 = 6,220 M−1 cm−1) per minute. For the assay of DCIP, one unit of activity was defined as the amount of enzyme that catalyzes the reduction of 1 µmol of DCIP (ε600 = 22,000 M−1·cm−1) per minute. The values for relative activities (%) and the specific activities (U/mg protein) are the average of two independent measurements that varied by less than 5%. The apparent Km values for NADH and NADPH were determined by varying the concentrations of both NAD(P)H in 50 mM potassium phosphate buffer, pH 6.5 using the Enzyme Kinetics Module 1.3 (SigmaPlot 11, SYSTAT Software, Chicago, IL). Initial rates were determined from linear plots of NAD(P)H reduction. Northern hybridization. Northern hybridization was performed as described previously8. Total RNA (5 µg) extracted from B. infantis cells harvested before or after the 5% O2 stress was loaded on agarose gels and blot- ted onto nylon membranes (Hybond N+, GE Healthcare, Cicago, IL, USA). The membrane was then probed with the 32P-labeled gene probe of B. infantis npoxA. The membrane was subsequently stripped of the probe and reprobed with a probe of B. infantis ahpC. www.nature.com/scientificreports/ Northern hybridization using the same membrane was repeated twice to confirm the results. The following oligonucleotide primer pairs were used to amplify the probes for B. infantis npoxA: 5′-ATGGTTACCAACGCAACAAT and 52-CTAATCCAGACGGAAGCCCT, B. infantis ahpC: 5′-ATGACTCTTCTGCAGCATGA and 52-TCACAGCTGGCCGACGAGGT. Before hybridization with a dif- ferent probe, the membrane was washed at 90 °C for 2 minutes in a stripping buffer (5% SDS in 50 mM Tris-HCl, pH7.5) and reused for the next northern analysis by reprobing with a different gene probe. Expression of B. infantis npoxA in B. minimum. B. infantis npoxA including its promoter region was amplified by PCR using the forward primer (5′-TAAAAGCTTTGGATGATGGTTTCTGTTGG) included a HindIII restriction site, and the reverse primer (5′-TAAGCGGCCGCCTAATCCAGACGGAAGCCCTG) included a NotI restriction site. The purified PCR product was digested with HindIII and NotI and ligated into HindIII- and NotI-digested pKKT427 vector54, resulting in the plasmid pBInpoxA, which was transformed into B. minimum by electroporation. B. minimum cells carrying pBInpoxA or cells carrying a control vector pKKT427 were tested the growth in MRS medium containing spectinomycin (Sp) (80 µg/ml) under several O2 concentrations. Gene knockout of npoxA. A plasmid pKO403Cm-∆npoxA for the knockout of the B. infantis npoxA gene was constructed as follows (Fig. S1). The PCR primers were designed according to the npoxA gene sequence (BLON_RS12650) and the flanking genomic regions of B. infantis JCM1222T (=ATCC15697) (GenBank Accession no. NC_011593). To obtain the npoxA deletion DNA fragments, upstream (1.35 kb) and downstream (1.36 kb) regions of the putative npoxA gene of B. infantis were amplified by PCR using the primers shown in Fig. S1B. The produced DNA fragments were connected to the upstream and downstream regions of the Sp resist- ance gene (Spr) by Golden Gate Assembly59 with SapI as the Type IIS restriction enzyme. The obtained fragments were cloned into pKO403Cm, a Bifidobacterium-Escherichia coli shuttle vector60, which carries a Golden Gate cloning site, chloramphenicol (Cm) resistance gene (Cmr), and temperature-sensitive replicon origin60. B. infantis cells were transformed with pKO403Cm-∆npoxA by electroporation55. After transformation, the cells were spread and cultured at 42 °C on MRS plates containing Sp (50 µg/ml). The transformants were transferred onto two MRS agar plates containing either Sp or Cm. The obtained Sp-resistant/Cm-sensitive transformants were selected as candidates for the double-crossover mutant. The deletion of npoxA was confirmed by PCR using Primer-1 and Primer-2 (Fig. S1C) and nucleotide sequencing to confirm the precise disruption. Scientific REPOrTS \| (2018) 8:10750 \| DOI:10.1038/s41598-018-29030-4 p , ( ) 2. Morris, J. G. Oxygen and the obligate anaerobe. J. Appl. Bacteriol. 40, 229–244 (1976). Methods The bound flavins were extracted and determined by HPLC analysis with fluorescence detector according to a previously described method using a CAPCELL PACK C18 column (4.6 mm I.D. × 150 mm L; SHISEIDO CO., LTD Tokyo Japan) with 5mM ammonium acetate in methanol as the mobile phase7 Riboflavin FAD and FMN y y g y The purified enzyme was subjected to SDS-PAGE and electroblotted onto polyvinylidene difluoride mem- branes (NIPPON GENETICS, Tokyo, Japan). The N-terminal amino acid sequence was determined by the Edman degradation method using the peptide sequencer described previously7. The UV-Vis absorption spectrum was recorded on a Hitachi U3300 spectrophotometer (Hitachi, Tokyo, Japan) using a 1 cm path length quartz cuvette.hll p p y p g p g q The bound flavins were extracted and determined by HPLC analysis with fluorescence detector according to a previously described method using a CAPCELL PACK C18 column (4.6 mm I.D. × 150 mm L; SHISEIDO CO., LTD. Tokyo, Japan) with 5 mM ammonium acetate in methanol as the mobile phase7. Riboflavin, FAD and FMN were used as standards. Enzyme properties. The optimal pH of the purified enzyme was determined in 50 mM potassium phos- phate buffer in the pH range of pH 5.0 – pH 8.0 at 37 °C, which is the optimum growth temperature for B. infantis. The temperature optimum of the purified enzyme was determined in 50 mM potassium phosphate buffer, pH 6.5, over a temperature range from 25 to 60 °C. p g H2O2 production by the B. infantis NAD(P)H oxidase reaction was detected while monitoring O2 production using an oxygen electrode by adding catalase into the reaction vessel8,33,44. Catalase catalyzes the stoichiometric Scientific REPOrTS \| (2018) 8:10750 \| DOI:10.1038/s41598-018-29030-4 Scientific REPOrTS \| (2018) 8:10750 \| DOI:10.1038/s41598-018-29030-4 7 www.nature.com/scientificreports/ 1. McCord, J. M., Keele, B. B. Jr. & Fridovich, I. 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Vibrio harveyi NADPH-flavin oxidoreductase: cloning, sequencing and overexpression of the gene and purification and characterization of the cloned enzyme. J. Bacteriol. 176, 3552–3558 (1994). Scientific REPOrTS \| (2018) 8:10750 \| DOI:10.1038/s41598-018-29030-4 9 www.nature.com/scientificreports/ Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-29030-4. Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-29030-4.h Competing Interests: The authors declare no competing interests. Competing Interests: The authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps an institutional affiliations. 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/W3140904289	https://ieeexplore.ieee.org/ielx7/7873788/9397409/09397418.pdf	English	null	Accurate evaluation of cooling system capability of three-phase IGBT-based inverter	Chinese journal of electrical engineering	2,021	cc-by	3,465	Accurate Evaluation of Cooling System Capability of Three-phase IGBT-based Inverter* Dan Zheng1, Puqi Ning1, 2, Xiaoguang Chai1, 2, Wei Sun1, Zhijie Qiu1, Yuhui Kang1, Han Cao1, 2 and Tao Fan1, 2 (1. Institute of Electrical Engineering Chinese Academy of Sciences, Beijing 100190, China; 2. School of Electronic, Electrical and Communication Engineering, University of Chinese Academy of Sciences, Beijing 100049, China) Abstract: In this paper, an offline evaluation method for the cooling capability of three-phase insulated-gate bipolar transistor (IGBT) inverters is presented, which can better emulate real working conditions. With a properly designed sudden-stop control sequence, the conventional junction temperature monitoring method at a low current is used to calculate the junction temperature before the sudden stop of an inverter. This can solve the challenging switching loss calculation issue in conventional methods. Finally, the feasibility, control sequence, and electrical behaviors of the proposed method are validated through experimental tests. Keywords: Junction temperature monitoring, IGBT, conduction voltage Keywords: Junction temperature monitoring, IGBT, conduction voltage the accuracy of thermal behavior estimated at the lower power may significantly decreases at the rated power. The non-ideal thermal imbalance, limited pump capability, and complex dynamic electrical load influence the loss calculation [8-9]. Therefore, it is important to explore a more advanced characterization method at the rated power, which does not involve too many assumptions in the thermal path and loss calculation. Letters Accurate Evaluation of Cooling System Capability of Three-phase IGBT-based Inverter Dan Zheng1, Puqi Ning1, 2, Xiaoguang Chai1, 2, Wei Sun1, Zhijie Qiu1, Yuhui Kang1, Han Cao1, 2 and Tao Fan1, 2 (1. Institute of Electrical Engineering Chinese Academy of Sciences, Beijing 100190, China; 2. School of Electronic, Electrical and Communication Engineering, University of Chinese Academy of Sciences, Beijing 100049, China) Chinese Journal of Electrical Engineering, Vol.7, No.1, March 2021 Chinese Journal of Electrical Engineering, Vol.7, No.1, March 2021 Manuscript received January 22, 2021; revised February 1, 2021; accepted March 1, 2021. Date of publication March 31, 2021; date of current version March 8, 2021. Corresponding Author, E-mail: npq@mail.iee.ac.cn * Supported by the National Key Research and Development Program of China (2016YFB0100600). Digital Object Identifier: 10.23919/CJEE.2021.000007 China (2016YFB0100600). Digital Object Identifier: 10.23919/CJEE.2021.000007 * Supported by the National Key Research and Development Program of China (2016YFB0100600). Corresponding Author, E-mail: npq@mail.iee.ac.cn China (2016YFB0100600). 2.1 Sudden-stop control procedure In this study, a three-phase inverter is loaded with three-phase inductors and resistors, as shown in Fig. 3. As indicated, T6 at the bottom of phase C represents the DUT IGBT. For motor drives and other three-phase power sources, the equivalent inductance (L) and resistance (R) must be carefully calculated and used. The switching frequency, power factor angle, and other working conditions must be considered. As current sensors are usually present in most inverters, only an on-state voltage sampling unit is required in this method. Fig. 2 Traditional cooling system capability evaluation using the method based on the on-state voltage at low current Fig. 3 Inverter topology Fig. 2 Traditional cooling system capability evaluation using the method based on the on-state voltage at low current To evaluate the thermal impedance of regular power modules, a widely used testing method is based on the on-state voltage with a very low current injection. However, the device under test (DUT) remains conductive in the heating phase, and the switching loss in real working conditions can only be estimated. To compensate for the issue of switching loss estimation, the heating current is usually set to a higher value [14]. This calculation-based setting may result in inaccuracies when operating at the rated power. Fig. 3 Inverter topology Fig. 4 shows the sudden-stop control sequence. Based on a set power target, all three phases can be powered with a regular control algorithm. After thermal equilibrium is reached, the load current is interrupted at time t2, without any further auxiliary switches. In this state, the DUT (T6) is activated and all the other IGBTs (T1–T5) are turned off. Ref. [15] introduced a method to estimate the junction temperature of an single-phase inverter under the switching mode, resulting in a thermal characterization that is closer to that of real operation cases. An H-bridge testing circuit and its corresponding control methods were presented. However, it is difficult to apply this method to a three-phase inverter system owing to control and current cutting reasons. t0-t3: run time; t3-t4: stop time; t2-t3: shutdown process; t3-t4: TJ measurement; t1-t3: compensate time; t2-t’: stage 1; t’-t3: stage 2. Fig. 4 Sudden-stop control sequence To solve the above issues, this paper presents an accurate and feasible offline cooling capability evaluation method for an IGBT-based inverter. 1 Introduction Cooling capability evaluation is a core step in three-phase inverter design and development [1-2]. In most studies, the junction temperature of the device is estimated using the loss model and thermal network model, as shown in Fig. 1. An accurate evaluation of the thermal dynamics can help to predict the stability of inverter systems [3-5]. In some conventional methods, the thermal impedance is derived from the datasheets of power modules and cooling systems; hence, an accurate calculation of the power loss is required. As an alternative method, a simplified working condition is assumed, and a detailed finite-element method simulation is used. However, currently, these methods are not sufficient for high-power-density inverters [6-7]. A method evaluates the junction temperature of insulated-gate bipolar transistors (IGBTs) under light load, half load, rated power, and overload conditions gradually is preferred, to prevent damage to power devices by directly operating at the maximum allowed power. Commonly used junction temperature monitoring methods can be classified as conduction contact-based, electro-thermal model-based, optical, and temperature-sensitive electrical parameter (TSEP)-based methods [10-13]. The TSEP-based method uses the chip itself as a temperature sensor, which has advantages in terms of accuracy, control modification, and hardware invasion [10-13]. Fig. 1 Conventional junction temperature estimation methods Fig. 1 Conventional junction temperature estimation methods ig. 1 Conventional junction temperature estimation metho In a high-power-density inverter, the cooling system usually operates in the saturation mode, and The method based on the on-state voltage at low current is widely used in junction temperature measurement, and the basic circuit is shown in Fig. 2. Among other TSEP methods, the peak gate current, Chinese Journal of Electrical Engineering, Vol.7, No.1, March 2021 74 74 leakage current, and overshoot voltage methods have relatively low resolutions, therefore requiring special sensors. In contrast, the threshold voltage, turn-off time, and turn-off delay methods have high nonlinearity, requiring complex calibration. Furthermore, the short-current and breakdown voltage methods may directly damage devices [10]. Owing to cost, linearity, complexity, and stability advantages, the on-state voltage at low current is the recommended method in the JESD51-14 and other standards when conducting thermal resistance tests. 2.1 Sudden-stop control procedure A control sequence is used to rapidly cut off all three-phase currents; thus, an approximate ideal sudden-stop situation can be formed. This contributes to a more accurate evaluation of thermal impedance. t0-t3: run time; t3-t4: stop time; t2-t3: shutdown process; Dan Zheng et al.: Accurate Evaluation of Cooling System Capability of Three-phase IGBT-based Inverter 75 75 In a real test, the forced discharge mode can be controlled by monitoring and comparing the phase currents. Every phase leg can be evaluated by repeatedly changing the control sequence and low-current injection circuit connection. If the phase C current (IC) flows from the IGBT module and its amplitude is larger than those of the currents of the other two phases at time t2, the load currents would flow through D1, D3, and D6. In this scenario, the inverter enters a forced discharge mode, as shown in Fig. 5a. In this mode, reverse voltages are applied to inductive loads. All phase currents quickly return to 0 A following a two-stage slope, generally within several milliseconds. The current discharge rate (di/dt) of the inductor LC is determined by Eq. (1). 2.2 Cooling down procedure To evaluate the impact of the shutdown process on the junction temperature, the FOSTER model of the inverter system, shown in Fig. 6, was established and is commonly used. Usually, the datasheet provides only the thermal impedance from the junction to the case. Therefore, the thermal impedance between the junction and fluid should be derived from experiments. When the cooling fluid is water with a flow rate of 70 L/min, the junction-to-fluid thermal impedance of a ABB module 5SNG1000X170300 is shown in Tab. 1, where Ri and τi are the thermal resistance and time constant pairs, respectively. × d d CN L L u i R i t L   (1) (1) where uCN is the instantaneous voltage of the phase C load, iL is the current flowing through the load, L is the inductance of LC, and R is the resistance of RC. In the first stage, uCN is 2/3UDC, where UDC is the DC-link voltage. When the minor currents of phases A and B return to 0 A, the current path will be disconnected instead of having a damped oscillation behavior due to the shutdown of T1–T5. Fig. 6 FOSTER network model of a thermal impedance The second stage is thereafter initiated, and uCN is set to 1/2UDC. The energy stored in the inductors is transferred to the DC-link capacitor as reactive power, resulting in a voltage increase (ΔU). Usually, the reactive power is already considered in the power stage design; therefore, ΔU is not sufficient to cause any damage. Fig. 6 FOSTER network model of a thermal impedance Fig. 6 FOSTER network model of a thermal impedance Tab. 1 Transient thermal impedance of an IGBT module Tab. 1 Transient thermal impedance of an IGBT module i Ri/(K/kW) τi/s 1 6.16 0.036 2 20.08 0.555 3 43.12 2.271 4 1.80 1.017 When the control sequence does not conform to the forced discharge mode, the natural discharge mode of the converter is activated, as shown in Fig. 5b. In this mode, the current flows between the three-phase loads, and the energy is only consumed by resistors. This process is relatively slow, usually performed in several seconds, and should be avoided during cooling evaluation procedures. Fig. 7 shows the relationship between the IGBT junction temperature and time based on a simulation. The IGBT was heated by an AC current flowing through it alternately. The heating power was set to 560 W. The junction temperature stabilized in approximately 10 s, and the fluctuation was approximately 3 ℃. The heating current was then interrupted at 15 s. In the following 10 ms, the temperature change was less than 0.5 ℃. Therefore, in this procedure, the junction temperature can be measured by the conduction voltage with a low current within 5 ms of the current interruption. The sampling time in this study was selected as 100 to 1 000 μs. Fig. 5 Inductor discharge mode Fig. 5 Inductor discharge mode Fig. 5 Inductor discharge mode Chinese Journal of Electrical Engineering, Vol.7, No.1, March 2021 76 76 76 Fig. 7 Simulation results of IGBT cooling curve Fig. 9 TJ -VCE calibration schematic In this study an ABB module (5SNG1000X170300 Fig. 7 Simulation results of IGBT cooling curve Fig. 9 TJ -VCE calibration schematic Fig. 7 Simulation results of IGBT cooling curve Fig. 9 TJ -VCE calibration schematic Fig. 7 Simulation results of IGBT cooling curve In this study, an ABB module (5SNG1000X170300) was tested considering IL ranging from 5 mA to 100 mA. The linear relation between TJ and VCE was confirmed for each IL, as shown in Fig. 10. From the data analysis, the slope is approximately 2.5 mV/℃. Furthermore, using the sudden-stop control procedure described in Section 2.1, the current after the cut-off moment is forced to flow through T5 instead of T6. Thus, the time between t1 and t3 can be measured. The IGBT junction temperature immediately before the cut-off can also be accurately calculated using a typical compensation algorithm, such as the square root t method [16] shown in Fig. 8. The relationship between Δ TJ and t is linear during the initial period of the shutdown. 3.2 Cooling capability evaluation case To demonstrate the feasibility of the cooling capability estimation, a comparison test was conducted, as shown in Fig. 11. The inverter was composed of ABB modules (5SNG1000X170300), and its main parameters are listed in Tab. 2. A three-phase RL test bank was used as the load. After estimating the ambient temperature and module rating, the inverter was safely tested using a DC bus voltage of up to 600 V and a peak current of 1 000 A. Fig. 8 Temperature compensation method Fig. 8 Temperature compensation method Tab. 1 Transient thermal impedance of an IGBT module This period, which is approximately hundreds of milliseconds, is related to the thermal characteristics of the module. Although this relationship is still based on some ideal assumptions, the error in practical applications is acceptable. Fig. 10 TJ -VCE relationship at low current injection Fig. 10 TJ -VCE relationship at low current injection Fig. 10 TJ -VCE relationship at low current injection Fig. 8 Temperature compensation method 3 On-state voltage measurement and a junction temperature estimation case Fig. 11 Cooling capability evaluation experimental platform 3.1 Junction temperature estimation with a very low current The junction temperature of IGBTs can be estimated using the on-state voltage under a very low current. As shown in Fig. 9, a very low current (IL) is injected into the DUT; therefore, its power loss can be neglected. To obtain the relation between the junction temperature (TJ) and on-state voltage (VCE), TJ was controlled by an ambient testing cabinet. Fig. 11 Cooling capability evaluation experimental platform Dan Zheng et al.: Accurate Evaluation of Cooling System Capability of Three-phase IGBT-based Inverter Dan Zheng et al.: Accurate Evaluation of Cooling System Capability of Three-phase IGBT-based Inverter 77 Tab. 2 Experimental platform parameters Parameter Value IGBT module 1 700 V/1 000 A DC-link capacitor 1 200 V/2 500 μF Load inductor/mH 0.36 Load resistor/mΩ 3 DC-link voltage/V 600 Fundamental frequency/Hz 50 Switching frequency/kHz 1.8-4.5 Flow rate/(L/min) 70 Tab. 2 Experimental platform parameters cooling fluid was water, the maximum flow rate was 70 L/min, and the ambient temperature was limited to 80 ℃. The proposed method was used considering different currents and switching frequencies to evaluate whether the cooling capability satisfied the design requirements. Furthermore, a commonly used thermal impedance method was selected for comparison of the measurements. The results are presented in Tab. 3, where TF is the fluid temperature, fZ is the switching frequency, and IF is the current amplitude. The results of the two methods agree with each other, and their errors are less than 5 ℃. Thus, the proposed cooling capability estimation method shows a good evaluation potential and can be used in future design and development of converters. As shown in Fig. 12, a peak current of 900 A was used, and then the inverter was suddenly stopped at 1.5 ms by performing the sudden-stop control sequence. Then, a very low current (6.25 mA) was injected into the IGBT T6 in phase C. The on-state voltage of T6 was measured at a sampling rate of 1.8 kHz. Fig. 13 TJ test value after shutting down Fig. 14 VCE measurement noise coupling path Fig 13 T test al e after sh tting do n Fig. 12 Sudden-stop process Fig. 13 TJ test value after shutting down Fig. 13 TJ test value after shutting down Fig. 13 TJ test value after shutting down Fig. 14 VCE measurement noise coupling path Fig. 12 Sudden-stop process Fig. 12 Sudden-stop process Fig. 13 shows TJ obtained after the shutdown procedure. 3.1 Junction temperature estimation with a very low current TJ at the shutdown moment was calculated by the t method. Although all other IGBTs were turned off, VCE on T6 was still affected by the noise from the DC side. The VCE noise coupling path is shown in Fig. 14. The on-resistor of the bottom IGBT T6 and the parasitic capacitor of the top IGBT T5 form a high-pass filter. Therefore, the high-frequency noise from the grid side is transmitted to the measuring circuit. It can be eliminated using a digital low-pass filter. Fig. 14 VCE measurement noise coupling path Tab. 3 Comparison of temperature monitoring TF/℃ fZ/Hz IF/A TJ/℃ TJ/℃ Test Thermal impedance method 80 1.8 1 000 133 131 80 2.5 900 134 132 80 4.5 700 139 136 70 2.5 1 000 130 130 50 4.5 1 000 138 135 Tab. 3 Comparison of temperature monitoring Tab. 3 Comparison of temperature monitoring The maximum allowed junction temperature of the IGBT module in the datasheet was 175 ℃. For safe and feasible operation, the average junction temperature was set to 140 ℃. In this experiment, the 4 Summary and conclusion An offline cooling capability evaluation method for three-phase IGBT inverters was proposed, which Chinese Journal of Electrical Engineering, Vol.7, No.1, March 2021 78 78 can emulate the working conditions of switching-mode devices. A current control sequence was used to stop all heating currents in a short time. Using the conventional method based on the on-state voltage at a low current and a related compensation method, the junction temperature could be accurately calculated to evaluate the cooling capacity of the inverter system. Compared with the traditional method, the proposed method can more accurately represent the actual working condition of the inverter. On the basis of the existing controller, only a simple voltage measuring unit was added, resulting in a low cost, small size, and simple operation. The proposed method can also be utilized in light load and overload conditions, as well as for single-phase inverters. Furthermore, it can be used to validate an online junction temperature monitoring system. model of high power IGBT modules considering heat coupling effects. Proceedings of the 2014 International Power Electronics and Application Conference and Exposition, 5-8 Nov. 2014, Shanghai, China, 2014: 1382-1387. [8] I Aranzabal, I M D Alegría, N Delmonte, et al. Comparison of the heat transfer capabilities of conventional single-and two-phase cooling systems for an electric vehicle IGBT power module. IEEE Transactions on Power Electronics, 2019, 34(5): 4185-4194. [9] M Wang, Y Mei, W Liu, et al. Reliability improvement of a double-sided IGBT module by lowering stress gradient using Molybdenum buffers. IEEE Journal of Emerging and Selected Topics in Power Electronics, 2019, 7(3): 1637-1648. [10] Y Avenas, L Dupont, Z Khatir. Temperature measurement of power semiconductor devices by thermo-sensitive electrical parameters: A review. IEEE Transactions on Power Electronics, 2012, 27(6): 3081-3092. References [11] H Luo, Y Chen, P Sun, et al. Junction temperature extraction approach with turn-off delay time for high-voltage high-power IGBT modules. IEEE Transactions on Power Electronics, 2016, 31(7): 5122-5132. [1] C Qian, A M Gheitaghy, J Fan, et al. Thermal management on IGBT power electronic devices and modules. IEEE Access, 2018, 6: 12868-12884. [2] S Yang, A Bryant, P Mawby, et al. An industry-based survey of reliability in power electronic converters. IEEE Transactions on Industry Applications, 2011, 47(3): 1441-1451. [12] C Chen, V Pickert, B Ji, et al. Comparison of TSEP performances operating at homogeneous and inhomogeneous temperature distribution in multichip IGBT power modules. IEEE Journal of Emerging and Selected Topics in Power Electronics, 2020, DOI: 10.1109/JESTPE.2020.3047738. [3] H Wang, C Chang, Z Liang, et al. Structure design and thermal simulation analysis of DBC substrate for high-power IGBT module. International Conference on Electronic Packaging Technology (ICEPT), August 12-15, 2020, Guangzhou, China, 2020: 1-4. [13] D Zheng, Y Kang, H Cao, et al. Monitoring of SiC MOSFET junction temperature with on-state voltage at high currents. Chinese Journal of Electrical Engineering, 2020, 6(3): 1-7. [4] Z Zhou, P M Holland, P Igic. Compact thermal model of a three-phase IGBT inverter power module. 26th International Conference on Microelectronics, 11-14 May 2008, Nis, Serbia and Montenegro. 26th International Conference on Microelectronics, 2008: 167-170. [14] P Panchal, T V Essen, M A Ras, et al. Accurate, versatile and compact transient measurement system for fast thermal package characterization and health monitoring. 2018 7th Electronic System-Integration Technology Conference (ESTC), 18-21 Sept. 2018, Dresden, Germany, 2018: 1-7. [5] H Cao, P Ning, X Wen, et al. An electrothermal model for IGBT based on finite differential method. IEEE Journal of Emerging and Selected Topics in Power Electronics, 2020, 8(1): 673-684. [15] Y Zhu, K Ma, X Cai. Thermal characterization method of power semiconductors based on H-bridge testing circuit. IEEE Transactions on Power Electronics, 2019, 34(9): 8268-8273. [6] B W Shook, A Nizam, Z Gong, et al. Multi-objective layout optimization for multi-chip power modules considering electrical parasitics and thermal performance. 2013 IEEE 14th Workshop on Control and Modeling for Power Electronics (COMPEL), 23-26 June 2013, Salt Lake City, UT, USA, 2013: 1-4. [16] JEDEC. Transient dual interface test method for the measurement of the thermal resistance junction to case of semiconductor devices with heat flow through a single path[2010-11]. JESD51-14. [7] A S Bahman, K Ma, F Blaabjerg. References Thermal impedance
https://openalex.org/W3047913931	https://link.springer.com/content/pdf/10.1007/s10482-020-01454-x.pdf	English	null	Streptobacillus felis, a member of the oropharynx microbiota of the Felidae, isolated from a tropical rusty-spotted cat	Antonie van Leeuwenhoek	2,020	cc-by	7,199	Antonie van Leeuwenhoek (2020) 113:1455–1465 https://doi.org/10.1007/s10482-020-01454-x (0123456789().,-volV)( 01234567 89().,-volV) ORIGINAL PAPER Streptobacillus felis, a member of the oropharynx microbiota of the Felidae, isolated from a tropical rusty-spotted cat Ahmad Fawzy . Jo¨rg Rau . Karin Riße . Nicole Schauerte . Christina Geiger . Jochen Blom . Can Imirzalioglu . Jane Falgenhauer . Alexa Bach . Christiane Herden . Tobias Eisenberg Received: 25 May 2020 / Accepted: 22 July 2020 / Published online: 9 August 2020 The Author(s) 2020 that this microorganism is common in the oropharynx, suggesting that S. felis is a member of their normal microbiota. Due to unawareness, fastidiousness, antibiotic sensitivity and lack of diagnostics the role of S. felis as a cat and human pathogen might be under- reported as with other Streptobacillus infections. More studies are necessary to elucidate the role of S. felis in domestic cats and other Felidae in order to better estimate its zoonotic potential. Abstract Streptobacillus felis is a fastidious microorganism and a novel member of the potentially zoonotic bacteria causing rat bite fever. Since its description, this is the second isolation of S. felis in a diseased member of the Felidae. Interestingly, the strain from this study was isolated from a zoo held, rusty-spotted cat (Prionailurus rubiginosus), with pneumonia, thereby indicating a possible broader host range in feline species. A recent preliminary sampling of domestic cats (Felis silvestris forma catus) revealed Keywords Streptobacillus felis Rat bite fever Cat reservoir Zoonosis Immuno-histochemistry (IHC) Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10482-020-01454-x) con- tains supplementary material, which is available to authorized users. A. Fawzy Faculty of Veterinary Medicine, Department of Medicine and Infectious Diseases, Cairo University, Cairo, Egypt A. Fawzy Faculty of Veterinary Medicine, Department of Medicine and Infectious Diseases, Cairo University, Cairo, Egypt J. Blom Bioinformatics and Systems Biology, Justus-Liebig- University Giessen, Heinrich-Buff-Ring 58, 35392 Giessen, Germany A. Fawzy K. Riße T. Eisenberg (&) Department of Veterinary Medicine, Hessian State Laboratory (LHL), Schubertstr. 60, 35392 Giessen, Germany e-mail: Tobias.Eisenberg@lhl.hessen.de A. Fawzy K. Riße T. Eisenberg (&) Department of Veterinary Medicine, Hessian State Laboratory (LHL), Schubertstr. 60, 35392 Giessen, Germany e-mail: Tobias.Eisenberg@lhl.hessen.de C. Imirzalioglu J. Falgenhauer Institute for Medical Microbiology, Justus Liebig University Giessen, Schubertstr. 81, 35392 Giessen, Germany J. Rau Chemical and Veterinary Analysis Agency Stuttgart, Schaﬂandstr. 3/2, 70736 Fellbach, Germany A. Bach C. Herden Institute of Veterinary Pathology, Justus-Liebig- University Giessen, Frankfurter Str. 96, 35392 Giessen, Germany Case description A breeding group of the endangered rusty-spotted cat (Prionailurus rubiginosus phillipsi), a subspecies native to humid zones of Sri Lanka, is managed for ex situ breeding purposes in a German zoo. The cats have been bred in the same zoo or within the European studbook program and are housed individually or in breeding pairs. From the breeding group no signiﬁcant morbidities and mortalities have occured, but individ- ual animals have suffered from intermittant signs of kitty ﬂu like sneezing, epiphora, elevated respiratory rate, reduced appetite, corneal ulceration in the years before this study. In the actual case, a female displayed bilateral blepharitis, weakness, respiratory distress and anorexia. Intra vitam tests for feline parvovirus, coronavirus and protozoa revealed negative results. Due to disease progression, the animal was euthanized. J. Rau N. Schauerte C. Geiger Frankfurt Zoo, Bernhard-Grzimek-Allee 1, 60316 Frankfurt, Germany T. Eisenberg Institute of Hygiene and Infectious Diseases of Animals, Justus-Liebig-University Giessen, 35392 Giessen, Germany 12 3 Antonie van Leeuwenhoek (2020) 113:1455–1465 1456 Introduction Streptobacillus (S.) moniliformis (Leptotrichiaceae, Fusobacteriales) has been the longstanding unique species in this genus (Levaditi et al. 1925). This bacterium represents the most important causative microorganism of rat bite fever (RBF) and its food- borne variant, Haverhill fever (Eisenberg et al. 2018). RBF is typically characterized by a triad of fever, arthritis and a maculopapular, petechial or pustular rash, but severe causes of infection may include life- threatening sequelae (Eisenberg 2017; Eisenberg et al. 2017a; Gaastra et al. 2009). A number of studies have stated a risk for RBF even through contacts to various non-rodent animal species like dogs, cats, weasels and ferrets as well as livestock animals. However, the proper identiﬁcation of these microorganisms was not carried out and such isolates have not been stored. Recently, [S.] hongkongensis (Woo et al. 2014), S. felis (Eisenberg et al. 2014), S. notomytis (Eisenberg et al. 2015b), S. ratti (Eisenberg et al. 2016) and S. canis (Eisenberg et al. 2020b) were described as novel species. Whereas S. notomytis and S. ratti are closely associated with black rats (Rattus rattus), [S.] hongkongensis has exclusively been isolated from humans (Lau et al. 2016; Woo et al. 2014) and was recently found to belong to a novel genus, Pseu- dostreptobacillus (Eisenberg et al. 2020a). S. felis and S. canis were only once isolated from clinical disease in animals, i.e. from a cat with pneumonia and a dog with phlegmon, respectively (Eisenberg et al. 2015a, 2020b). However, with respect to zoonotic potential, S. notomytis has been found to also cause RBF in humans (Fukushima et al. 2017; Ogawa et al. 2018) and a similar case of RBF could recently be attributed to S. felis for the ﬁrst time (Matt et al. 2020). Interestingly, various Streptobacillus phylotypes con- sistent with 16S rRNA gene sequence based opera- tional taxonomic units (OTU) have been described from humans and various animal species (Fig. 1). We here report a second strain of S. felis, isolated from a tropical rusty-spotted cat (Prionailurus rubiginosus), one of the smallest members of Felidae, that suc- cumbed to infection. Pathological investigation A gross pathology examination and histology were performed. For histopathological examination, speci- mens of multiple organs were ﬁxed in buffered 4% formalin, processed by standard methods and embed- ded in parafﬁn. Microtome sections were stained with hematoxylin–eosin (HE). Immuno-histochemistry (IHC) for S. moniliformis Immuno-histochemistry (IHC) for S. moniliformis The IHC examination of the formalin ﬁxed parafﬁn embedded (FFPE) samples taken for histopathological examination was performed using a recently estab- lished and not yet published protocol. Brieﬂy, this protocol utilizes a standard IHC procedure with the use of heat induced antigen demasking in target retrieval solution (Dako Cytomation Denmark AS, Glostrup, Denmark), followed by goat serum (Life Technologies Corporation, Paisley, UK) and avidin/ biotin blocking agent (Linaris Biologische Produkte GmbH, Dossenheim, Germany) in order to block non- speciﬁc binding and reactions, respectively. The primary antibody used was an afﬁnity puriﬁed poly- clonal rabbit-anti-S. moniliformis antibody supplied by Davids Biotechnologie GmbH (Regensburg, Ger- many). A goat-anti-rabbit IgG biotinylated antibody 3 12 Antonie van Leeuwenhoek (2020) 113:1455–1465 1457 (Vector Laboratories, Burlingame, USA) served as a secondary antibody and allowed the detection of the antigen–antibody complex using the Vectastain ABC- Elite Kit (Linaris). Diaminobenzidine (DAB; Sigma- Aldrich Chemie GmbH, Steinheim, Germany) was added, resulting in a brown-colored precipitate form- ing where antibody have bound. FFPE samples of the lung of a C57BL/6 mouse that was experimentally infected with S. moniliformis (Fornefett et al. 2017) underwent the same protocol and served as positive controls. For a negative control, FFPE samples of the rusty-spotted cat underwent the described protocol with only the primary antibody being replaced with negative control rabbit Fig. 1 UPGMA consensus tree depicting phylotypes and species of the family Leptotrichiaceae. The data set was based on 16S rRNA gene sequences and processed in Geneious vers. 8.1.9 (Kearse et al. 2012) using a Clustal W nucleotide alignment with standard settings and rapid bootstrap analysis (1,000 bootstraps). GenBank accession numbers are given in parentheses. Numbers at branch nodes refer to bootstrap values; Fusobacterium nucleatum is used as outgroup. ‘‘T’’ indicating type strain; Bar, 0.02 nucleotide substitutions per site Fig. 1 UPGMA consensus tree depicting phylotypes and species of the family Leptotrichiaceae. The data set was based on 16S rRNA gene sequences and processed in Geneious vers. 8.1.9 (Kearse et al. 2012) using a Clustal W nucleotide alignment with standard settings and rapid bootstrap analysis (1,000 bootstraps). GenBank accession numbers are given in parentheses. Numbers at branch nodes refer to bootstrap values; Fusobacterium nucleatum is used as outgroup. ‘‘T’’ indicating type strain; Bar, 0.02 nucleotide substitutions per site (Vector Laboratories, Burlingame, USA) served as a secondary antibody and allowed the detection of the antigen–antibody complex using the Vectastain ABC- Elite Kit (Linaris). Immuno-histochemistry (IHC) for S. moniliformis Diaminobenzidine (DAB; Sigma- Aldrich Chemie GmbH, Steinheim, Germany) was added, resulting in a brown-colored precipitate form- ing where antibody have bound. FFPE samples of the lung of a C57BL/6 mouse that was experimentally infected with S. moniliformis (Fornefett et al. 2017) underwent the same protocol and served as positive controls. For a negative control, FFPE samples of the rusty-spotted cat underwent the described protocol with only the primary antibody being replaced with negative control rabbit 12 3 Antonie van Leeuwenhoek (2020) 113:1455–1465 1458 immunoglobulin fraction (Dako). Evaluation of the immune-histochemical examination was performed using a transmission light microscope. (Eisenberg et al. 2020b; Rau et al. 2016). Identiﬁcation was done with the commercial Bruker database, and with the extended database. Phenotypic characterization Phenotypic characterization Whole genome sequencing Whole genome sequencing (WGS) was carried out to get insight into a core genome based phylogeny and compare the rusty-spotted cat’s strain with established type strain genomes from the same family. The genome sequence of strain LHL191014123 was generated by de-novo assembly with reads from Illumina technology. In brief, DNA was isolated from cells grown for 3 days at 37 C on TSA supplemented with 20% horse serum using a PureLink genomic DNA kit (Thermo Fisher). The library was prepared with a Nextera XT library preparation kit (Illumina) and sequenced on NextSeq 500 (mid output kit v2, 2 9 150 bp) instruments. The genome assembly was carried out by SPAdes (version 3.10.1), resulting in 163 contigs with 179 9 average coverage. Matrix-assisted laser desorption/ionization-time of ﬂight mass spectrometry (MALDI-TOF MS) Bacterial isolation and physiological properties PCR analysis Two earlier designed PCR assays for the detection of S. moniliformis were employed to detect characteristic amplicon sizes of approximately 269 and 1,190 bp also for the rusty-spotted cat strain LHL191014123 (primers S5: 50-CAT ACT CGG AAT AAG ATG G-30 and AS2: 50-GCT TAG CTC CTC TTT GTA C-30) (Kimura et al. 2008) and [primers SbmF: 50-GAG AGA GCT TTG CAT CCT-30 and SbmR: 50-GTA ACT TCA GGT GCA ACT-30; Nicklas, cited in (Rohde et al. 2008)]. It was recently found that these PCR assays are rather genus than species speciﬁc (Eisenberg et al. 2015c). Therefore, we have recently designed primers (forward: 50- AGT ATG GGA AAT AGT AGA TAA TAG-30 and reverse 50- ACT GTA GAT TGT GAG TTC TT-30) that could speciﬁcally amplify a partial sequence of the gyrB gene (732 bp) of the S. felis genome (Matt et al. 2020). The PCR reaction components and cycling conditions were carried out as previously described in Fawzy et al. (2016) with minor modiﬁcations (annealing temperature was 53 C and elongation time was 90 s). Bacterial isolates were obtained and isolates were identiﬁed using standard microbiological examina- tions. Brieﬂy, native tissue samples were processed for microbial culture by inoculating ﬂame sterilized, freshly cut tissue surfaces onto culture media (Colum- bia agar with 5% sheep blood [SBA; Oxoid, Wesel, Germany] and Gassner agar [VWR, Darmstadt, Ger- many]). Agar plates were incubated for up to 48 h at 20 C using aerobic and microaerobic culture condi- tions. Phenotypic characterization of streptobacilli is known to yield only few weakly positive reactions (Eisenberg et al. 2015c), however, standard microbi- ological procedures included tests for hemolysis on SBA, catalase activity with 3% H2O2 on microscopic slides and for presence of cytochrome oxidase with the BBL DrySlide oxidase system (Becton–Dickinson, Heidelberg, Germany). Urease, hydrogen sulﬁde, indole, motility and oxidative and fermentative glu- cose assimilation were tested on Christensen agar, SIM and OF medium in slant agar tubes, respectively (all Merck, Darmstadt, Germany). Microscopic exam- inations of ﬁxed smears were performed using Gram’s stain. For further identiﬁcation attempts, the Omnilog GEN III plate identiﬁcation system (Biolog, Hayward, USA) was utilized for the ﬁrst time using the most sensitive protocols for fastidious bacteria (C1 and C2) with and without addition of 10% bovine serum according to manufacturer’s recommendations. Immuno-histochemistry The IHC examination using a method designed for the detection of S. moniliformis in tissue samples revealed negative results in all examined tissues of the rusty- spotted cat. Positive and negative controls were successfully showing the expected results, reafﬁrming sufﬁcient speciﬁcity for the detection of S. moniliformis. Biochemical identiﬁcation Due to the fastidious growth, physiological reaction patterns are generally very weak in members of the genus Streptobacillus [Eisenberg et al. 2015c]. The physiological characterization of strain LHL191014123 with the Omnilog GEN III plate identiﬁcation system did not reveal a superior resolu- tion compared to other standard tests, although a panel Gross pathology A coproscopy ﬂotation technique was negative. The rusty-spotted cat weighed approx. 1.9 kg, which constitutes a normal weight of this small wildcat. The female was born in 2010 and was 9-years old at the time of death. During post mortem examination a light creamy fur, possibly indicating signs of chronic cat ﬂu, was found in the left cavum nasi, accompanied by a light hemorrhagic exudate. Phenotypic characterization of isolate LHL191014123 obtained from liver tissue Phenotypic characterization of isolate LHL191014123 obtained from liver tissue Phenotypic characterization of isolate LHL191014123 obtained from liver tissue General microbiology Bacterial culture revealed growth of S. felis in all tissues, except intestine and intestinal lymph node. The semiquantitative number of streptobacilli as obtained by counting colonies on the directly inocu- lated agar surface was found to be low (\ 20) in spleen and kidney, moderate (20–50) in liver and lung and high ([ 50) in the nasal cavity. Varied growths of other Gram-positive and Gram- negative microbiota were cultivated from other tissues and identiﬁed as Enterococcus faecalis, Vagococcus teuberi and Escherichia coli using MALDI-TOF MS. Selective veriﬁcation procedures for purely microaer- obic bacteria, Chlamydia spp., Mycoplasma spp. or Salmonella spp. and herpesviruses revealed negative results throughout (data not shown). Matrix-assisted laser desorption/ionization-time of ﬂight mass spectrometry (MALDI-TOF MS) Mass spectrometry procedure has been recently described in detail (Eisenberg et al. 2018, 2020b). The commercial database used (DB 8,468; Bruk- erDaltonics) comprised 24 spectra each from 10 S. moniliformis strains. Reference spectra from well- characterized, quality-controlled strains of all other Streptobacillus/Pseudostreptobacillus species and most other members of the Leptotrichiaceae were added to the database from previous studies Phylogenetic and phylogenomic analyses For a ﬁrst phylogenetic placement, a tree based on nearly full- 123 Antonie van Leeuwenhoek (2020) 113:1455–1465 1459 length 16S rRNA gene sequences was constructed with Geneious vers. 8.1.9 (Kearse et al. 2012) using a Clustal W nucleotide alignment with standard settings and a Neighbor-Joining (NJ) tree (data not shown). Therefore, the 16S rRNA gene sequences of all type strains of the Leptotrichiaceae were obtained from GenBank and for strain LHL191014123 deduced from the full genome sequence (s. below). For a more detailed view into the phylogenetic relationship of strain LHL191014123 and all other Streptobacillus species the criteria of Woo et al. (2014) were considered. Phylogenetic analyses based on near full-lengths nucleotide sequences of the groEL, gyrB and recA genes were performed for all Streptobacillus species and the type species of all other genera of the Leptotrichiaceae. Respective nucleotide sequences were aligned using ClustalW implemented in Geneious vers. 8.1.9 (Kearse et al. 2012) and visualized as unweighted pair group method with arithmetic mean (UPGMA) phylogenetic trees (based on 1,000 replications [bootstrap analysis]). A representative tree for the gyrB gene is shown in Suppl. Fig. S1. The average nucleotide identity (ANI) values and core genome phylogeny were calculated for strain LHL191014123 in comparison with type strain genomes of the family Leptotrichiaceae using the EDGAR 2.3 platform (Blom et al. 2016). ANI values were computed as described by Goris et al. (2007) and as implemented in JSpecies (Richter and Rossello-Mora 2009). desquamated alveolar macrophages and neutrophil granulocytes with occasional phagocytized bacteria were detected in bronchioles and pulmonary alveoli. Single cysts were found in the kidneys. Histo-pathology A moderate follicular hyperplasia was noted in the spleen. Focal edema and emphysema were found in histological sections of lung tissue. Focally, ﬁbrin, 12 3 3 Antonie van Leeuwenhoek (2020) 113:1455–1465 1460 strain LHL191014123 to S. felis (score values up to 2.719). Custom-made MSP of all Streptobacillus species were prepared from known type strains based on Bruker quality criteria and used in this study; respective MSP are available for exchange via the MALDI-user platform MALDI-UP (https://maldi-up. ua-bw.de) (Rau et al. 2016). A dendrogram depicting the topologic position of the reference spectrum of strain LHL191014123 from the rusty-spotted cat from this study to closely related strains of the other known streptobacilli and related taxa is shown in Fig. 2. of 94 different reactions was assessed. Even the most sensitive protocols for fastidious bacteria (C1 [24 h] and C2 [48 h incubation]) with and without addition of 10% bovine serum resulted in very weak reactions. Slightly positive reactions were found only for L- aspartic acid, L-glutamic acid, D-glucuronic acid, glucuronamide, L-lactic acid, citric acid, a-keto- butyric acid, sodium butyrate and sodium bromate (data not shown). MALDI-TOF MS MALDI-TOF MS spectra of strain LHL191014123 show the m/z signals typical of Streptobacillus genus at 3,631.2 ± 3.6, 7,262.0 ± 7.3, and 7,392.0 ± 7.4 (data not shown, Eisenberg et al. 2018). Nevertheless, the strain LHL191014123 had score values lower than 1.5 with the used commercial Bruker database version and was, therefore, not identiﬁed. The application of the enlarged database, extended by reference entries for each of the known members of the genus Strep- tobacillus, including the type strain of S. felis 131000547T, allowed the unequivocal assignment of PCR analysis PCR analysis Both earlier published PCR protocols for the detection of S. moniliformis are based on the Streptobacillus 16S rRNA gene. As expected, strain LHL191014123 from this study gave a positive ampliﬁcation in these two PCR assays. The recently designed gyrB gene PCR was also positive for strain LHL191014123. Streptobacillus canis IHIT1603-19T Streptobacillus ra OGS16T Streptobacillus felis 191014123 Streptobacillus felis 131000547T Distance Level 1000 800 600 400 200 0 Pseudostreptobacillus hongkongensis DSM 26322T Streptobacillus notomys AHL370-1T Streptobacillus moniliformis DSM 12112T Sneathia sanguinegens CCUG 38322 Pseudoleptotrichia goodfellowii DSM 19756T Oceanivirga miroungae ES3154-GLUT Caviibacter abscessus CCUG 39713T Oceanivirga salmonicida AVG 2115T Leptotrichia hofstadii ENR-0619 Leptotrichia trevisanii 150423-0534 Leptotrichia wadei ENR-0483 Sebaldella termidis ATCC 33386T Fig. 2 Dendrogram including reference main spectra (MSP) of the family Leptotrichiaceae available in the Bruker Taxonomy Database; spectra of Streptobacillus canis IHIT1603-19T, S. felis 131000547T, S. notomytis AHL 370-1T, S. ratti OGS16T, Pseudostreptobacillus hongkongensis DSM26322T, Caviibac- ter abscessus CCUG39713T, Oceanivirga salmonicida AVG2115T, Oceanivirga miroungae ES3154-GLUT, Sebaldella termitidis NCTC11300T, Sneathia sanguinegens CCUG41628T reference strains were recorded using an acetonitrile-formic acid extraction protocol. The dendrogram was generated using the MBT Compass Explorer MSP Dendrogram Creation Standard Method (v1.4) of the MALDI Biotyper OC Software (v3.1, build 66). The database used (DB 8,468, BrukerDaltonics) comprised only strains of Streptobacillus moniliformis including the the type strain DSM 12112T as well as spectra of the depicted Leptotrichia spp.; T, type strain; ENR, European Network for the Rapid Identiﬁcation of Anaerobes (ENRIA) Streptobacillus canis IHIT1603-19T Streptobacillus ra OGS16T Streptobacillus felis 191014123 Streptobacillus felis 131000547T Distance Level 1000 800 600 400 200 0 Pseudostreptobacillus hongkongensis DSM 26322T Streptobacillus notomys AHL370-1T Streptobacillus moniliformis DSM 12112T Sneathia sanguinegens CCUG 38322 Pseudoleptotrichia goodfellowii DSM 19756T Oceanivirga miroungae ES3154-GLUT Caviibacter abscessus CCUG 39713T Oceanivirga salmonicida AVG 2115T Leptotrichia hofstadii ENR-0619 Leptotrichia trevisanii 150423-0534 Leptotrichia wadei ENR-0483 Sebaldella termidis ATCC 33386T Fig. 2 Dendrogram including reference main spectra (MSP) of the family Leptotrichiaceae available in the Bruker Taxonomy Database; spectra of Streptobacillus canis IHIT1603-19T, S. felis 131000547T, S. notomytis AHL 370-1T, S. ratti OGS16T, Pseudostreptobacillus hongkongensis DSM26322T, Caviibac- ter abscessus CCUG39713T, Oceanivirga salmonicida AVG2115T, Oceanivirga miroungae ES3154-GLUT, Sebaldella termitidis NCTC11300T, Sneathia sanguinegens CCUG41628T reference strains were recorded using an acetonitrile-formic acid extraction protocol. The dendrogram was generated using the MBT Compass Explorer MSP Dendrogram Creation Standard Method (v1.4) of the MALDI Biotyper OC Software (v3.1, build 66). extraction protocol. The dendrogram was generated using the MBT Compass Explorer MSP Dendrogram Creation Standard Method (v1.4) of the MALDI Biotyper OC Software (v3.1, build 66). The database used (DB 8,468, BrukerDaltonics) comprised only strains of Streptobacillus moniliformis including the the type strain DSM 12112T as well as spectra of the depicted Leptotrichia spp.; T, type strain; ENR, European Network for the Rapid Identiﬁcation of Anaerobes (ENRIA) PCR analysis The database used (DB 8,468, BrukerDaltonics) comprised only strains of Streptobacillus moniliformis including the the type strain DSM 12112T as well as spectra of the depicted Leptotrichia spp.; T, type strain; ENR, European Network for the Rapid Identiﬁcation of Anaerobes (ENRIA) 12 3 Antonie van Leeuwenhoek (2020) 113:1455–1465 1461 Phylogenetic and phylogenomic analyses The 16S rRNA gene sequence of strain LHL191014123 was derived from WGS and repre- sents a stretch of 1,514 unambiguous nucleotides. This sequence was blasted against the quality-controlled database EzBioCloud (Yoon et al. 2017) and highest similarities to the type strains of S. felis (99.93%), S. canis (98.68%), S. notomytis (98.26%), S. ratti (97.85%), S. moniliformis (97.64%) and P. hongkon- gensis (94.23%), followed by Oceanivirga salmoni- cida (91.10%) and ‘Sneathia amnii’ (90.58%) were found. In a 16S rRNA gene sequence phylogenetic tree (ML algorithm), strain LHL191014123 clustered most closely and in a separate branch together with the type strain of S. felis. The next closely related species was S. canis that grouped as a sister clade to S. felis with high bootstrap support (data not shown). Based on partial nucleotide sequences of the groEL, gyrB Accession numbers and strain deposition Accession numbers and strain deposition The GenBank/ENA/DDBJ accession numbers for the 16S rRNA, groEL, gyrB and recA gene sequences of strain LHL191014123 as well as for the complete genome sequence are MN764137, MN793979, MN793980, MN793981 and genome acc. no. (JABMKT000000000; BioSample SAMN14996772; BioProject PRJNA634464), respectively. Further accession numbers of gyrB sequences from mouth swabs from cats are MT498840-MT498846 and have been published in Matt et al. (2020). Strain LHL191014123 has been deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ), the Culture Collection of the University Gotenburg (CCUG), the Collection of Institute Pasteur (CIP) and the strain collection of the Hessian State Laboratory (LHL) under identiﬁers DSM110500, CCUG74119, CIP111794 and LHL191014123. Genomic features (Suppl. Fig. S1) and recA genes, this topologic position was also identical for the investigated house- keeping genes. A core genome phylogeny of strain LHL191014123 and 20 genomes of the family Lep- totrichiaceae was calculated in EDGAR 2.3 based on MUSCLE alignment as previously described (Eisen- berg et al. 2017b). This resulted in one multiple alignment of 267 core genes per genome (5,607 genes in total), with 95,146 amino acid residues per genome (1,998,066 in total). The Neighbor-Joining algorithm (Fig. 3) as well as the approximately-Maximum Likelihood phylogeny (data not shown) both con- ﬁrmed the taxonomic position of strain LHL191014123 as a member of Streptobacillus felis with S. canis being the closest relative, still with a larger phylogenetic distance. Species identity between the rusty-spotted cat’s strain to S. felis was once more conﬁrmed by mean average nucleotide identity (ANI) values of 99.32% (reciprocal 99.24), which is clearly above the [ 95–96% proposed boundary for identical species (Goris et al. 2007). The draft genome (1,386,907 bp) consists of 163 contigs and possesses 1,345 CDS, 2 rRNA and 38 tRNA. Analysis of further genomic features revealed one prophage (PHAGE_Gor- don_Smoothie_NC_030696, (Zhou et al. 2011)) and 96 tandem repeats (Benson 1999). However, screening for CRISPR regions was negative in contrast to S. felis type strain (131000547T) that was found to possess a relatively large CRISPR region (2,078 bp) with 31 spacers (Grissa et al. 2007). Five genomic islands with a size range 3,220 to 66,556 bp were also identiﬁed (Bertelli et al. 2017). Four islands possess mainly hypothetical proteins and none seems to express pathogenic factors. However, one island (26,565 bp) seems to be associated with transport and metabolism of different substrates including carbohydrates and minerals. Interestingly, genome analysis with Pathogenﬁnder (Cosentino et al. 2013) suggested that S. felis (LHL191014123 as well as 131000547T) has a probability of 0.976 to be a human pathogen since it harbors seven pathogenic families, all of which originate from S. moniliformis, the classical pathogen of the RBF zoonosis. This web-tool predicts the pathogenicity of a submitted genome based on a model that compares its sequence data to a protein family database containing proteins known to be associated with pathogenic or non-pathogenic bacteria. Discussion It is evident from the depicted molecular results and from MALDI-TOF MS analysis that LHL191014123 is an additional strain of S. felis. To our knowledge, this is the second available strain with proper species 12 3 Antonie van Leeuwenhoek (2020) 113:1455–1465 1462 genome in total. GenBank accession numbers are given in parentheses. ‘‘Sneathia amnii’’ and ‘‘Leptotrichia massiliensis’’ were included, however, these taxonomic names have been effectively published but not validly published under the rules of the International Code of Nomenclature of Bacteria. Fusobac- terium nucleatum is used as outgroup. ‘‘T’’ indicating type strain; Bar, 0.01 amino acid substitutions per site Fig. 3 Core genome phylogenetic tree depicting strain LHL191014123 within the family Leptotrichiaceae. Core genes of these genomes were computed in EDGAR 2.3 based on MUSCLE alignments and the Neighbor-Joining algorithm as implemented in the PHYLIP package. The core genome analysis was based on of 267 genes per genome in 17 type species genomes (5,607 in total) of the family Leptotrichiaceae. The core has 95,146 amino acid residues and 1,998,066 bp per Fig. 3 Core genome phylogenetic tree depicting strain LHL191014123 within the family Leptotrichiaceae. Core genes of these genomes were computed in EDGAR 2.3 based on MUSCLE alignments and the Neighbor-Joining algorithm as implemented in the PHYLIP package. The core genome analysis was based on of 267 genes per genome in 17 type species genomes (5,607 in total) of the family Leptotrichiaceae. The core has 95,146 amino acid residues and 1,998,066 bp per The species S. felis has been described from cats as well as from a human patient with contact to cats, suggesting that this microorganism may be a member of the cats’ microbiota with the potential to cause zoonotic infections (Eisenberg et al. 2015a; Matt et al. 2020). The high occurrence (50% found in Matt et al. (2020)) should be considered with respect to the potential role of S. felis both as a cat pathogen and a potentially zoonotic microorganism because cats rep- resent the most popular pet animal species and streptobacillosis is considered a signiﬁcantly under- reported disease. Discussion However, possible reasons why streptobacilli have been infrequently diagnosed may include a lack of awareness of the disease among clinicians, an absence of pathognomic signs of disease in animals, a lack of reliable diagnostics, fastidious growth of the pathogen, susceptibility to most antibi- otics used for empiric therapy, unnoticed animal contact and that this is a non-notiﬁable disease worldwide (Eisenberg 2017). identiﬁcation and an extended genetic and phenotypic knowledge base. Although we could recently show that approximately 50% of randomly selected, mostly healthy domestic cats harbor S. felis (Matt et al. 2020), the isolation of these streptobacilli from cats is a rare exceptional case. A number of studies have indicated dogs and cats as possible vectors of S. moniliformis to humans, especially after mouthing wild rats (Gascard et al. 1967; Maynard et al. 1986; Mollaret 1969; Peel 1993; Wouters et al. 2008). However, isolates have not been stored and phylotypes are not available for most of the mentioned studies. Therefore, these microor- ganisms cannot be veriﬁed as S. moniliformis. Con- versely, 16S rRNA gene phylotypes from one former and additional studies in dogs suggested a much closer relationship of their Streptobacillus OTUs to S. canis than to S. moniliformis (Dewhirst et al. 2012; Xenoulis et al. 2008) (Fig. 1). Relatively few dogs have suffered from streptobacillosis (Das 1986; Ditchﬁeld et al. 1961). identiﬁcation and an extended genetic and phenotypic knowledge base. Although we could recently show that approximately 50% of randomly selected, mostly healthy domestic cats harbor S. felis (Matt et al. 2020), the isolation of these streptobacilli from cats is a rare exceptional case. A number of studies have indicated dogs and cats as possible vectors of S. moniliformis to humans, especially after mouthing wild rats (Gascard et al. 1967; Maynard et al. 1986; Mollaret 1969; Peel 1993; Wouters et al. 2008). However, isolates have not been stored and phylotypes are not available for most of the mentioned studies. Therefore, these microor- ganisms cannot be veriﬁed as S. moniliformis. Con- versely, 16S rRNA gene phylotypes from one former and additional studies in dogs suggested a much closer relationship of their Streptobacillus OTUs to S. canis than to S. moniliformis (Dewhirst et al. 2012; Xenoulis et al. 2008) (Fig. 1). Relatively few dogs have suffered from streptobacillosis (Das 1986; Ditchﬁeld et al. 1961). OTU sequences of cats that are most closely related to S. 123 Conclusion Open Access This article is licensed under a Creative Com- mons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any med- ium 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/. This is the second isolation of S. felis in a diseased cat species. A preliminary sampling of cats revealed that this microorganism is frequently found in the orophar- ynx and that cats represent a reservoir for S. felis. However, this is further evidence that this species is cat speciﬁc but with a broader distribution in feline hosts than previously thought. Further studies are necessary to elucidate the role of S. felis in domestic and other cat species in order to better estimate its zoonotic potential. Acknowledgements Open Access funding provided by Projekt DEAL. We like to thank all cat owners for their participation in the prevalence assessment. For excellent technical assistance, we thank Mersiha Curic, Matrin Dyk, Katharina Engel, Jens Heinba¨cher, Ulrike Kling and Marie- Luise Sonneborn. The Hessian Ministry for the Environment, Climate Change, Agriculture and Consumer Protection (HMUKLV) supports the Hessian State Laboratory. Conﬂicts of interest Not applicable. Conﬂicts of interest Not applicable. Ethics approval There are no ethical issues associated with this manuscript. Animal husbandry fulﬁlled ethical standard guidelines according to the code of ethics and animal welfare of the World Association of Zoos and Aquariums (WAZA; https:// www.waza.org/priorities/animal-welfare/). Sampling of the rusty-spotted cat followed veterinary euthanasia of the diseased animal. Consent to participate All authors gave their consent to participate in this study. Consent for publication All authors gave their consent to publish results from this study and to be listed as a co-author. Availability of data and material All data have been made fully available to the public. Interestingly, a novel diagnostic immune-histology tool for the detection of S. moniliformis turned out to reveal negative results throughout. The method was found to successfully detect experimentally infected mice (data not shown). Because the lack of suitable di- agnostics for the detection of streptobacillosis is often referred to as a diagnostic dilemma (Mahmoodi et al. 2016; Rumley et al. 1987), the novel IHC assay represents a promising diagnostic tool to improve this situation and to identify S. moniliformis in situ. The lack of binding in the here presented case suggests deviant epitopes in S. felis. Discussion felis were previously lacking, but have recently been found in half of the investigated cats (Matt et al. 2020) and also closely related bacterial species (uncultured ‘Leptotrichia’, ‘Leptotrichiaceae’) have been detected at various body sites (Older et al. 2019; Sturgeon et al. 2014). One study mentions two Streptobacillus isolates but without any further iden- tiﬁcation or disease association (Whyte et al. 2017). S. felis has ﬁrst been isolated from a cat with acute bronchopneumonia and a myocardium with multifocal haemorrhages on the endo- and epicardium (Eisenberg et al. 2014). In this second report, again lungs were predominantly affected, but streptobacilli were iso- lated from all major organs pointing towards an agonal spread of these bacteria or an early septicemia. Likewise, the next closely related species, S. canis, has recently been found to also constitute a member of 123 12 123 Antonie van Leeuwenhoek (2020) 113:1455–1465 1463 canine oral microbiota (Matt et al. 2020) and has been isolated from a phlegmon on a dog’s hindleg (Eisen- berg et al. 2020b). Hypothesizing that bite wounds are often caused by oral microbiota (Abrahamian and Goldstein 2011), one can speculate that streptobacilli from cats might occasionally also be involved in wound infections. Funding This research received no speciﬁc grant from any funding agency in the public, commercial, or not-for-proﬁt sectors. The Hessian Ministry for the Environment, Climate Change, Agriculture and Consumer Protection supports the Hessian State Laboratory. 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W2566618086.txt	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0168405&type=printable	en		Using the Three Delays Model to Examine Civil Registration Barriers in Indonesia	PloS one	2,016	cc-by	7,968	RESEARCH ARTICLE Using the Three Delays Model to Examine Civil Registration Barriers in Indonesia Cyril Bennouna1, Brooke Feldman2, Rahmadi Usman1, Rama Adiputra1, Santi Kusumaningrum1, Lindsay Stark2* 1 Center on Child Protection and Wellbeing, Universitas Indonesia (PUSKAPA), School of Social and Political Sciences, Depok, Indonesia, 2 Program on Forced Migration and Health (PFMH), Mailman School of Public Health, Columbia University, New York, NY, United States of America * ls2302@cumc.columbia.edu a11111 Abstract OPEN ACCESS Citation: Bennouna C, Feldman B, Usman R, Adiputra R, Kusumaningrum S, Stark L (2016) Using the Three Delays Model to Examine Civil Registration Barriers in Indonesia. PLoS ONE 11 (12): e0168405. doi:10.1371/journal. pone.0168405 Editor: Jacobus P. van Wouwe, TNO, NETHERLANDS Received: August 7, 2016 Accepted: November 29, 2016 Published: December 19, 2016 Copyright: © 2016 Bennouna 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 Three Delays Model has proven a useful framework for examining barriers to seeking obstetric care and preventing maternal and child mortality. This article demonstrates the applicability of the Three Delays Model to the case of civil registration in rural Indonesia and examines ways that efforts to strengthen civil registration services can draw on lessons from maternal and child health programming. Twenty focus group discussions were conducted using a participatory ranking exercise in four Indonesian districts. Focus groups were stratified into four groups: (1) government officials involved in civil registration, (2) civil society organization members that assist communities in civil registration, and (3) female and (4) male community members. Transcripts were analyzed using constant comparative method and thematic analysis, revealing barriers that communities commonly faced in accessing civil registration services. In examining the categories and themes related to these barriers, the research team found a significant overlap with the factors and phases of the Three Delays Model. Participants were delayed from seeking registration services by a range of sociocultural factors and by the perceived inaccessibility and poor quality of services. Once they decided to seek care, long distances to services and poor transportation options delayed their access to registration offices. Finally, a series of bottlenecks in service provision created extended delays once applicants reached registration offices. Ownership of civil registration documents in Indonesia remains exceptionally low, with just over half of children and youth possessing a birth certificate. To strengthen civil registration and health systems more generally, it is important to understand the factors that enable and constrain civil registration, how these factors relate to one another, and how they change over a child’s life. Data Availability Statement: All relevant data is located in a zip file at Figshare with the following DOI: https://dx.doi.org/10.6084/m9.figshare. 4224204 The zip file is password-protected with the password: PlosOne. Introduction Funding: This research was supported by the Australian Government’s Australia-Indonesia Partnership for Justice (AIPJ) [grant number AC43600/AIPJ/PUSKAPA/SL-18-05-14]. The URL is http://www.aipj.or.id/en/main. The funder had no role in the study design, data collection and Around the world, nearly one in three children under the age of five do not have a birth certificate [1]. Researchers at the World Health Organization have referred to the enormous gap in civil registration coverage globally as “the single most critical development failure over the past 30 years” [2]. Countries such as Indonesia, with decentralized authority, large rural population, and remote communities, have struggled to expand access to quality civil registration services. PLOS ONE \| DOI:10.1371/journal.pone.0168405 December 19, 2016 1 / 15 The Three Delays Model and Civil Registration Barriers analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. As a result, large domestic inequities in birth certificate ownership persist, depending on factors such as urbanization, income, and education [3, 4]. A 2013 review, for example, found that the proportion of Indonesian children without birth certificates in rural areas was twice that of children in urban areas [4]. Living in the poorest quartile also significantly decreased children’s chances of having a birth certificate, in some provinces by a factor of nine. Another study reported that application costs, distance from services, and the complexity of application procedures were the three most critical barriers to obtaining birth certificates in Indonesia [5]. Addressing these inequities in civil registration is a key component of the Indonesian government’s Medium Term Development Plan, which aims to increase birth certificate coverage to 85 percent of all children under 18 years old by 2019, with a specific focus on vulnerable populations [6]. According to the Indonesian Central Statistics Agency (BPS)’s most recent national estimate, the current coverage for children across the country is 63 percent [7]. Narrowing this coverage gap, and strengthening the country’s civil registration system more generally, will require a more comprehensive understanding of the dynamic factors that enable and constrain birth registration for Indonesia’s vulnerable populations. In addition to being a development priority, strengthening civil registration is increasingly seen as a core investment in public health as well [2]. Civil registration documents can enable individuals to access basic services, and in turn civil registries can feed into national vital statistics systems, which allow government bodies to plan health programs and monitor progress more effectively. Without comprehensive and timely birth registration, for example, it can be challenging to surveil fertility and fertility-related trends, such as child mortality [8]. Countries with stronger civil registration and vital statistics tend to have better health outcomes, including lower maternal mortality ratios and child mortality risk [9]. In Indonesia, although the health sector has no formal role in birth registration, studies have found children’s birth certificate ownership to be positively associated with being delivered in a health facility or by a skilled health attendant, and use of postnatal care [10]. With the Indonesian government’s renewed commitments to improving civil registration and maternal and child health under its National Medium Term Development Plan and the Sustainable Development Goals (SDGs), locating birth registration within a healthcare framework can help to reinterpret the challenges the country faces and can inform new strategies that strengthen both systems at once [6]. Thaddeus and Maine’s Three Delays Model may provide a useful conceptual framework for understanding the relationships between the barriers to civil registration in Indonesia [11]. The model was originally developed to characterize factors contributing to maternal mortality between the onset of an obstetric complication and its outcome. Similar to the case of birth registration, maternal health studies that preceded the Three Delays Model had identified cost, distance, and quality of care as barriers to seeking obstetric care, but lacked a broader framework to explain the relationships between these barriers, and to contextualize them within the larger system failures that made quality care inaccessible to many community members. In addition to providing a useful heuristic for interpreting the barriers to civil registration, the Three Delays Model also offers an opportunity to locate intersections between Indonesia’s civil registration and vital statistics system and its healthcare system and to identify ways these systems can mutually reinforce one another. Guiding Framework: The Three Delays Model The Three Delays Model is comprised of three phases. Each phase is modified by factors affecting utilization and outcomes, including socioeconomic and culture factors, accessibility, and the quality of services. Phase I centers on the decision to seek care, which can be influenced by societal norms and poor knowledge. Resources such as time and money often limit the ability PLOS ONE \| DOI:10.1371/journal.pone.0168405 December 19, 2016 2 / 15 The Three Delays Model and Civil Registration Barriers of those who intend to use social services. Phase II includes identifying and traveling to facilities, and is influenced by the distance and transportation infrastructure between villages and the appropriate facilities. Phase III involves receiving adequate and appropriate care at facilities. Factors in this phase affect both utilization of services and outcomes. Provider competency, availability of commodities, and communication influence this third phase. The Three Delays Model has been widely used across development settings from Malawi to Haiti to Indonesia to explain high rates of maternal mortality [12–16]. In India, Tanzania, and Uganda, the model was expanded to inform interventions to reduce neonatal and child mortality [17–19]. Evidence has shown the great value of this model in a variety of contexts. Although it has usually been applied to understand factors related to emergency medical services, many similar social, behavioral, and systems factors constrain access to non-emergency services, such as civil registration. This study drew on focus group discussions to explore the applicability of the Three Delays Model to understand shortcomings in Indonesia’s birth registration system. The application of this model to the context of civil registration may contribute to theorizing solutions for Indonesia’s enduring birth registration gap. Methods Setting Qualitative data were collected from four districts across North Sumatra (SUMUT) and West Nusa Tenggara (NTB) in June and July 2015. SUMUT, a large, mountainous, and heavily forested area, is the fourth most populous province in Indonesia, with over 13.5 million people [20]. About 10 percent of SUMUT’s population lives below the village-based Indonesian poverty line, slightly better than the national average of 14 percent. Langkat is the northernmost district in the province with a population of just under one million. Asahan is southeast, located on the coast of the Strait of Malacca, with a slightly smaller population of about 670,000. Both districts are predominantly Muslim with a diverse ethnic makeup comprised of Malays, Chinese, Batak, and Nias. Rice, coffee, and palm oil cultivation account for a large portion of the economy. Asahan also depends largely on its fishing industry. The province of NTB lies just east of Bali with a population of about 4.7 million, of which 16 percent live below Indonesia’s village poverty line [20]. Lombok Utara is the northernmost district of NTB’s main island with a population of about 200,000. Lombok Tengah is directly below it and has a population of about 900,000. Both districts have a Muslim, ethnically Sasak majority. Rice farming is the principal source of income for most households, though many supplement their income by fishing, weaving, or working in the tourism industry. NTB is also home to many migrant workers, usually with jobs in domestic labor in countries such as Malaysia and Saudi Arabia. Poor road networks, together with seasonal flooding and inadequate public transportation, restricted access to the district capitals for many community members in the four districts. Data collection and sample Data collection was led by the Center on Child Protection and Wellbeing at Universitas Indonesia (PUSKAPA) and Columbia University. Focus group discussions were conducted in Bahasa Indonesia by a PUSKAPA researcher with a lead researcher and a note taker present. All sessions were also recorded digitally. After each session, the team reviewed the recordings and field notes and held a debriefing session to address issues during data collection. Focus groups included an adapted participatory ranking methodology (PRM) exercise to engage participants more actively during data collection while generating rich and contextualized data [21]. The framing question was “what barriers to obtaining a legal identity document do PLOS ONE \| DOI:10.1371/journal.pone.0168405 December 19, 2016 3 / 15 The Three Delays Model and Civil Registration Barriers communities identify as being the most critical to overcome.” In the PRM, participants ranked these barriers in order of priority. Focus groups in each district were stratified into four groups. The first consisted of district officials from the Office of Population and Civil Registration, the Office of Religious Affairs, and the Religious Court. Officials were included in the study if their primary job responsibility involved delivering legal identity services. The second group consisted of members from an all-female civil society organization (CSO) who provided information and support to community members applying for birth and marriage certificates, as well as marriage legalization for religiously married couples who had their children before being legally married. These CSO members also helped the research team to identify participants for the other two strata: male and female community members who were married with children. Ethical considerations Atma Jaya University’s Ethical Review Board reviewed and approved the study protocols. After the purpose of the study was explained to them, all participants provided written, informed consent agreeing to participate in the study and have their voices recorded. Participants were not paid, but were thanked with small gift bags including school supplies or groceries worth USD 5, which was deemed appropriate for the context. Analysis All of the recordings were transcribed verbatim. Research assistants who were present during data collection verified and corrected all transcripts. Half of the transcripts were translated into English to allow the Columbia researchers to guide the PUSKAPA researchers through the first phases of analysis. In-country researchers also received training on Nvivo10 [22]. The analysis team, which consisted of three researchers from Columbia and five researchers from PUSKAPA, reviewed the English transcripts first to resolve any issues with the translation and then to familiarize themselves with the data. The team then open-coded a sample of the English transcripts in Nvivo10. Using constant comparative method, the team reviewed their opencodes, held discussions, wrote memos, and developed an initial codebook [23]. The team then applied the initial codebook to the entirety of the English transcripts, refining the codes and definitions iteratively. After resolving discrepancies in the codes and the application of the codebook, the PUSKAPA team coded the remaining Indonesian transcripts. Without reference to a preexisting theory or model, the codes were then classified into different categories corresponding to larger themes in the data. In examining the categories and themes, the research team found significant overlap with the factors and phases of the Three Delays Model. The data were then revisited using thematic analysis to inform the results presented in this article. Results A total of 20 focus group discussions (FGDs) were conducted in 11 villages across the four districts. The research team met with four groups of district-level government officials, including providers of birth and marriage certificates and judges who were responsible for presiding over marriage legalizations and divorces. Most of these officials were men. The two CSO focus groups consisted entirely of female paralegals with a median age of 43. Most participants in the seven FGDs with the female stratum of community members identified as housewives. Their median age was 36. Of the seven FGDs with male community members, most were farmers, with some working as fishermen, particularly in SUMUT. Their median age was 44. Despite generally low birth certificate ownership in these villages, the majority of the participants’ children had birth certificates. Results from the FGDs indicated that financial issues, access to information, cultural beliefs and practices, logistics, and perceived poor quality of services posed major challenges for PLOS ONE \| DOI:10.1371/journal.pone.0168405 December 19, 2016 4 / 15 The Three Delays Model and Civil Registration Barriers Table 1. Most Common Barriers to Civil Registration in SUMUT and NTB, Based on PRM. Government Officials Categories Freq CSO Freq Female Community Members Freq Male Community Members Freq Totals Freq % Financial 5 1 7 4 17 85 Procedures and requirements 0 2 5 3 10 50 Lack of information 1 1 3 4 9 45 Quality of services 1 1 2 2 6 30 Time needed for application 0 0 3 2 5 25 Cultural practices 1 0 1 3 5 25 Distance 1 0 0 1 4 20 Perception and motivation 3 0 0 1 4 20 doi:10.1371/journal.pone.0168405.t001 communities to register their children’s births (Table 1). Of all the barriers to registration, participants across strata mentioned financial issues the most frequently, with the exception of male community members. The complex procedures and requirements for registration were also major concerns for community members and CSO members. Participants also mentioned the lack of information about registration procedures as a major barrier, especially in NTB. The following sections describe how these categories contribute to themes and specific delays in birth registration (Fig 1). Phase I: Deciding to Apply for Birth Certificates The first delay phase, the decision to apply for a birth certificate, reportedly occurred for a number of socioeconomic and cultural reasons, such as the availability of resources, local traditions, and the requirement of a birth certificate to access services, such as schooling. Fig 1. Adaptation of the Three Delays Model for Civil Registration. doi:10.1371/journal.pone.0168405.g001 PLOS ONE \| DOI:10.1371/journal.pone.0168405 December 19, 2016 5 / 15 The Three Delays Model and Civil Registration Barriers Across strata, most participants believed that birth certificates were important for their children’s futures. They attributed this importance to the use of certificates for specific administrative activities, typically school enrollment and passport applications for the hajj pilgrimage or migratory work. Parents in all study areas, however, often did not begin applying for birth certificates until they were needed for one of these activities. As one female participant from SUMUT explained, “Children need to have a birth certificate in order to continue their education. . . They only start applying for the certificate when they are about to enroll in high school.” Knowing the tendency for community members to delay their marriage and birth registration applications, religious court officials in SUMUT even had a policy of expediting marriage legalization cases that could enable family members to go on hajj. As one official confirmed, “The ones applying for hajj pilgrimage are sometimes prioritized.” Parents had several reasons for delaying the application process. Many participants claimed they could not afford the costs associated with registration, such as transportation to registration offices, duty stamps for official letters, and photocopies of required documents. With insufficient income to pay for their family’s various needs, parents weighed the opportunity costs of these purchases against more immediate needs. As one participant narrated, “My daughter needed to get medical treatment. She had an ear infection and a tonsil problem. Which one is more important? Surely getting the medical treatment is more important than getting the certificate.” (Female community member, SUMUT). Community members also reported finding the registration process complex, confusing, and time-intensive. Many did not know how to start an application or what was required. As one CSO member from SUMUT described, “Some parents have no idea how or where to acquire certificates.” Participants often attributed the lack of knowledge about registration procedures to low educational attainment in their communities. According to one male participant from NTB, “Many village people don’t go to school, they don’t dare to [apply for documents] by themselves, and nor do we. Most of the people in the sub-villages are like that.” Others thought the problem resulted from insufficient efforts by civil registration officials and other authorities to inform communities about the official registration process. As one male participant from SUMUT opined, “They should explain the process well and make it easy. If they did that, then maybe all of us would have certificates.” Obtaining a marriage certificate–which until early 2016 was required in order to include the father’s name on a child’s birth certificate–was particularly challenging for many parents. This challenge was especially significant in NTB, where polygamy is common and remarrying several times is customary. Rather than paying the costs of divorce and remarriage, couples simply forego registering their marriage. One male participant from NTB explained, “I’ve gotten married five times, but no marriage certificate at all. Not even one.” PLOS ONE \| DOI:10.1371/journal.pone.0168405 December 19, 2016 6 / 15 The Three Delays Model and Civil Registration Barriers Without the parents’ marriage certificate, children were provided birth certificates without a father’s name, which many community members found stigmatizing. Not having a marriage certificate delayed birth registration because many unregistered couples did not want their children’s birth certificates to exclude the father’s name. One CSO member recounted the following story about a second wife in a polygamous marriage who had asked for help registering her child: “She wanted to obtain a birth certificate for her child because her child wanted to go to school. When the birth certificate was issued, the child was enrolled in the school, but the teacher asked why the father’s name was not on the birth certificate. The wife came to me and asked me [why that was the case] and I said because she had no marriage certificate as required for a [complete] birth certificate, and told her to explain that to the teacher. The officer from registration office does not want to issue birth certificates with the mother and father’s name if the parents do not have a marriage certificate. [The mother] looked agitated, upset, and disappointed.” (CSO member, SUMUT). Many participants also mistakenly believed that marriage certificates were mandatory for registering their children’s births. As one male participant from SUMUT explained, “[S]ome people only have a religious wedding and thus do not have a marriage certificate, and cannot apply for [their children’s] birth certificates.” The legalization process that couples without marriage certificates had to undergo at the religious court in order to produce a birth certificate with both parents’ names required them to bring witnesses, visit the court several times, and, in many cases, spend additional money. As one female participant from SUMUT explained, “They tell us to bring a witness. That means we have to pay for the witness. I understand the need for a witness but I don’t like the idea of having to pay for the witness to go to court. I need that money.” Marriage certificates were not the only problematic requirement. Many parents had expired identity cards or had not procured the appropriate documentation, such as a birth notification letter. In SUMUT, for example, a migrant family originally from Lombok was turned away when applying for their family card, which is required for a child’s birth certificate. They had not known to update their residential status with an official migration letter before travelling to the registration office. As a CSO member recounted, “So this person migrated from Lombok and wanted to apply for a family card. But this person was asked to return to Lombok to obtain a statement letter for relocation. This person does not have any money; thus, the person does not own any family card up to this day.” (CSO member, SUMUT). These additional complications factored heavily into decisions to seek birth registration services. Phase II: Identifying and Reaching Civil Registration Offices Once parents decided to register their children’s births, they had difficulty accessing registration offices in the district capital. The delay in accessing services at government offices was primarily due to a combination of factors regarding information, infrastructure, and resources. PLOS ONE \| DOI:10.1371/journal.pone.0168405 December 19, 2016 7 / 15 The Three Delays Model and Civil Registration Barriers Residents in rural areas rarely had contact with the district capital. Many did not know which offices to visit, let alone where they were located. As one male participant from NTB noted, “We lived in the sub-village and [birth certificates] are made in the city. We don’t even know where the office is.” Participants in all study areas reported that the distance to the registration offices and the time needed for the commute delayed birth registration. One community in NTB even held demonstrations to request services closer to their village. Applicants in Langkat had to drive up to three hours each way to reach the registration offices. As one male participant from SUMUT elaborated, “The problem is that the distance is too great. We don’t have the money, and we are too busy to make the time.” Poor roads and a lack of public transportation made trips to civil registration offices an uncomfortable and costly endeavor. As one CSO member from NTB reported, “Some roads there are still in a poor condition, and also, [applicants] have to go back and forth by motorbike taxi. People who don’t have motorbikes take days to process [their certificates].” To avoid commuting to district offices, communities submitted their applications to one individual, usually the village leader, who then delivered the applications to the registration office. These individuals usually charged a transportation fee, but, in order to reduce costs, tended not to submit the applications until they had several at once. This created a further registration delay. As one male participant from SUMUT reported, “I had to pay money for [the village leader’s] transportation and accommodations. The official here had to wait until he had enough applicants before he could go to [the district capital].” In addition to belaboring birth registration, these obstacles also fed back into the first phase delay, as they discouraged people from wanting to apply in the first place. Phase III: Submitting the Application and Receiving Document Those who managed to overcome the challenges above often faced additional challenges after arriving at district registration offices. Errors in required documents were common, such as a misspelled name or an incorrect date. Because of strict policy regulations and input procedures within the civil registration management information system, civil registration providers rejected applications with spelling or date inconsistencies between documents. As one participant recalled, “I once saw this at a civil registration office, far away. This person was there from morning to evening and yet her certificate wasn’t finished. She cried and begged. She only had a little mistake and they refused to help her.” (Female community member, SUMUT). PLOS ONE \| DOI:10.1371/journal.pone.0168405 December 19, 2016 8 / 15 The Three Delays Model and Civil Registration Barriers According to participants, many of these inconsistencies resulted from simple clerical errors in the documents required for registration applications, while others were influenced by cultural practices. In Lombok, parents traditionally add their first child’s name to their own, and also change their names when they make the pilgrimage to Mecca. If parents use their new names on any of the documents required for the newborn’s birth certificate, the application can be rejected. Furthermore, if this happens and the officer does not catch the discrepancy, birth certificates may be printed with the parents’ non-legal names, potentially creating problems in the future. Acceptance of child marriage by local leaders was another cause of inconsistent documents. One CSO participant described an underage couple that had wedded without registering their marriage. When they wanted to produce a birth certificate for their child, they falsified their age to comply with Indonesia’s legal age of marriage. As one CSO member from NTB explained, if child applicants are already religiously married, sometimes officer “mark up their age—even if the kids are still underage—to get [officially] married.” Many community members reported that they could not submit their applications even when they had all the requirements. Offices were often understaffed and did not have enough blank certificates available, and several reportedly closed early. One CSO member from NTB shared a typical experience: “Even to submit the requirements, we have to queue for the whole day, and we would still not necessarily get our turn on that day. Sometimes we have to wait until the day after, and go back and forth again.” Participants reported that, once they submitted their applications, they waited weeks and sometimes months before picking them up. Registration officials reportedly had inadequate human resources and budget available for processing the volume of applications they received. Officials felt the practice of waiting until a birth certificate was required to begin the application made community members rush unnecessarily through the process. As one official from SUMUT said, “[t]here are always people demanding immediate services every day. If possible,” the official continued, applicants want their birth certificates “processed in a day. It would be even better for them if we were able to process [their applications] within half an hour.” Community members reported that there was no system to notify applicants when documents were ready. Applicants had to check their status with the officer repeatedly, often in person. As one female community member from SUMUT said, “I don’t mind waiting if there is a certainty that the birth certificate will be issued. Whether it’s three days or one week. What I don’t like is having to go to the office all the time [to check on the status of my application].” Bribes, solicited and unsolicited, were reported as being used to expedite services across study sites. One male participant from NTB expressed a common sentiment that community members were PLOS ONE \| DOI:10.1371/journal.pone.0168405 December 19, 2016 9 / 15 The Three Delays Model and Civil Registration Barriers “afraid their documents would not be processed if they don’t give a tip.” Participants in SUMUT reported the same tendency. As one male community member explained, “You need to pay the money first. All the other requirements such as copies of marriage certificates, family cards, and such things can follow later, so long as you pay them some money.” Poorly communicated information, errors in printed documents, long wait times, and a lack of transparency in the application process were interpreted by many as poor quality service. This inadequate service also compounded the Phase II delay, as applicants were forced to travel to registration offices several times to complete their applications or to check their application status. The expectation of poor quality service and the costly and lengthy travel times in turn fed back into Phase I delays, in which parents decided to forestall registering their children’s births. Discussion Applicability of the Three Delays Model to Civil Registration These results demonstrate the relevance of Thaddeus and Maine’s model to civil registration in rural SUMUT and NTB. The focus group discussions confirmed findings from previous studies of civil registration in Indonesia, where cost, distance, application information, and a complex application process obstructed access to birth certificates for children in poor and rural settings [5]. Unsurprisingly, financial barriers were the most frequently mentioned and often the highest ranked factor in the PRM exercise. By examining these barriers as part of an integrated framework, the study also found that the significance of the different barriers varied not only according to the applicant’s socioeconomic standing, but across the child’s life course as well. Without question, the cost of birth certificates discouraged many parents in low-income households from registering births during the first few years of a child’s life. Because Indonesian authorities and service providers rarely required young children to have birth certificates to benefit from government programs, parents often preferred to spend their money on more immediate concerns, such as medical care. In fact, many participants said that they delayed registering their children’s births until the certificate became valuable to them, such as during school enrollment, even if it meant paying more money. Parents’ willingness to spend more money for delaying birth registration may help to explain why previous attempts to remove application fees and impose late-registration fines did not lead to rapid increases in birth registration [4, 20]. As children grow older, their birth certificates confer more benefits, and so the monetary investment becomes more worthwhile for their parents. A UNICEF review of data from 161 countries found that birth certificate coverage tended to increase with age around the world, in part because schools and health providers required them [1]. A recent study of three sub-districts in Indonesia found a similar trend, though it also noted that there was no sizeable increase in the proportion of children receiving a birth certificate between the ages of four and six, as would be expected if initial school enrollment were the principal motivating factor for registration [24]. While birth certificates are not required to access healthcare or social protection benefits in Indonesia, their use for creating school diplomas, and for scholarship applications, marriage certificates, and passports contribute to their increasing value as children age. This finding suggests that in order to address Phase I delays and achieve improvements in the demand for timely birth registration, authorities should focus on enhancing the value of PLOS ONE \| DOI:10.1371/journal.pone.0168405 December 19, 2016 10 / 15 The Three Delays Model and Civil Registration Barriers birth certificates earlier in children’s lives rather than on deterrence policies that increase the financial burden of applying, such as late registration fees. Connecting birth certificates more closely to programs that directly benefit young children would ensure that registration becomes valuable to parents earlier in their children’s lives. At the same time, requiring that children present birth certificates to benefit from basic services risks excluding the most marginalized populations from those services, especially considering the challenges that many families face in accessing civil registration services [3, 24]. In an effort to incentivize early registration without adversely affecting children’s access to basic services, some initiatives in Indonesia have created special benefits for children with birth certificates, such as discounts on goods related to school, health, and extracurricular activities, though it is still unclear whether these benefits have contributed to increased demand for registration [25]. Applying the Three Delays Model also helps to parse the multiple effects that single factors may have on birth registration. The long distance to services for instance directly created delays in registration. If an applicant had to drive three hours to the district registration office to submit an application, rather than submitting it locally, that was three hours that the registration official was delayed in reviewing the application. The expectation of long drives also led parents to delay preparing or submitting their applications. By implication, an investment in reducing the distance between applicants and services should have a dual benefit: reducing the delay in applying for birth certificates and in processing them. The finding that parents’ distance from registration services had multifold effects on their registration delays provides support for the model of delivering civil registration to the sub-district and village levels through integrated and mobile services. It also supports policies that provide authority for registration to officials below the district level [4, 24]. Similarly, the framework of delays helps to reveal feedback effects between barriers. In Phase I, the decision to delay birth registration until the moment a child needed the certificate led parents to rush the application process. This in turn led to complications with Phase III, including errors in application forms, dissatisfaction with processing times, and negative exchanges with registration officials. These experiences then discouraged future applications, contributing to the Phase I delays. Examining the relationship between these barriers provides a deeper understanding of how communities experienced the registration process, with important insights for civil registration strengthening programs. One implication is that efforts to build demand for birth registration, for instance by increasing community awareness about the uses of birth certificates, are likely to be hamstrung without commensurate efforts to improve the supply of capable and friendly registration staff. Applying Approaches in Primary Health Programs to Improve Civil Registration Applying the Three Delays Model to birth registration encourages cross-sector learning. While this article does not intend to compare obstetric care with civil registration, the several decades of experience tackling the barriers of distance, cost, and quality of care in the reproductive health field can be adapted to inform effective interventions for civil registration. Using a shared framework highlights the natural overlap between birth delivery and birth registration and promotes leveraging existing health approaches, such as voucher systems to overcome transportation barriers for the poor [26]. Crossover already occurs between primary health and civil registration. In Ghana and Ethiopia, community health workers were trained to register births to reduce the transportation burden in remote areas [27–29]. In Bangladesh, birth registration was incorporated into the Expanded Program for Immunization, and health workers helped to collect and transfer birth PLOS ONE \| DOI:10.1371/journal.pone.0168405 December 19, 2016 11 / 15 The Three Delays Model and Civil Registration Barriers certificate application materials [27]. UNICEF has worked with dozens of countries in similar ways to integrate birth registration into primary health facilities and the provision of routine health services [27]. Primary health interventions are also relevant to the Indonesian civil registration system. The country’s civil registration system parallels and often intersects with the primary healthcare system, creating opportunities for the two systems to mutually reinforce one another. As a result of efforts to reduce rural, maternal mortality dating back to 1989, midwives are often the most accessible healthcare provider for households in Indonesia [24, 30]. In addition to attending and reporting births and deaths within health facilities as well as in private homes, midwives are also responsible for signing birth notification letters, which are required for birth certificate applications. Despite their convenient access to birth certificate applicants, health staff are not authorized to register births and do not have any official responsibility for supporting birth registration [24]. Still, mothers sometimes ask their attending midwives for counsel or assistance with birth registration procedures as there are typically no civil registration service providers available in villages or even sub-districts. One study found that in-facility births, births delivered by a skilled health attendant and accessing postnatal care in Indonesia were all associated with child birth certificate ownership [10]. In many districts and cities across Indonesia, local governments have started to formalize the relationship between the health and civil registration sectors, whether through local regulations or formal cooperation agreements. In the city of Surakarta, staff at hospitals and birthing centers facilitate birth registration and input birth data into the population registry [25]. In the district of Bireuen in Aceh, health workers help patients prepare certificate applications, and completed birth certificates are distributed through health facilities [31]. The Australian Department of Foreign Affairs and Trade’s Governance for Growth (KOMPAK) program is collaborating with the Ministry of National Development Planning (BAPPENAS) and the Center on Child Protection and Wellbeing at the University of Indonesia to learn from these examples and develop reproducible models for institutionalizing civil registration and vital statistics within basic services systems, including the healthcare system [24]. For the moment, however, the Ministry of Health in Indonesia remains completely independent of the Ministry of Home Affairs’ civil registration activities. Further fostering relationships between these ministries could be particularly useful for the Indonesian context and should be explored further. The applicability of the Three Delays Model to connect primary healthcare and civil registration does have limitations. While the Three Delays Model in this study looks at birth certificate ownership, registration is generally an intermediary output to access other social services like education. Thus, improving the accessibility and the quality of the civil registration system does not automatically translate to increasing the utilization and positive outcomes of health or other basic services. This diverges from the healthcare context, where improving the quality of obstetric services directly affects the ultimate outcome of reducing maternal mortality. As such, reducing delays such as Phase I’s “decision to seek care or services” may be more difficult to achieve in the case of birth certificate ownership, whose negative outcome is not as severe as delaying obstetric care. The different implications of the results found by using the Three Delays Model should be accounted for when determining solutions to overcome barriers and similarly to adjust expected outcomes for reducing maternal mortality versus increasing birth certificate ownership. Limitations Limitations for this study are primarily due to our selection of study sites and participants. The CSO members who helped the research team identify study participants also assisted PLOS ONE \| DOI:10.1371/journal.pone.0168405 December 19, 2016 12 / 15 The Three Delays Model and Civil Registration Barriers community members to apply for birth certificates and played a role in the delivery of these services. This selection method likely left out more vulnerable community members, who were unknown to the CSO workers, and whose participation may have identified additional barriers. The majority of the study participants had registered their children’s births. Participants were thus fairly aware of the civil registration system and how to navigate it successfully. Part of this knowledge may have been due to the site selection. All of the communities visited had received civil registration awareness campaigns and integrated mobile service events to register children. Although none of the participants had directly accessed the mobile service units, there may have been a spillover effect making them more aware of the importance of and requirements for birth certificates. Our sample may have had greater knowledge of the legal identity process and perceived importance of documents. Attempts were made to reduce this bias by also including randomly selected community members who participated in a household survey simultaneous to this research. Finally, this study cannot be generalized to the broader Indonesian civil registration system as it excluded important aspects of that system, most notably death registration. The Three Delays Model may be useful in characterizing the challenges related to death registration, especially considering the health sector’s traditional responsibility of ascertaining and certifying cause of death. Such an analysis is beyond the scope of this article. Conclusions Identifying and understanding the relationship between the barriers to accessing high quality social services is crucial. The Three Delays Model, originally developed to conceptualize factors contributing to maternal mortality, helps capture this complexity in the Indonesian civil registration system. Dividing the process of applying for civil registration documents into three phases offers useful insight for policies to promote civil registration ownership. Ultimately, in order to reach its ambitious target of registering 85 percent of children’s births by 2019, Indonesia’s government will likely need to formalize the role of the primary healthcare system in working with the civil registration sector [6, 24]. Not only do their shared factors affecting utilization and outcome of public services help inform programs, but the facilities and staff supporting maternal, neonatal, and child care also present an indispensable opportunity to incorporate birth registration as a basic service. As Indonesia renews its commitment to civil registration and population health under its National Medium Term Development Plan and the SDGs, this inter-sectorial work may consolidate efforts to achieve both of these objectives. Acknowledgments The authors would like to thank PEKKA (an NGO working on empowerment of female heads of households) for its contribution to data collection, as well as PUSKAPA’s research team Wenny Wandasari, Prisilia Riski, and Harriz Jati. We also thank Kathryn Davis, Yeray Novoa Medina, and Meutia Aulia for their contributions to data collection and analysis. Lastly, we are thankful to Adhi Ariebowo and Marina Mary for their help with interpretation and translation. Author Contributions Conceptualization: SK LS CB. Data curation: CB BF. Formal analysis: LS CB BF. PLOS ONE \| DOI:10.1371/journal.pone.0168405 December 19, 2016 13 / 15 The Three Delays Model and Civil Registration Barriers Funding acquisition: SK. Investigation: RU RA CB BF. Methodology: SK LS CB BF. Project administration: RU RA CB. Resources: SK. Supervision: LS SK. Validation: SK RU RA CB. Visualization: BF. Writing – original draft: CB BF LS. Writing – review & editing: SK RU RA LS CB BF. References 1. 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https://openalex.org/W2969821421	https://europepmc.org/articles/pmc6706430?pdf=render	English	null	Properties of echoic memory revealed by auditory-evoked magnetic fields	Scientific reports	2,019	cc-by	7,189	OPEN gi Tomoaki Kinukawa 1, Nobuyuki Takeuchi 2, Shunsuke Sugiyama3, Makoto Nishihara4, Kimitoshi Nishiwaki1 & Koji Inui5,6 Received: 28 February 2019 Accepted: 12 August 2019 Published: xx xx xxxx We used auditory-evoked magnetic fields to investigate the properties of echoic memory. The sound stimulus was a repeated 1-ms click at 100 Hz for 500 ms, presented every 800 ms. The phase of the sound was shifted by inserting an interaural time delay of 0.49 ms to each side. Therefore, there were two sounds, lateralized to the left and right. According to the preceding sound, each sound was labeled as D (preceded by a different sound) or S (by the same sound). The D sounds were further grouped into 1D, 2D, and 3D, according to the number of preceding different sounds. The S sounds were similarly grouped to 1S and 2S. The results showed that the preceding event significantly affected the amplitude of the cortical response; although there was no difference between 1S and 2S, the amplitudes for D sounds were greater than those for S sounds. Most importantly, there was a significant amplitude difference between 1S and 1D. These results suggested that sensory memory was formed by a single sound, and was immediately replaced by new information. The constantly-updating nature of sensory memory is considered to enable it to act as a real-time monitor for new information. According to the model by Atkinson and Shiffrin1, human memory is divided into three components: the sensory register, short-term store, and long-term store. The sensory register, or sensory memory, is thought to hold sen- sory information briefly for use in the next step of the multi-store memory system. The mechanisms of memory build-up and decay have been examined for many years2–5, and psychological studies have revealed the existence of brief storage6,7. For example, by using the method known as partial report procedures, Sperling demonstrated that subjects could remember many briefly-presented characters using short-lasting visual memory if the cue to recall the characters was given within one second after the visual display8. By using a similar paradigm, Darwin et al. demonstrated the existence of a brief storage of several seconds in the auditory system9. Psychological studies have revealed unique properties of sensory memory, including its decay10, vulnerability to interference stimuli11,12, and the effects of stimulus repetition13. www.nature.com/scientificreports www.nature.com/scientificreports Received: 28 February 2019 Accepted: 12 August 2019 Published: xx xx xxxx 1Department of Anesthesiology, Nagoya University Graduate School of Medicine, Nagoya, 466-8550, Japan. 2Neuropsychiatric Department, Aichi Medical University, Nagakute, 480-1195, Japan. 3Department of Psychiatry and Psychotherapy, , Gifu University, Gifu, 501-1193, Japan. 4Multidisciplinary Pain Center, Aichi Medical University, Nagakute, 480-1195, Japan. 5Department of Functioning and Disability, Institute for Developmental Research, Kasugai, 480-0392, Japan. 6Department of Integrative Physiology, National Institute for Physiological Sciences, Okazaki, 444-8585, Japan. Correspondence and requests for materials should be addressed to T.K. (email: t-kinukawa@med.nagoya-u.ac.jp) Scientific Reports \| (2019) 9:12260 \| https://doi.org/10.1038/s41598-019-48796-9 Results I h i In the main experiment (Experiment 1), there were two sounds (Sound), lateralized to the left (Right-delay) and right (Left-delay). The sounds were labelled according to whether the sound was preceded by the same sound or different sound, and grouped into S trials and D trials. The D sounds were further grouped into 1D, 2D, and 3D, according to the number of preceding different sounds. The S sounds were similarly grouped to 1S and 2S. The effect of the preceding sound (Event) on auditory-evoked middle-latency component, N100m, was investigated in this study. The mean latency and amplitude of N100m for each condition are listed in Table 1. Three-way ANOVA showed that Sound (F1,12 = 12.7, p = 0.004) and Event (F4,48 = 25.2, p = 2.8 × 10−11) but not Hemisphere (F1,12 = 0.40, P = 0.84) were significant factors in determining the amplitude. The overall amplitude was greater for the Left-delay (L) sound (20.4 nAm) than for the Right-delay (R) sound (16.2). Although Hemisphere was not a significant factor, there was a significant Hemisphere x Sound interaction (F1,12 = 15.3, p = 0.002). Additionally, there was a main effect of Sound for the left hemisphere (F1,12 = 27.5, p = 2.1 × 10−4). That is, the amplitude of the response in the left hemisphere was greater for L (21.5 nAm) than for R (14.8). On the other hand, there was no significant difference between L (19.4 nAm) and R (17.7) for the right hemisphere. In other words, the contralat- eral bias was clear for R, but not for L. Similar findings have been reported in previous studies using MEG37 and fMRI38. These findings appear to show hemispheric differences for auditory spatial processing.f hi g pp pf y p p g As for the effects of the preceding event, the results of the post hoc tests showed that, in general, the ampli- tudes of D trials were greater than those of S trials: the amplitude for 1S was significantly smaller than those for 1D, 2D, and 3D; 2S was significantly smaller than 2D and 3D. It is noteworthy that the difference was significant between 1D and 1S (p = 0.039). Among the D trials, the amplitude was greater for 3D, 2D, and 1D, in that order, with significant differences between 1D and 2D (p = 0.011) and between 1D and 3D (p = 0.004). OPEN In a previ- ous study24, it was shown that sensory memory acts as a real-time sensory monitor, as a single presentation of a sound was able to build up memory and affected the cortical response to the next sound. However, in order to act as a real-time monitor, there must be active forgetting, in order to update information in real-time. To clarify this, three experiments were conducted in the present study. In addition, peripheral contribution to such ERP components remain possible36. Therefore, we used sounds with an interaural time difference (ITD) in this study to rule out these peripheral contributions. Scientific Reports \| (2019) 9:12260 \| https://doi.org/10.1038/s41598-019-48796-9 OPEN However, it is not easy for psychological studies to deal with sensory memory because it is outside of our cognitive control. In addition, the duration of storage appears to be too short for standard memory and recall procedures, given that the lifetime is considered to be a few seconds6,9,14. y p g Mismatch negativity (MMN)15–18, a component of event-related potentials (ERPs), has been used as an objec- tive method to observe sensory memory. When rare and frequent sensory stimuli are presented randomly, the former elicits MMN with a maximum negativity at Fz and positivity at the mastoid. MMN is considered to reflect the process of automatically detecting deviant stimuli based on short-term memory trace. Therefore, it is believed that MMN can index the sensory memory. By using MMN, for example, several researchers have measured the lifetime of sensory memory19–22. As the brain has to form a representation of the repetitive aspects of auditory stimulation before the occurrence of the rare stimulus for elicitation of MMN23, the sequence of stimulus pres- entation is limited. In order to study sensory memory, we have used the change-related cortical response – a kind of event-related response that is specifically elicited when the brain detects sensory information different from the preceding 1Department of Anesthesiology, Nagoya University Graduate School of Medicine, Nagoya, 466-8550, Japan. 2Neuropsychiatric Department, Aichi Medical University, Nagakute, 480-1195, Japan. 3Department of Psychiatry and Psychotherapy, , Gifu University, Gifu, 501-1193, Japan. 4Multidisciplinary Pain Center, Aichi Medical University, Nagakute, 480-1195, Japan. 5Department of Functioning and Disability, Institute for Developmental Research, Kasugai, 480-0392, Japan. 6Department of Integrative Physiology, National Institute for Physiological Sciences, Okazaki, 444-8585, Japan. Correspondence and requests for materials should be addressed to T.K. (email: t-kinukawa@med.nagoya-u.ac.jp) www.nature.com/scientificreports/ Left-delay Right-delay Left Right Left Right Amplitude 1D 21.0 (7.4) 19.3 (8.8) 14.4 (3.9) 17.3 (7.2) 2D 25.1 (7.2) 21.3 (9.0) 16.5 (3.5) 18.1 (6.6) 3D 26.3 (8.1) 22.1 (10.5) 16.6 (5.0) 20.4 (8.5) 1S 17.5 (5.1) 16.3 (7.3) 12.8 (4.0) 15.8 (6.8) 2S 17.6 (6.3) 18.0 (8.5) 13.4 (3.6) 16.8 (6.8) Peak latency 1D 123.8 (14.0) 131.2 (13.7) 134.7 (18.6) 127.1 (14.6) 2D 127.3 (16.0) 130.2 (11.9) 130.2 (18.2) 121.9 (14.0) 3D 126.5 (16.1) 127.6 (15.5) 128.9 (16.0) 123.7 (17.0) 1S 123.0 (15.1) 124.8 (13.8) 128.2 (14.0) 120.0 (15.2) 2S 129.5 (20.5) 130.1 (16.7) 127.5 (15.8) 117.7 (14.7) Table 1. Peak latency and amplitude of N100m. Data are shown as the mean (SD). Table 1. OPEN Previous studies showed that the amplitude of the change-related cortical response depends on the degree of the sensory change24,27,28, length of the stimulus to be stored26, length of the preceding sensory status to be compared28–30, length of the decay time of the storage of previous events26,31,32, and the probability of the test stimulus under an oddball paradigm33. These findings indicate that the sensory storage and comparison processes are involved in generating the response. The storage is capable of retaining details of the sensory stimulus, like a snapshot34. Taken together, it appears that the storage involved in the change-related cortical response is sensory memory, according to the lifetime-based standard classification of memory35. The advantage of this method is that it requires no task of the subjects, and thereby subjects need not pay attention to, remember, or recall the stimulus. This allows us to observe sensory memory objectively. In this study, we used the change-related cortical response to investigate the properties of echoic memory in the brain, particularly its nature of decay. In a previ- ous study24, it was shown that sensory memory acts as a real-time sensory monitor, as a single presentation of a sensory status24, and is clearly observed by magnetoencephalography (MEG) or electroencephalography. Unlike MMN, it can be elicited without repetition of a frequent stimulus24–26. Therefore, it is easy to use for various stimu- lation paradigms. Previous studies showed that the amplitude of the change-related cortical response depends on the degree of the sensory change24,27,28, length of the stimulus to be stored26, length of the preceding sensory status to be compared28–30, length of the decay time of the storage of previous events26,31,32, and the probability of the test 33hi stimulus under an oddball paradigm33. These findings indicate that the sensory storage and comparison processes are involved in generating the response. The storage is capable of retaining details of the sensory stimulus, like a snapshot34. Taken together, it appears that the storage involved in the change-related cortical response is sensory memory, according to the lifetime-based standard classification of memory35. The advantage of this method is that it requires no task of the subjects, and thereby subjects need not pay attention to, remember, or recall the stimulus. This allows us to observe sensory memory objectively. In this study, we used the change-related cortical response to investigate the properties of echoic memory in the brain, particularly its nature of decay. OPEN Peak latency and amplitude of N100m. Data are shown as the mean (SD). Table 1. Peak latency and amplitude of N100m. Data are shown as the mean (SD). sensory status24, and is clearly observed by magnetoencephalography (MEG) or electroencephalography. Unlike MMN, it can be elicited without repetition of a frequent stimulus24–26. Therefore, it is easy to use for various stimu- lation paradigms. Previous studies showed that the amplitude of the change-related cortical response depends on the degree of the sensory change24,27,28, length of the stimulus to be stored26, length of the preceding sensory status to be compared28–30, length of the decay time of the storage of previous events26,31,32, and the probability of the test stimulus under an oddball paradigm33. These findings indicate that the sensory storage and comparison processes are involved in generating the response. The storage is capable of retaining details of the sensory stimulus, like a snapshot34. Taken together, it appears that the storage involved in the change-related cortical response is sensory memory, according to the lifetime-based standard classification of memory35. The advantage of this method is that it requires no task of the subjects, and thereby subjects need not pay attention to, remember, or recall the stimulus. This allows us to observe sensory memory objectively. In this study, we used the change-related cortical response to investigate the properties of echoic memory in the brain, particularly its nature of decay. In a previ- ous study24, it was shown that sensory memory acts as a real-time sensory monitor, as a single presentation of a sound was able to build up memory and affected the cortical response to the next sound. However, in order to act as a real-time monitor, there must be active forgetting, in order to update information in real-time. To clarify this, three experiments were conducted in the present study. In addition, peripheral contribution to such ERP components remain possible36. Therefore, we used sounds with an interaural time difference (ITD) in this study to rule out these peripheral contributions. sensory status24, and is clearly observed by magnetoencephalography (MEG) or electroencephalography. Unlike MMN, it can be elicited without repetition of a frequent stimulus24–26. Therefore, it is easy to use for various stimu- lation paradigms. Results I h i Grand-averaged waveforms of each event are shown in Fig. 1. There was no significant difference between 1S and 2S (p = 0.94). We consider that the lack of a significant difference between 1D and 2S was due to the relatively small effect of the 1D condition. A comparison between 1D and 2S with a paired t-test showed a significant difference (p = 0.004) when correction for multiple comparisons was not applied. In addition, the amplitudes for 1D and 2S are highly correlated (r2 = 0.74) with a regression line with a slope of 0.9 indicating that the amplitude is reliably greater for 1D by approximately 10%.fi Regarding latency, none of the main effects were significant. Although there was a tendency for the laten for 1S (121.0 ms) and 2S (123.1) to be shorter than those for 1D (126.1), 2D (124.3), and 3D (123.6), the differe www.nature.com/scientificreports/ Figure 1. Grand-averaged waveforms of auditory-evoked cortical activity. Waveforms in response to the Left- delay sound (L) and Right-delay sound (R) are shown in the upper and lower panels, respectively. For each sound and hemisphere, there were five conditions, 1D, 2D, 3D, 1S, and 2S, which indicate how many different (D) or same (S) sounds preceded the probe sound. Figure 1. Grand-averaged waveforms of auditory-evoked cortical activity. Waveforms in response to the Left- delay sound (L) and Right-delay sound (R) are shown in the upper and lower panels, respectively. For each sound and hemisphere, there were five conditions, 1D, 2D, 3D, 1S, and 2S, which indicate how many different (D) or same (S) sounds preceded the probe sound. Figure 1. Grand-averaged waveforms of auditory-evoked cortical activity. Waveforms in response to the Left- delay sound (L) and Right-delay sound (R) are shown in the upper and lower panels, respectively. For each sound and hemisphere, there were five conditions, 1D, 2D, 3D, 1S, and 2S, which indicate how many different (D) or same (S) sounds preceded the probe sound. was not significant (F4,48 = 2.38, p = 0.065). In contrast, there was a significant Hemisphere x Sound interaction (F1,12 = 16.7, p = 0.002). The results of subsequent analyses showed that in both the left (F1,12 = 5.01, p = 0.045) and right (F1,12 = 7.6, p = 0.018) hemispheres, there were significant main effects of Sound. Results I h i In the left hemisphere, the latency of the response to L (122.9 ms) was shorter than that to R (126.8). In the right hemisphere, the latency for R (119.0) was shorter than that for L (125.7). That is, the peak latency of N100m was shorter and the amplitude was greater for the side with the interaural time difference (ITD) or the hemisphere contralateral to sound later- alization, confirming a previous study39.h i g p y These results suggested the existence of cortical activities sensitive to the sound sequence. In order to quantify these activities, the source strength waveform of 1S was subtracted from those of other events, and the amplitude and latency were measured using the difference waveforms. Because no clear peak at around the N100m latency was seen for the subtracted 2S waveform in most of the subjects, data for the D trials were analyzed. The mean peak latencies and amplitudes are listed in Table 1. The amplitude was greater for 3D, 2D, and 1D, in that order. Three-way ANOVA showed a significant main effect of Event (F2,24 = 9.37, p = 0.001). Post hoc tests revealed significant differences between 1D and 2D (p = 0.002), and 1D and 3D (p = 0.012), but not between 2D and 3D (p = 0.78). The latency did not differ among events (F2,24 = 0.029, p = 0.97). Grand-averaged difference waveforms across hemispheres and sounds are illustrated in Fig. 2. As shown in Table 2 and Fig. 2, the N100m peak latency for the difference waveforms was slightly longer than that for the original waveforms. When the latency for the difference waveform was compared with that for the original 1S using a paired t-test, the difference was significant for 1D (p = 0.042, corrected for multiple comparisons) and 3D (p = 0.01). The latency for 2D also tended to be longer than that for the original 1S (p = 0.11). Discussion h In the present study, we sought to clarify the properties of echoic memory by using auditory-evoked cortical responses. As the subjects did not need to pay attention to, memorize, or recall the stimuli, we could evaluate echoic memory objectively through its cortical responses. Our results revealed a significant difference in the amplitude of the evoked cortical responses for 1D and 1S, suggesting that a single presentation of the sound (R or L) was sufficient to store the information and that the storage was replaced immediately with another when the brain detected a different sound. Therefore, echoic memory can be considered to act as a monitor of the current sensory status. Scientific Reports \| (2019) 9:12260 \| https://doi.org/10.1038/s41598-019-48796-9 www.nature.com/scientificreports/ Figure 2. Difference waveforms with original 1S. Difference waveforms obtained by subtracting the 1S waveform are shown. Thus, the waveforms indicate cortical activity due to the presence of the prior different sounds. For comparison, the original waveform for 1S is also shown. Waveforms of both hemispheres are combined. Note the slightly later peak of the difference waveform than the original 1S waveform. Figure 2. Difference waveforms with original 1S. Difference waveforms obtained by subtracting the 1S waveform are shown. Thus, the waveforms indicate cortical activity due to the presence of the prior different sounds. For comparison, the original waveform for 1S is also shown. Waveforms of both hemispheres are combined. Note the slightly later peak of the difference waveform than the original 1S waveform. Left-delay Right-delay Left Right Left Right Amplitude 1D 7.94 (3.4) 7.50 (3.9) 6.17 (2.2) 5.56 (3.6) 2D 10.2 (3.8) 9.88 (6.0) 7.79 (3.9) 6.78 (3.8) 3D 12.0 (4.2) 10.3 (7.4) 8.36 (3.81) 7.23 (3.1) Peak latency 1D 126 (25.4) 130.0 (22.7) 129.7 (23.4) 132.3 (12.1) 2D 131.3 (40.1) 134.8 (17.6) 124.5 (16.7) 124.8 (18.0) 3D 126.1 (21.0) 129.8 (17.4) 129.5 (18.6) 131.8 (20.0) Table 2. Peak latency and amplitude of difference waveforms. Left-delay Right-delay Left Right Left Right Amplitude 1D 7.94 (3.4) 7.50 (3.9) 6.17 (2.2) 5.56 (3.6) 2D 10.2 (3.8) 9.88 (6.0) 7.79 (3.9) 6.78 (3.8) 3D 12.0 (4.2) 10.3 (7.4) 8.36 (3.81) 7.23 (3.1) Peak latency 1D 126 (25.4) 130.0 (22.7) 129.7 (23.4) 132.3 (12.1) 2D 131.3 (40.1) 134.8 (17.6) 124.5 (16.7) 124.8 (18.0) 3D 126.1 (21.0) 129.8 (17.4) 129.5 (18.6) 131.8 (20.0) Table 2. Peak latency and amplitude of difference waveforms. Table 2. Peak latency and amplitude of difference waveforms. Discussion h Methodological consideration: In the present study. we used the change-related cortical response26, which is specifically evoked when the brain detects any kind of novel sensory event. Although this response is typically evoked by an abrupt change to a continuous sensory stimulus, it is also evoked by a stimulus preceded by another with different features, after a gap26. In the present study, the different waveforms showed N100m peaking at 120–130 ms, which appears to correspond to the change-related N100m evoked by an abrupt change in sound location peaking at 120–135 ms21,30,33. The current study used a simple train of clicks as the test stimulus. As the two sounds, L and R, differed only in their phase, they were identical at each ear for every trial, which excluded the possible contribution of the periphery to the present results. The subjects’ task during the experiments was to watch a silent movie and ignore the sound, meaning that their cortical responses were automatic. Therefore, the present results suggested that the information about the sound was automatically stored in the processing pathway of the brain. Characteristics of echoic memory revealed in the present study. As the cortical response was sig- nificantly affected by a single event preceding the test stimulus, the information was stored during presentation of a single sound of 500 ms and was used during processing of the next sound. This indicates that the single event of the train of clicks was sufficient to retain the information for later use, which is in agreement with the instan- taneous nature of sensory memory. In a psychological study by Sperling8, a stimulus of 25 ms was shown to be sufficient to establish visual sensory memory. In a study using dichotic listening, a single word could be stored in memory14. Therefore, it is reasonable that the single 500-ms sound was sufficient to be stored in an available form. However, it is significant that the present study confirmed this instantaneous nature of echoic memory without the subjects being aware of it. This relatively long duration of 500 ms was used to strengthen the storage and to reduce its decay.hfi y The response amplitudes to 1S and 2S did not differ significantly, suggesting that once the memory for a sound was established, a change-related cortical response did not occur in response to the next sound if it was identical. Scientific Reports \| (2019) 9:12260 \| https://doi.org/10.1038/s41598-019-48796-9 Discussion h If the storage for the first L in LRL (1D) had remained at the presentation of the next L, no change-related cortical response would have occurred. Therefore, for one auditory sub-modality, such as sound location, only one instance of the latest status is stored. In order to confirm this notion, Experiment 2 was performed in seven subjects (Supplemental Fig. 1), in which a brief click train of 50 ms composed of L or R was inserted between the original 500-ms sounds. The results showed that the brief click train had only a weak effect on processing of the next different sound; it did not elicit a signif- icant change-related cortical response for the next sound (p = 0.15), but it could reset the memory for preceding sounds (p = 0.0026). (p ) As the occurrence probability of the two sounds was even and the storage of each sound lasted longer than the trial-trial interval in the present study, there must be a specific mechanism that shortens the memory of each sound. The results of Experiment 3 (Supplemental Fig. 2) showed that the lifetime of the memory trace in the present study was 4–6 s. Without such mechanisms, the memory would increase up to its limit during the record- ing, and differences among 1D, 2D, and 3D would not arise. We consider that replacement of the memory trace by new information is the main responsible mechanism. Figure 3 explains the present results using a model. In this model24, the strength of sensory memory is increased during stimulus presentation or by repetition of the stimulus in a positively-accelerated fashion, decreased during a blank in a negatively-accelerated fashion, and is abolished/replaced by a new stimulus. Conclusions There is a debate surrounding the contributions of time-dependent decay and interference to forgetting in short-term or working memory10. Echoic memory, indexed by the change-related cortical response, is clearly dependent on the passage of time26. In the present study, a different sound preceding the probe sound was an interference stimulus, particularly the brief train in Supplemental Experiment 1. Therefore, both decay and interference contribute to forgetting in echoic memory. Under the present paradigm using only two sounds, the interference effect was powerful, almost abolishing the previous storage. It can thus be functionally regarded as replacement. The change-related cortical response is considered to be a subtype of a defense reaction41, playing an important role in the prompt detection of changes in the sensory environment. Sensory memory is the basis of the change-related cortical response, and therefore plays a role in survival. For this purpose, forgetting/replace- ment is important in order to update the current sensory status. We consider that these properties of sensory memory enable it to play a role as a real-time monitor, identifying new events to which attention may be required. Discussion h As the sequences were LRR and LRRR, respectively, this means that the memory for L in these sequences was lost when the probe R was presented. This mechanism is considered to exist to avoid excessive responses to irrelevant information, and to reduce energy consumption. In contrast, repeated presentations of a sound increased the N100m amplitude following the next different sound, suggesting that memory is strengthened by repetition. Similar effects were reported in a psychological study40. This indicates that the present 500-ms click train was not sufficient for full storage, which is in agreement with previous findings that the amplitude of the change-related cortical response was influenced by the duration of the sensory status, when compared up to 3–6 s30,32. Taken together, the present results suggest that the short storage established during the presentation of a sound was replaced immediately by short storage for the next different sound. In sequences of LRL (1D) and RLL (1S), www.nature.com/scientificreports/ Figure 3. A model for echoic memory to Left-delay (L) and Right-delay (R) sounds. The Y axis indicates the strength of memory. At the breaking point, the change-related cortical response, whose amplitude is determined by the strength of the memory, is elicited. Figure 3. A model for echoic memory to Left-delay (L) and Right-delay (R) sounds. The Y axis indicates the strength of memory. At the breaking point, the change-related cortical response, whose amplitude is determined by the strength of the memory, is elicited. the amplitude of 1D was clearly larger than that of 1S (p = 0.039). If the storage for the first L in LRL (1D) had remained at the presentation of the next L, no change-related cortical response would have occurred. Therefore, for one auditory sub-modality, such as sound location, only one instance of the latest status is stored. In order to confirm this notion, Experiment 2 was performed in seven subjects (Supplemental Fig. 1), in which a brief click train of 50 ms composed of L or R was inserted between the original 500-ms sounds. The results showed that the brief click train had only a weak effect on processing of the next different sound; it did not elicit a signif- icant change-related cortical response for the next sound (p = 0.15), but it could reset the memory for preceding sounds (p = 0.0026). the amplitude of 1D was clearly larger than that of 1S (p = 0.039). Methodsh (C) Two sequences consisting of RLL and LRR. (D) Labelling of each sound by preceding events. sound twice (2S) (LLL and RRR). Therefore, there were five types of events in this study, 1D, 2D, 3D, 1S, and 2S, with an occurrence probability of 1:1:1:2:1 for each of L and R (Fig. 4D). None of the subjects could identify the sequence of sounds even when they listened to them carefully after the experiment. sound twice (2S) (LLL and RRR). Therefore, there were five types of events in this study, 1D, 2D, 3D, 1S, and 2S, with an occurrence probability of 1:1:1:2:1 for each of L and R (Fig. 4D). None of the subjects could identify the sequence of sounds even when they listened to them carefully after the experiment. Recordings. The subjects sat in a chair and watched a silent movie on a screen placed 2 m in front of them, and were instructed to ignore all stimuli throughout the experiment. Magnetic signals were recorded using a 306-channel whole-head type MEG system (Vector-view, ELEKTA Neuromag, Helsinki, Finland), which com- prised 102 identical triple sensor elements. Each sensor element consisted of two orthogonal planar gradiometers and one magnetometer coupled to a multi-superconducting quantum interference device, and thus provided three independent measurements of the magnetic fields. In the present study, we analyzed MEG signals recorded from 204 planar-type gradiometers, which were sufficiently powerful to detect the largest signal just over local cerebral sources. Signals were recorded with a bandpass filter of 0.1–330 Hz and digitized at 1000 Hz. Analyses were con- ducted from 100 ms before, to 400 ms after, the onset of the stimulus. Epochs with MEG signals larger than 2.7 pT/cm were excluded from averaging. The waveform was digitally filtered with a bandpass filter of 1.0–100 Hz. Analysis. We performed single dipole analysis using the brain electric source analysis (BESA) software pack- age (GmbH, Grafefling, Germany) for the main component peaking at approximately 120 ms (N100m), as previ- ously described24. First, all five conditions were added for L and R. The equivalent current dipole for N100m was estimated in the auditory cortex of each hemisphere. The two-dipole model was then applied to waveforms for all conditions, and obtained source strength waveforms were used to measure the amplitude and latency of the cortical response. Methodsh The study protocol was designed in accordance with the Declaration of Helsinki (World Medical Association, 2008), and was approved in advance by the Ethics Committee of the National Institute for Physiological Sciences, Okazaki, Japan. All subjects provided written informed consent prior to participation. Thirteen healthy volun- teers (3 women, 10 men; aged 25–55 years, mean 37 years) participated in the study. None had a history of mental or neurological disorders, nor substance abuse, in the most recent five years, and all were free of medication at the time of testing. Stimulation. The sound used in the present study was a train of clicks, 100 Hz in repetitive frequency, 70 dB SPL in sound pressure, and 500 ms in total duration. Clicks were generated as single cycles of a 1-ms sine wave (Fig. 4A,B). By inserting an ITD of 0.49 ms between sides, two sounds, Left-delay (L) and Right-delay (R), were created (Fig. 4C). Next, we made two sequences composed of LRR and RLL, as previously described26, with a blank of 300 ms between sounds. The two sequences, LRR and RLL, were randomly presented at an identical probability with a trial-trial interval of 800 ms during the experiment. Under this paradigm, the probability of each sound (L and R) was even. The sounds were labelled according to whether a sound was preceded by the same sound or different sound, and grouped into S trials and D trials. Among the D trials, three types appeared at an identical probability: a trial with a sound preceded by a different sound (1D), a trial preceded by two different sounds (2D) (LLR and RRL), and a trial preceded by three different sounds (3D) (LLLR and RRRL). Among the S trials, there were two types: a trial with a sound preceded by the same sound (1S), and a trial preceded by the same Scientific Reports \| (2019) 9:12260 \| https://doi.org/10.1038/s41598-019-48796-9 www.nature.com/scientificreports/ Figure 4. Schematic illustration of auditory stimuli. (A) Repetition of a 1-ms click at 100 Hz. (B) Left lateralized sound (R) created by inserting a 0.49-ms interaural time delay to the right side. (C) Two sequences consisting of RLL and LRR. (D) Labelling of each sound by preceding events. Figure 4. Schematic illustration of auditory stimuli. (A) Repetition of a 1-ms click at 100 Hz. (B) Left lateralized sound (R) created by inserting a 0.49-ms interaural time delay to the right side. 1. Atkinson, R. C. & Shiffrin, R. M. Human memory: a proposed system and its control processes. Psychol Learn Motiv. 2, 89–195 (1968). 2. Baddeley, A. D. & Hitch, G. J. Working memory. Psychol Learn Motiv. 8, 47–90 (1974). 3. Dick, A. O. Iconic memory and its relation to perceptual processing and other memory mechanisms. Perception & Psychophysics. 16, 575–596 (1974). Methodsh The latency at the point of maximum amplitude within the range of 90 to 150 ms was defined as the peak latency of N100m. 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Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-48796-9.h Competing Interests: The authors declare no competing interests. Competing Interests: The authors declare no competing interests. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Scientific Reports \| (2019) 9:12260 \| https://doi.org/10.1038/s41598-019-48796-9 www.nature.com/scientificreports e.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. 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W4387952433.txt	https://periodicos.saude.sp.gov.br/bis/article/download/35380/33816	pt		Síntese de Evidências sobre Estratégias para Redução da Mortalidade Materna no Município de Franco da Rocha, São Paulo	BIS. Boletim do Instituto de Saúde	2,016	cc-by	4,550	Síntese de Evidências sobre Estratégias para Redução da Mortalidade Materna no Município de Franco da Rocha, São Paulo Evidence Brief for Policy about Strategies to Reduce Maternal Mortality in the Municipality of Franco da Rocha, Sao Paulo. Carolina Médici de FigueiredoI, Inayá da Silva DuarteII, Luciana C. Alves dos SantosIII, Luciana de Mendonça FreireIV, Tatiane Aparecida Rocha MarceloV, Sonia Isoyama VenancioVI Resumo Abstract O Brasil ainda está longe de atingir uma das Metas do Milênio assumidas em 1990, de redução da mortalidade materna (MM). Atualmente, morrem no país cerca de 69 mulheres a cada 100 mil nascidos vivos, mas pelas metas da Organização das Nações Unidas esse número deveria ser de no máximo 35 mulheres. Nesse contexto, é possível ressaltar que as principais causas dessas mortes são a hipertensão arterial, hemorragia, complicações de aborto em condições inseguras e infecção pós-parto. Ademais, notam-se índices elevados de mortalidade materna no Brasil em regiões periféricas, onde o acesso às redes de saúde é quase inexistente e/ou precário. No município de Franco da Rocha, as altas taxas de MM vêm preocupando os gestores da saúde, especialmente por estarem relacionadas a causas consideradas evitáveis. Este artigo apresenta alguns resultados de uma Síntese de Evidências elaborada por alunas do Programa de Aprimoramento Profissional em Saúde Coletiva do Instituto de Saúde, com apoio do Núcleo de Evidências do Instituto de Saúde, o qual integra a Rede para Políticas Informadas por Evidências – EVIPNet Brasil, com o objetivo de apoiar a gestão municipal de saúde de Franco da Rocha na tomada de decisões para o enfrentamento da MM. Brazil is still far from achieving the Millennium Development Goals undertaken in 1990 to reduce maternal mortality (MM). Currently, in the country about 69 women die per 100 thousand live births, but by the goals of the United Nations, that number should be a maximum of 35 women. In this context, it is possible to emphasize that the main causes of these deaths are hypertension, hemorrhage, complications of abortion in unsafe conditions and postpartum infections. Moreover, we note high rates of MM in Brazil in remote areas, where access to health networks is almost non-existent and / or precarious. In the city of Franco da Rocha, the high MM rates have been worrying policy makers, especially because it is related to causes considered preventable. This article presents some of the results of a Policy Brief prepared by students of the Professional Program in Public Health at Instituto de Saúde, with the support of its Evidence Center, which is part of the Evidence Informed-Policy Network - EVIPNet Brazil in order to support municipal policy makers in Franco da Rocha in decision making for reducing MM. Keywords: Maternal Mortality, Health Policies, Evidence Brief for Policy. Palavras-chave: Mortalidade Materna, Políticas de Saúde, Síntese de Evidências. I Carolina Médici de Figueiredo (carolina_medici@hotmail.com) é obstetriz formada pela Escola de Artes, Ciências e Humanidades (EACH-USP), com aprimoramento em Saúde Coletiva pelo Instituto de Saúde – SES/SP. IV II Inayá da Silva Duarte (inaya.d@terra.com.br) é obstetriz formada pela Escola de Artes, Ciências e Humanidades (EACH-USP), tem aprimoramento em Saúde Coletiva pelo Instituto de Saúde – SES/SP. V Luciana C. Alves do Santos (lu.cristinaalves@hotmail.com) é obstetriz formada pela Escola de Artes, Ciências e Humanidades (EACH-USP), com aprimoramento em Saúde Coletiva pelo Instituto de Saúde – SES/SP. VI Sonia Isoyama Venancio (soniav@isaude.sp.gov.br) é médica, pesquisadora e vice-diretora do Instituto de Saúde- SES/SP. III Luciana de Mendonça Freire (luciana.m.freire23@gmail.com) é graduada em Saúde Coletiva pela Universidade Federal do Acre (UFAC), com aprimoramento em Saúde Coletiva pelo Instituto de Saúde – SES/SP. Tatiane Aparecida Rocha Marcelo (tati-aprm@hotmail.com) é obstetriz formada pela Escola de Artes, Ciências e Humanidades (EACH-USP), com aprimoramento em Saúde Coletiva pelo Instituto de Saúde – SES/SP. \|105 46042001 miolo.indd 105 04/11/16 16:38 Políticas de Saúde Informadas por Evidências Introdução e Justificativa Mortalidade Materna (MM) é definida como a morte de uma mulher durante a gestação ou dentro de um período de 42 dias após o término da gestação, independentemente da duração ou da localização da gravidez, devida a qualquer causa relacionada ou agravada pela gravidez ou por medidas em relação a ela, porém não a causas acidentais ou incidentais25. O número de mortes maternas de um país constitui excelente indicador de sua realidade social, estando inversamente relacionado ao grau de desenvolvimento humano. Nesse aspecto, estudos nacionais confirmam que os desfechos maternos são influenciados pelas condições de assistência ao pré-natal e segundo recomendações de organismos oficiais de saúde este deve ter início precoce, ter cobertura universal, ser realizado de forma periódica, estar integrado com as demais ações preventivas e curativas e deve ter um número mínimo de consultas2. Para investigar as causas de MM no Brasil conta-se com os índices calculados a partir das MM declaradas, obtidas das declarações de óbitos do Sistema de Informação sobre Mortalidade e pelo Sistema de Informação sobre Nascidos Vivos, ambos geridos pelo Ministério da Saúde. A partir dos dados coletados pelos serviços de saúde, é possível identificar diversos aspectos relevantes para o enfrentamento da MM, ou seja, as principais causas de morte, quando estas ocorrem e se poderiam ser evitadas. Nesse contexto, o problema da MM no Brasil tornou-se relevante, pois os dados disponíveis e suas respectivas análises apontam que o país está acima da meta definida pelos Objetivos de Desenvolvimento do Milênio para 2015 – o valor desejável seria igual ou inferior a 35 óbitos maternos por grupo de 100.000 NV –, estipulada pelas Nações Unidas. A Região de Saúde (RS) de Franco da Rocha (que engloba os municípios de Cajamar, Caieiras, A Francisco Morato, Franco da Rocha e Mairiporã) apresentou, no ano de 2010, taxa de 71,06 por 100.000 NV e em 2013, 46,37 por 100.000 NV. Restringido o olhar para o município de Franco da Rocha, em consulta à Fundação Seade sobre Informações dos Municípios Paulistas, nota-se que esta razão de mortalidade era 47,87 em 2009, 90,21 em 2010, e 141,18 em 2011. Ressalta-se aqui que os dados referentes ao ano de 2012 e 2013 não estavam disponíveis para pesquisa (www.imp.sade.gov.br) Em Franco da Rocha os dados mostram ainda que a cobertura do pré-natal, com sete ou mais consultas, encontra-se em queda no período analisado (índice de 72% no ano de 2010, 69,7% em 2011, 68% em 2012 e 65,5% em 2013); além disso, quando comparando com o Estado de São Paulo e a RS de Franco da Rocha, o município mostra menor cobertura de pré-natal. Tais dados podem sugerir diferentes causas, como: menor adesão das gestantes, redução das equipes que prestam assistência à mulher, ou ainda, estagnação dos serviços e recursos. Um aspecto que merece destaque é que o município não possui leitos obstétricos, tendo como referência para partos de baixo risco o município de Caieiras. Quando analisadas as causas de óbitos maternos no município percebe-se que essas estão relacionadas à mortalidade obstétrica direta (em sua maioria pré-eclâmpsia, eclampsia e hemorragias) podendo indicar pré-natal de baixa qualidade, baixa qualidade da atenção ao parto ou até mesmo a falta de acesso das gestantes aos serviços de saúde. A evolução da MM e os indicadores relacionados à cobertura do pré-natal vêm preocupando os gestores de saúde do município de Franco de Rocha e motivou a definição deste problema para o desenvolvimento de uma Síntese de Evidências para Políticas de Saúde, a qual reúne evidências de pesquisa global (a partir de revisões \|106 46042001 miolo.indd 106 04/11/16 16:38 Políticas de Saúde Informadas por Evidências sistemáticas) e evidências locais para as deliberações sobre as políticas e programas de saúde. A Síntese de Evidências foi desenvolvida por um grupo de alunas do Programa de Aprimoramento Profissional em Saúde Coletiva do Instituto de Saúde, sob supervisão de pesquisadoras do Núcleo de Evidências do Instituto de Saúde, o qual integra a Rede para Políticas Informadas por Evidências – EVIPNet Brasil. Este artigo tem por objetivo apresentar alguns resultados desta Síntese de Evidências, incluindo as opções de políticas identificadas e alguns aspectos relacionados à sua implementação, visando apoiar a gestão municipal de saúde de Franco da Rocha na tomada de decisões para o enfrentamento da MM. O detalhamento sobre as opções, custo-efetividade e percepção dos sujeitos envolvidos poderão ser consultadas no texto completo da Síntese de Evidências.VII Metodologia A busca de evidências científicas foi realizada nos repositórios da Biblioteca Virtual em Saúde, Health Systems Evidence e PubMed. A estratégia de busca se deu com os seguintes termos e resultados: na BVS, “mortalidade materna” or “maternal mortality” or “mortalidad materna” or “maternal deaths” e aplicação do filtro “Tipo de Estudo” e seleção de ‘Revisões Sistemáticas’, retornando 68 estudos, dos quais, após a leitura dos títulos, foram selecionados 20 para leitura dos resumos; VII Disponível em: http://www.saude.sp.gov.br/resources/instituto-de-saude/ homepage/acesso-rapido/sintesedeevidencias-mm.pdf. no HSE, “mortalidade materna” or “maternal mortality” or “mortalidad materna” or “maternal deaths”, retornando 20 estudos, sendo 14 selecionadas para leitura dos resumos (seis revisões sistemáticas concluídas, uma avaliação econômica e sete documentos de política); no PubMed, (“mortalidade materna” or “maternal mortality” or “mortalidad materna” or “maternal deaths”) or ((postpartum or “posparto” or puerperio) and (mortali$ and Matern$)) or ((antenatal or “ante natal” or prenatal or “pre natal”) and (mortali$ and Matern$)), aplicando o filtro “Article type” e selecionando “Systematic reviews” e “Meta-analysis”, retornaram 1076 estudos; após realizar a leitura dos títulos, 68 foram selecionados para leitura dos resumos. Após a leitura dos resumos selecionados e identificação das questões de interesse, excluíram-se os estudos duplicados, as revisões sistemáticas voltadas a aspectos de manejo clínico e aquelas relacionadas a intervenções hospitalares, pelo fato de o município não contar com leitos obstétricos sob sua gestão, restando para leitura completa o total de 45 artigos, dos quais foram selecionadas seis revisões sistemáticas sobre efeitos de intervenções para a redução da mortalidade materna, que atendiam ao escopo de identificar opções de políticas desta síntese. A qualidade das revisões sistemáticas foi avaliada utilizando-se o instrumento AMSTAR19. Resultados O Quadro a seguir apresenta um resumo das seis revisões sistemáticas que apoiaram a definição das opções de políticas. \|107 46042001 miolo.indd 107 04/11/16 16:38 Não foram encontrados ensaios clínicos controlados que avaliassem os efeitos dos processos de auditoria e feedback na MM. Porém, o autor afirma que não há dúvidas com relação aos benefícios que tais processos trazem e que o processo de auditoria por si só não mostra grandes benefícios, é necessário que seja realizado juntamente com o processo de feedback, onde os profissionais de saúde teriam um retorno sobre seu desempenho. A revisão incluiu 118 estudos. Na análise primária 88 comparações de 72 estudos foram incluídas (qualquer intervenção em que auditoria e feedback fosse um componente em relação a nenhuma intervenção). Para desfechos dicotômicos a diferença de risco ajustado de conformidade com a prática desejada variou de - 0,16 (16% de redução absoluta em conformidade) para 0,70 (um aumento de 70% em conformidade). Para resultados contínuos a variação percentual ajustada relativamente ao controle variou de -0,10 (uma diminuição de 10% em conformidade absoluta) para 0,68 (um aumento de 68% em conformidade). Conformidade baixa com a prática recomendada na linha de base e maior intensidade de auditoria e feedback foram associados com taxas de risco ajustado maiores (maior eficácia) em todos os estudos. Foram selecionados sete estudos, com base nos critérios de inclusão e exclusão estabelecidos, em Bangladesh, Índia, Malawi e Nepal. Em Malawi foram realizados grupos de mulheres para empoderamento social através de 20 encontros. Os resultados indicam que a intervenção foi fator de proteção para o grupo de mulheres 0,26 (0,10 - 0,70). No Nepal em um estudo onde foram realizados grupos de mulheres, aconteceram 10 reuniões mensais com abordagem de aprendizado participativa. A participação dos grupos foi um fator de proteção para MM 0,20 (0,04 - 0,91). Avaliar se os processos de auditoria e feedback são efetivos para diminuição da MM. Avaliar os efeitos da auditoria e feedback sobre a pratica dos profissionais de saúde e os resultados sobre os pacientes. Avaliar o efeito de grupos de discussão com gestantes em relação ao cuidado habitual para redução da MM e neonatal em locais com poucos recursos. Benefícios dos processos de auditoria e feedback para profissionais de saúde. Influência dos processos de auditoria e feedback sobre a prática profissional. Pattinson et al.15, 2008. Jamtvedt et al.5, 2003. Grupos de discussão e ações participativas com gestantes para Prost et al.17, empoderar mulheres 2013. quanto ao autocuidado, reconhecendo quando e onde procurar ajuda. 1 1 2 Principais achados Objetivo do estudo Elemento da opção Opção Estudo 46042001 miolo.indd 108 8/11 9/11 6/11 AMSTAR Políticas de Saúde Informadas por Evidências \|108 04/11/16 16:38 46042001 miolo.indd 109 Avaliar a eficácia dos programas de capacitação profissional. Buscar evidências para a eficácia dos Cuidados Obstétricos de Emergência como estratégia para reduzir a MM em países em desenvolvimento. Benefícios dos cuidados obstétricos de emergência para reduzir a MM em países em desenvolvimento. Paxton et al.16, 2005. 4 5 Esta revisão identificou que o acesso aos Cuidados Obstétricos de Emergência reduz a MM em até 50% dos casos. Mostra também que quanto maior a distância de um centro de referência Obstétrico de Emergência, maior a probabilidade de ocorrerem mortes maternas. Conclui-se que cuidados obstétricos de emergência têm sido uma boa estratégia para a prevenção da MM. Estes resultados fortalecem a justificativa para a implementação e o fortalecimento de redes de atenção à saúde. Revisão sistemática para explorar as evidências disponíveis de intervenções para reduzir MM e dos fatores que influenciam sua implementação em países com recursos limitados. Cursos de capacitação para profissionais de saúde para melhorar na qualidade do atendimento obstétrico. Nyamtema et al.12 buscaram, em uma revisão sistemática de alta qualidade, as evidências científicas disponíveis de intervenções para reduzir mortalidade materna e dos fatores que influenciam sua implementação em países com recursos limitados. Os resultados dos estudos trazem o planejamento familiar como intervenção importante na redução da mortalidade materna. Estudo realizado em Bangladesh mostrou o planejamento familiar como fator de proteção OR 0,99 (0,66 - 1,50). Os resultados indicaram que o planejamento familiar é uma intervenção eficaz para a redução da mortalidade materna, mas deve ser realizada inserida em programas de intervenções integrados e baseados em evidências científicas. As conclusões do estudo em relação à redução da MM, através de intervenção em nível comunitário, basearam-se em um estudo randomizado controlado no Nepal, no qual a participação nos grupos na comunidade foi um fator de proteção para a MM 0,20 (0,04 - 0,91) e em outros estudos quase experimentais em Bangladesh. Essa revisão identificou 24 artigos sobre cursos e seus efeitos para profissionais de saúde. Estudos sobre cursos de curta duração apontam que profissionais que participam desses cursos melhoram o atendimento prestado aos pacientes, tendo melhor percepção dos riscos à vida. Alguns estudos sobre cursos de longa duração que verificaram o impacto destes cursos e que houve pouca ou nenhuma melhora em relação ao conhecimento do pré-natal, parto, nascimento, porém, melhoram a comunicação e o trabalho em equipe. As lacunas nas evidências quanto aos impactos desses cursos de aperfeiçoamento permanecem, logo devem ser consideradas ao decidir por sua adoção no sistema de saúde. Principais achados Objetivo do estudo Van Lonkhuijzen et al.27, 2010. 2e3 Elemento da opção Intervenção em nível comunitário através de grupos de discussão Nyamtema et para conscientização a 12 al. , 2011. respeito dos sinais de perigo de complicações na gravidez. Opção Estudo 8/11 8/11 10/11 AMSTAR Políticas de Saúde Informadas por Evidências \|109 04/11/16 16:38 Políticas de Saúde Informadas por Evidências Discussão Existem muitas opções para enfrentar a MM, mas nem todas apresentam o mesmo nível de certezas, efetividade ou são condicionadas pelos mesmos fatores de implementação. Além disso, as opções de política podem incluir desde ações isoladas até intervenções muito complexas, exigindo a consideração sobre a potência dos benefícios e riscos, além das barreiras e aspectos facilitadores nos diversos níveis afetados pela implementação de uma política, do sistema de saúde ao indivíduo. Com base nas evidências identificadas, foram formuladas cinco opções de políticas, descritas a seguir. Opção 1 - Auditoria dos óbitos maternos e feedback para profissionais de saúde: Os estudos mostraram que o processo de auditoria de todos óbitos maternos, juntamente com um feedback para os profissionais de saúde, pode auxiliar a redução da MM, uma vez que conhecer a causa da morte é tão ou mais importante quanto apenas quantificá-la, sendo fundamental que os profissionais de saúde possam ter um retorno sobre seu trabalho15. Além disso, uma parte das mortes maternas ocorridas no mundo poderia ser evitada se os profissionais de saúde estivessem capacitados para atender as necessidades básicas das mulheres durante o ciclo gravídico puerperal. Jamtvedt et. al5, avaliaram, em sua revisão sistemática de alta qualidade, os efeitos dos processos de auditora e feedback sobre a prática dos profissionais de saúde e os resultados sobre os pacientes, mostrando que essa estratégia pode ser efetiva para melhorar a prática dos profissionais. Tem-se assim que a implementação dessa opção traz melhora da comunicação entre os profissionais, melhoria nos cuidados com a paciente e satisfação profissional6, o que pode auxiliar no processo de redução da MM, embora os efeitos provocados sejam pequenos ou moderados15. Como incertezas em relação aos benefícios têm-se que o processo de auditoria por si só não mostra grandes benefícios15, a coleta de informações e as reuniões de feedback demandam muito tempo, podendo ocasionar conflitos entre funcionários, departamentos e instituições; e os processos de auditoria e retroalimentação podem ocasionar nos trabalhadores medo de repressões, impedindo assim, a eficácia de seu trabalho6. Opção 2 - Mobilização da Comunidade e Ações Educativas: Grupos permanentes de empoderamento da comunidade permitem que haja melhor compreensão, confiança e suporte para o autocuidado, tornando seu público mais atento para “quando” e “onde” buscar cuidados em saúde, o que pode ajudar a reduzir a MM. A literatura aponta que a implementação dessa opção em locais com poucos recursos apresentou através de programas de intervenções integrados, baseados em evidências científicas, resultados satisfatórios12. Além disso, nos países onde a intervenção por meio de grupos com as gestantes foi adotada, notou-se que quando ao menos 30% das gestantes participavam, havia redução de 49% da MM17. Com relação às incertezas dos benefícios, os autores não relatam danos potenciais nessa opção, e apontam a necessidade, em países com baixos recursos, que os governos e as instituições de saúde se unam em seus compromissos e responsabilidades na implementação de pacotes de intervenções baseadas em evidências científicas12. Opção 3 - Qualificação das Ações de Planejamento Familiar: O planejamento familiar acompanhado de um investimento gradual na qualidade dos serviços é capaz de auxiliar na redução da MM, em áreas rurais e urbanas4, dando suporte às escolhas das mulheres de decidir quando ou não engravidar e também ofertando contraceptivos de barreira na prevenção de doenças sexualmente transmissíveis. Para essa ação ser efetiva em sua implementação para redução da MM deve ser realizada por meio de programas e intervenções integradas, baseados em evidências científicas12. As incertezas \|110 46042001 miolo.indd 110 04/11/16 16:38 Políticas de Saúde Informadas por Evidências dos benefícios referem-se à religião, que pode ser uma forma de impedimento na utilização dos métodos; e a distribuição de métodos contraceptivos para jovens. Essa deve ser realizada de modo com que os pais não sejam motivo de constrangimento ou recusa para seus filhos19. Opção 4 - Capacitação para Profissionais de Saúde: A capacitação para profissionais de saúde consiste em um programa de Educação Permanente em saúde, que visa um processo dinâmico-pedagógico de desenvolvimento e qualificação de ações que alberguem conhecimento nas dimensões técnico-científica, ético-política e socioeducativa da assistência realizada por esses trabalhadores. Também preza pela melhora da capacidade de prestar cuidados à mulher, através de intervenções que considerem todas as dimensões do ser humano a fim de melhorar a saúde materna. Van Lonkhuijzen et. al (2010)27 em uma revisão sistemática de alta qualidade, avaliaram a eficácia dos programas de formação que visam melhorar os cuidados obstétricos de emergência em ambientes com poucos recursos. Identificou assim que, cursos de capacitação profissional contribuem positivamente no aumento do conhecimento e, consequentemente, no comportamento das habilidades após treinamento; assim, profissionais que participam desses cursos apresentam maior feedback com a equipe, gerenciamento de tarefas e comunicação, competência para realizar suas tarefas no serviço, consciência da deficiência no atendimento, melhor identificação das condições que ocasionam risco de morte e maior educação pelos pares quando comparados com aqueles que não participaram de nenhum curso de treinamento profissional. Como danos potenciais e incertezas em relação aos benefícios dessa opção tem-se que, o controle excessivo do desempenho dos profissionais, no caso da recertificação parece ter efeitos negativos sobre esses11. Assim, apesar dessa opção apresentar inúmeras melhorias, o sucesso da estratégia está ligado ao método de avaliação e monitoramento da ação, sujeitos e efeitos no orçamento27. Opção 5 - Referenciamento aos Serviços de Emergências Obstétricas: A detecção precoce e, consequentemente, o referenciamento a serviços de atendimento especializado de emergência obstétrica são essenciais para evitar e diminuir a MM. A literatura aponta que, em áreas onde há cobertura de serviços obstétricos de emergências e o acesso é facilitado, a redução da MM é de até 50% 16. Ressalta-se aqui que, para auxiliar nesta redução é necessário que haja boa qualidade nos serviços3, criação de normas e protocolos de transferência rápida entre as unidades de atendimento11 e formação de equipes multidisciplinares para atuar, dentro de instituição e da comunidade, distribuídos de forma equivalente em todos os estabelecimentos que oferecem serviços de atenção obstétrica24. Como incertezas em relação aos benefícios tem-se que a falta de compromisso dos tomadores de decisão e outros atores chave no financiamento dos órgãos provedores, que poderiam dar sustento aos programas, podem prejudicar o financiamento desta opção14. Conclusão Embora as opções apresentadas não tenham que necessariamente ser implementadas de forma conjunta e completa, a aplicação prática deve considerar a viabilidade local, inserindo-se na governabilidade da tomada de decisão, independentemente da dimensão do sistema de saúde (nacional, regional ou local). Nesse sentido as opções de políticas com foco na Atenção Básica poderiam ser priorizadas, uma vez que o município não conta com leitos obstétricos sob sua gestão. Também é importante considerar as barreiras de implementação das opções, especialmente as localizadas no campo da cultura e representações sociais dos usuários e trabalhadores de saúde. \|111 46042001 miolo.indd 111 04/11/16 16:38 Políticas de Saúde Informadas por Evidências As opções de políticas foram apresentadas e discutidas amplamente em um Diálogo Deliberativo (que consiste em uma discussão estruturada centrada em uma síntese de evidências para políticas), com a participação de gestores municipais, profissionais de saúde que participam do Comitê de Investigação da MM e Infantil do município, representantes do Conselho Municipal de Saúde, do Comitê Estadual de Investigação da MM e Infantil e pesquisadores. Espera-se que o resultado desse processo seja a definição de um plano de ação municipal de enfrentamento à MM no município de Franco da Rocha, construído com base em evidências científicas e no contexto local. Referências 1. Blackwell SP, Breen M, Hinshaw K. Learning from low income countries: what are the lessons? Hands on course may help deliver obstetric care. BMJ.2004;329(7475):1184. 2. Coimbra CL, Silva AMA, Mochel EG, Alves MTSSB, Ribeiro VS, Aragão VMF, et al. Fatores associados à inadequação do uso da assistência pré-natal. Rev Saúde Pública. 2003;37:456-62. 3. Fournier P, Dumont A, Tourigny C, Dunkley G, Dramé S. Improved access to comprehensive emergency obstetric care and its effect on institutional maternal mortality in rural Mali. Bull World Health Organ. 2009;87:30–38. 4. Goldie SJ, Sweet S, Carvalho N, Natchu UCM, Hu D. Alternative strategies to reduce maternal mortality in India: a cost-effectiveness analysis. PLoS Med. 2010;7(4):e1000264. 5. Ivers N, Jamtvedt G, Flottorp S, Young JM, Odgaard-Jensen J, French SD, et al. Audit and feedback: effects on professional practice and health care outcomes. The Cochrane Database of Systematic Reviews, 3, 2003. 6. Johnston G, Crombie IK, Davies HTO, Millard A. Reviewing audit: barriers and facilitating factors for effective clinical audit. Qual Health Care. 2000;9(1):23–36. 7. Jonas, E. Improving provider performance: an exploration of the literature, Box 3. Bolivia In-Service Training. Mother Care Matters.2000;9:10–1. 8. Kroeger M, Kaphagawani N, Kafulafula UK, Maluwa VM. Final evaluation of the malawi safe motherhood project life saving skills training programme. Blantyre: DFID/Safe Motherhood Project. 2003. 9. McDermott J, Beck D, Buffington ST, Annas J, Supratikto G, Prenggono D, et al. Two models of in-service training to improve midwifery skills: how well do they work? J Midwifery Womens Health. 2001;46:217–25. 10. Merkur S, Mladovsky P, Mossialos E, Mckee M. Policy brief. Do lifelong learning and revalidation ensure that physicians are fit to practise? Copenhagen: WHO; 2008. 11. Ministère de la Santé Publique et de la Population. Normes et procédures cliniques des services de santé de la reproduction en République Centrafricaine. Haiti; 2003. 84p. 12. Nyamtema AS, Urassa DP, Roosmalen JV. Maternal health interventions in resource limited countries: a systematic review of packages, impacts and factors for change. BMC Pregnancy and Childbirth. 2011; 11:30. 13. O’rourke K. The effect of hospital staff training on management of obstetrical patients referred by traditional birth attendants. Int J Gynaecol Obstet.1995;48(Suppl):S95-102. 14. Pattinson R, Kerber K, Waiswa P, Day LT, Mussell F, Asiruddin SK, et al. Perinatal mortality audit: counting, accountability, and overcoming challenges in scaling up in low-and-middle-income countries. International Jornal of Gynaecology and Obstetrics.2009;107:113–21,121–2. 15. Pattinson RC, Say L, Makin JD, Bastos MH. Auditoría de acontecimientos críticos y retroalimentación (“feedback”) para disminuir la mortalidad y la morbilidad perinatales y maternas (Revisión Cochrane traducida). Biblioteca Cochrane Plus. 2008;4. 16. Paxton A, Maine D, Freedman L, Fry D, Lobis S. The evidence for emergency obstetric care International Journal of Gynecology and Obstetrics. 2005; 88:181-193. 17. Prost A, Seward N, Azad K, Coomarasamy A, Copas A, Houweling TAJ, et al. Women’s groups practising participatory learning and action to improve maternal and newborn health in low-resource settings: a systematic review and meta-analysis. Lancet. 2013;381:1736–46. 18. Ronsmans C, Vanneste A, Chakraborty J, Van Ginneken J. Decline in maternal mortality in Matlab, Bangladesh: acautionary tale. Lancet.1997;350:1810-4. 19. Sepou A, Serdouma E, Komas NP, Gody C, Abeye J, Koffi B, et al. Strategies for reducing maternal mortality in Central African Republic. Evidence-Informed Policy Network (EVIPNet);2011. 20. Shea JB, Grimshaw JM, Wells GA, Boers M, Andersson N, Hamel C, et al. Development of AMSTAR: a measurement tool to assess the methodological quality of systematic reviews. BMC Medical Research Methodology, London. 2007;7:10. 21. Sloan NL, Nguyen TN, Do TH, Quimby C, Winikoff B, Fassihian G. Effectiveness of lifesaving skills training and improving institutional emergency obstetric care readiness in Lam Dong, Vietnam. J Midwifery Womens Health. 2005;50:315–2. 22. Thairu A, Schmidt K. Training and author is ingmid level providers in life saving skills in Kenya case study No 8. In: Crump S, editor. Shaping policy for maternal and newborn health: a compendium of case studies. Baltimore, MD: JHPIEGO.2003. 23. Warren C, Liambila W. Safe motherhood demonstration project western province. approaches to providing quality maternal care in Kenya. Nairobi: Population Council. 2004. 24. World Health Organization. Monitoring emergency obstetric care: a handbook. Geneva; 2009. 25. World Health Organization. Trends in maternal mortality: 1990 to 2008. Estimates developed by WHO, UNICEF, UNFPA and The World Bank. Geneva; 2010. [acesso em 13 jun. 2015]. Disponível em: http:// whqlibdoc.who.int/ publications/2010/9789241500265_eng.pdf 26. Woods DL. An innovative programme for training in maternal and newborn care. Seminars in Fetal & Neonatal. 1999;4(3):209-216. 27. Van Lonkhuijzen L, Dijkman A, Van Roosmalen J, Zeeman G, Scherpbier A. A systematic review of the effectiveness of training in emergency obstetric care in low-resource environments. BJOG. 2010; 117(7):777-87. \|112 46042001 miolo.indd 112 04/11/16 16:38
https://openalex.org/W4248024640	https://opus.bibliothek.uni-wuerzburg.de/files/5641/Kleinschnitz_journal.pbio.1000479.pdf	English	null	Post-Stroke Inhibition of Induced NADPH Oxidase Type 4 Prevents Oxidative Stress and Neurodegeneration	SciVee	2,010	cc-by	11,615	Abstract Ischemic stroke is the second leading cause of death worldwide. Only one moderately effective therapy exists, albeit with contraindications that exclude 90% of the patients. This medical need contrasts with a high failure rate of more than 1,000 pre-clinical drug candidates for stroke therapies. Thus, there is a need for translatable mechanisms of neuroprotection and more rigid thresholds of relevance in pre-clinical stroke models. One such candidate mechanism is oxidative stress. However, antioxidant approaches have failed in clinical trials, and the significant sources of oxidative stress in stroke are unknown. We here identify NADPH oxidase type 4 (NOX4) as a major source of oxidative stress and an effective therapeutic target in acute stroke. Upon ischemia, NOX4 was induced in human and mouse brain. Mice deficient in NOX4 (Nox42/2) of either sex, but not those deficient for NOX1 or NOX2, were largely protected from oxidative stress, blood-brain-barrier leakage, and neuronal apoptosis, after both transient and permanent cerebral ischemia. This effect was independent of age, as elderly mice were equally protected. Restoration of oxidative stress reversed the stroke-protective phenotype in Nox42/2 mice. Application of the only validated low-molecular-weight pharmacological NADPH oxidase inhibitor, VAS2870, several hours after ischemia was as protective as deleting NOX4. The extent of neuroprotection was exceptional, resulting in significantly improved long-term neurological functions and reduced mortality. NOX4 therefore represents a major source of oxidative stress and novel class of drug target for stroke therapy. Citation: Kleinschnitz C, Grund H, Wingler K, Armitage ME, Jones E, et al. (2010) Post-Stroke Inhibition of Induced NADPH Oxidase Type 4 Prevents Oxidative Stress and Neurodegeneration. PLoS Biol 8(9): e1000479. doi:10.1371/journal.pbio.1000479 Academic Editor: Malcolm McLeod, University of Edinburgh, United Kingdom Citation: Kleinschnitz C, Grund H, Wingler K, Armitage ME, Jones E, et al. (2010) Post-Stroke Inhibition of Induced NADPH Oxidase Type 4 Prevents Oxidative Stress and Neurodegeneration. PLoS Biol 8(9): e1000479. doi:10.1371/journal.pbio.1000479 Academic Editor: Malcolm McLeod, University of Edinburgh, United Kingdom Received February 19, 2010; Accepted July 28, 2010; Published September 21, 2010 nschnitz 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: 2010 Kleinschnitz et al. This is an open-access article distributed under the terms of the Creative Commons Attribut unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Funding: This work was supported by the NHMRC, Australia, the Deutsche Forschungsgemeinschaft (DFG), Germany (to HHHWS and CK), and by the Bundesministerium fur Bildung und Forschung within the framework of the NGFN-Plus and the European Commission (EUMODIC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: HHHWS and KW declare a potential competing interest as shareholder and previous employee, respectively, of Vasopharm GmbH, which develops NADPH oxidase inhibitors such as VAS2870. All authors declare that they adhere to all PLoS Biology policies on sharing data and materials as detailed in the PLoS Biology guide for authors. Abbreviations: CISS, constructive interference in steady state; KO, knock out; pMCAO, permanent middle cerebral artery occlu species; rt-PA, recombinant tissue plasminogen activator; tMCAO, transient middle cerebral artery occlusion; TTC, 2,3,5-triphenyltet type. onstructive interference in steady state; KO, knock out; pMCAO, permanent middle cerebral artery occlusion; ROS, reactive oxygen nt tissue plasminogen activator; tMCAO, transient middle cerebral artery occlusion; TTC, 2,3,5-triphenyltetrazolium chloride; WT, wild * E-mail: h.schmidt@farmaco.unimaas.nl (HHHWS); christoph.kleinschnitz@mail.uni-wuerzburg.de (CK) Post-Stroke Inhibition of Induced NADPH Oxidase Type 4 Prevents Oxidative Stress and Neurodegeneration Christoph Kleinschnitz1, Henrike Grund2, Kirstin Wingler2,3,4,5, Melanie E. Armitage3,5, Emma Jones3, Manish Mittal2, David Barit6, Tobias Schwarz1, Christian Geis1, Peter Kraft1, Konstanze Barthel7, Michael K. Schuhmann1,8, Alexander M. Herrmann1,8, Sven G. Meuth1,8, Guido Stoll1, Sabine Meurer3, Anja Schrewe9, Lore Becker9,10, Vale´rie Gailus-Durner9, Helmut Fuchs9, Thomas Klopstock10, Martin Hrabe´ de Angelis9,11, Karin Jandeleit-Dahm6, Ajay M. Shah12, Norbert Weissmann2, Harald H. H. W. Schmidt2,3,4,5 1 Neurologische Klinik und Poliklinik, Universita¨t Wu¨rzburg, Wu¨rzburg, Germany, 2 Rudolf-Buchheim-Institut fu¨r Pharmakologie & Medizinische Klinik, Justus-Liebig- Universita¨t, Gießen, Germany, 3 Department of Pharmacology and Centre for Vascular Health, Monash University, Melbourne, Australia, 4 Department of Pharmacology and Toxicology and Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, The Netherlands, 5 National Stroke Research Institute, Florey Neuroscience Institutes, Melbourne, Australia, 6 Baker IDI Heart and Diabetes Institute, Juvenile Diabetes Research Foundation (JDRF) International Center for Diabetic Complications Research, Melbourne, Australia, 7 Abteilung Neurologie, Georg-August Universita¨t Go¨ttingen, Go¨ttingen, Germany, 8 Universita¨tsklinik Mu¨nster, Klinik und Poliklinik fu¨r Neurologie—Entzu¨ndliche Erkrankungen des Nervensystems und Neuroonkologie, Mu¨nster, Germany, 9 Institute of Experimental Genetics, Helmholtz Zentrum Mu¨nchen, German Research Center for Environmental Health, Mu¨nchen, Germany, 10 Friedrich-Baur-Institut an der Neurologischen Klinik, Klinikum der Ludwig- Maximilians-Universita¨t Mu¨nchen, Mu¨nchen, Germany, 11 Lehrstuhl fu¨r Experimentelle Genetik, Technische Universita¨t Mu¨nchen, Freising-Weihenstephan, Germany, 12 King’s College London School of Medicine, The James Black Centre, Cardiovascular Division, London, United Kingdom PLoS Biology \| www.plosbiology.org NOX4 Is Induced during Ischemic Stroke in Mice and Humans Because NOX4 mRNA is expressed at higher levels in cerebral than in peripheral blood vessels [13] and is induced in stroke [14], we first sought to validate these data not only at the mRNA but also at the protein level. In all experiments, we followed current guidelines defining methodological standards for experimental stroke studies [4,6,7,16,17]. Here we chose a model of acute ischemic stroke in which mice are subjected to transient middle cerebral artery occlusion (tMCAO). This disease model is thought to involve oxidative stress and an induction of Nox4 expression [18]. Indeed, expression of NOX4 mRNA was significantly higher 12 h and 24 h after tMCAO in the basal ganglia and neocortex of wild-type mice than in sham-operated controls, in which basal NOX4 expression was low (Figure 1A). This result was validated by immunohistochemistry using a specific NOX4 antibody. We detected a stronger staining in neurons and cerebral blood vessels in wild-type mice subjected to tMCAO than in sham-operated controls. Although immunohistochemistry is not quantitative, this finding suggests higher levels of NOX4 protein (Figure 1B). Importantly, NOX4 staining was also stronger in brain samples from stroke patients. Although NOX4 was barely detectable in healthy brain regions, clear positive labeling of NOX4 was seen in neurons and vascular endothelial cells from the forebrain cortex of stroke patients. This finding was confirmed by double labeling for NeuN (a neuronal marker) or von Willebrand factor (an endothelial marker) and NOX4 in brain tissue (Figure 1B). These data indicate that NOX4 protein is induced during brain ischemia in mice, and this observation would be in agreement with a major functional role for NOX4 in ischemic stroke. Our limited observations in a small number of human cases provide some support to the hypothesis that these processes are also important in human stroke. Although a plethora of drugs for the treatment of acute stroke are effective in animal models, their translation into clinical practice has completely failed [3,4]. As a result, many pharma- ceutical companies have withdrawn from drug discovery in this area. To overcome this lack of clinically effective neuroprotective drugs, innovative strategies are urgently needed to identify pathways that can be targeted with innovative therapies [5]. Higher quality study designs are also required [6,7]. One such high-potential pathway in ischemic stroke may be the occurrence of oxidative stress, i.e., the increased occurrence of reactive oxygen species (ROS) above physiological levels. NOX4 Is Induced during Ischemic Stroke in Mice and Humans Oxida- tive stress has been suggested for many years to cause tissue damage and neuronal death. The toxicity of ROS can be further increased by nitric oxide to produce reactive nitrogen species such as peroxynitrite (ONOO2), a molecule that causes oxidation and nitration of tyrosine residues on proteins [8]. Disappointingly, there is no conclusive evidence of a causal link between oxidative stress and the development of disease, and there is no successful therapeutic application targeting oxidative stress. To date, clinical attempts to scavenge ROS by applying antioxidants did not result in clinical benefit [9] or even caused harm [10,11]. However, the characterization of the relevant enzymatic sources of oxidative stress may allow therapeutic targeting of oxidative stress by preventing the formation of ROS in the first place, instead of scavenging ROS after they have been formed. Nox42/2 but Neither Nox1y/2 nor Nox2y/2 Mice Are Protected in Both Transient and Permanent Ischemic Stroke We first subjected 6- to 8-wk-old male Nox42/2 mice to tMCAO and, after 24 h, assessed infarct volumes by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) (Figure 2A). Infarct volumes were significantly smaller, by approximately 75%, in male Nox42/2 mice than in sex-matched wild-type controls (25.5614.8 mm3 versus 78.7619.5 mm3, respectively). The smaller infarct volume was functionally relevant: compared with wild-type mice, Nox42/2 mice had significantly better overall neurological function (Bederson score 1.260.7 in Nox42/2 mice versus 3.761.1 in wild-type mice) as well as better basal motor function and coordination (grip test score 4.360.7 in Nox42/2 mice versus 1.761.3 in wild-type mice) 24 h after tMCAO (Figure 2B). Gender can significantly influence stroke outcome in rodents [4,16,17]. Therefore, we also subjected female Nox42/2 mice to 60 min of tMCAO. In line with the results in male mice, Nox4-deficient female mice also developed significantly smaller infarctions A potential source of ROS are NADPH oxidases, the only known enzyme family that is only dedicated to ROS production [12]. Four rodent genes of the catalytic subunit NOX, Nox1, Nox2, Nox3, and Nox4, have been identified, of which Nox1, Nox2, and Nox4 are expressed in the vasculature. NOX4 is the most abundant vascular isoform; its expression is even higher in cerebral than in peripheral blood vessels [13] and, further, induced in stroke [14]. Therefore, we hypothesized that NOX4 is the most relevant source of ROS in stroke. Author Summary Stroke is the second leading cause of death worldwide. Today, only one approved therapy exists—a drug that breaks down blood clots—the effectiveness of which is moderate, and it can only be used in about 10% of patients because of contraindications. New therapeutic strategies that are translatable to humans and more rigid thresholds of relevance in pre-clinical stroke models are needed. One candidate mechanism is oxidative stress, which is the damage caused by reactive oxygen species (ROS). Antioxidant approaches that specifically target ROS have thus far failed in clinical trials. For a more effective approach, we focus here on targeting ROS at its source by investigating an enzyme involved in generating ROS, known as NADPH oxidase type 4, or NOX4. We found that NOX4 causes oxidative stress and death of nerve cells after a stroke. Deletion of the NOX4-coding gene in mice, as well as inhibiting the ROS-generating activity of NOX with a pharmacological inhibitor, reduces brain damage and improves neurological function, even when given hours after a stroke. Importantly, neuroprotection was preserved in old male and female Nox42/2 mice as well as in Nox42/2 mice subjected to permanent ischemia. NOX4 thus represents a most promising new therapeutic target for reducing oxidative stress in general, and in brain injury due to stroke in particular. Introduction [3], only one therapy approved by the United States Food and Drug Administration is available, i.e., thrombolysis using recom- binant tissue plasminogen activator (rt-PA). However, the efficacy of rt-PA on functional outcomes is moderate at best, and more than 90% of all stroke patients must be excluded from rt-PA treatment because of over 25 labeled contraindications. Therefore, there is an unmet need for more effective therapies in acute stroke. Ischemic stroke has outstanding medical relevance as it is the second leading cause of death in industrialized countries [1]. Due to the aging of the population, the incidence of stroke is projected to rise even further in the future [2]. Despite tremendous research activity, with more than 100 clinical trials in human stroke patients PLoS Biology \| www.plosbiology.org September 2010 \| Volume 8 \| Issue 9 \| e1000479 September 2010 \| Volume 8 \| Issue 9 \| e1000479 1 Role of NOX4 in Stroke has been implicated in the regulation of systemic and hypoxic vascular responses. Therefore, we had to exclude systemic vascular effects of NOX4 deletion on blood pressure, which may affect stroke outcome independent of a specific neuronal or neurovas- cular mechanism. Finally, to examine the therapeutic potential of NOX4 as a drug target, we infused the specific NADPH oxidase inhibitor VAS2870 [15] after ischemia, thus mirroring the clinical scenario. PLoS Biology \| www.plosbiology.org September 2010 \| Volume 8 \| Issue 9 \| e1000479 NOX4 Is Induced during Ischemic Stroke in Mice and Humans However, in contrast to Nox42/2 mice, we observed no protection in these animals, neither in terms of infarct volumes nor on functional outcomes on day 1 after tMCAO, even with large subject sample sizes (n = 19 for Nox2y/2 mice, p.0.05; Figure 2A). Ischemic stroke is usually a disease of the elderly and, consequently, one should verify any stroke-protective effects observed in young adult laboratory animals also in an older cohort [4,16,17]. Indeed, 18- to 20-wk-old Nox42/2 mice also developed significantly smaller brain infarctions (27.8615.1 mm3 versus 81.8619.0 mm3, respectively) and less severe neurological deficits than age-matched controls, thereby confirming our results in young animals (Figure 2B). We also determined the functional outcome and mortality of 6- to 8-wk-old male Nox42/2 mice and matched wild-type controls over a longer time period after ischemic stroke (Figure 2D). Five days after 60 min of tMCAO, 15 of 15 wild-type mice (100%) had died, which is in line with previous reports [19]. In contrast, seven of ten Nox42/2 mice (70%) survived until day 5, and five of these were still alive after 1 wk (p = 0.0039) (Figure 2D). In line with these findings, Nox4- deficient mice showed significantly better Bederson scores than controls over the whole observation period, and neurological deficits remained low until day 7 (Figures 2D and S4). (30.166.7 mm3 versus 89.5622.2 mm3, respectively) and less severe neurological deficits (p,0.001) than female controls (Figure 2A and 2B). Histological analysis revealed that all infarcts in Nox42/2 mice were restricted to the basal ganglia (arrow in Figure 2A and 2C), whereas in wild-type mice, the neocortex was also consistently affected. Serial magnetic resonance imaging of living mice up to 6 d after stroke showed that in Nox42/2 mice the infarct volume did not increase over time, thus indicating that deletion of the Nox4 gene provides sustained protection against stroke (Figure 2C). Moreover, infarcts always appeared hyperin- tense on blood-sensitive constructive interference in steady state (CISS) sequences. Hypointense areas, which typically indicate intracerebral hemorrhage, were absent from Nox42/2 mice and wild-type controls. This finding excludes the possibility of an increased rate of bleeding complications caused by Nox4 deficiency. are restricted to the cortex and highly reproducible in size and location. Moreover, photothrombosis has been shown to induce early and profound ROS formation and blood-brain-barrier leakage [20,21], two key readout parameters of the present investigation. No Apparent Vascular Phenotype of Nox42/2 Mice Other Than in Stroke In line with these findings, Nox4- deficient mice showed significantly better Bederson scores than controls over the whole observation period, and neurological deficits remained low until day 7 (Figures 2D and S4). Protection from Ischemic Stroke in Nox42/2 Mice Is a Result of Reduced Oxidative Stress, Neuronal Apoptosis, and Blood-Brain-Barrier Leakage NOX4 Is Induced during Ischemic Stroke in Mice and Humans Importantly, photothrombosis-induced infarct vol- umes were as reduced in Nox42/2 mice relative to wild-type mice (3.364.6 mm3 versus 25.0612.8 mm3, respectively, a difference of 86.8%; Figure 2F) as they were in the tMCAO model. PLoS Biology \| www.plosbiology.org Protection from Ischemic Stroke in Nox42/2 Mice Is a Result of Reduced Oxidative Stress, Neuronal Apoptosis, and Blood-Brain-Barrier Leakage Next we sought to elucidate the underlying mechanisms of this NOX4-specific neurotoxicity in stroke. NOX4 can form superox- ide or H2O2, which can interact with nitric oxide to form reactive nitrogen species. Therefore, we stained brain sections with broad- spectrum indicators of oxidative/nitrative stress, i.e., dihydroethi- dium [28] and nitrotyrosine [8]. At 12 h and 24 h after tMCAO, brains from wild-type mice exhibited a significantly larger amount (by a factor of 2.5–3.5) of ROS in neurons than brains from sham- operated animals, as quantified by dihydroethidium staining (Figure 3A). Neurons from Nox42/2 mice, in contrast, showed only very small ischemia-induced increases in ROS relative to those in sham-operated controls (p.0.05). ROS formation from neurons after 24 h was also significantly reduced in Nox42/2 mice subjected to pMCAO (Figure S6). Because the dihydroethidium stain may also indicate oxidative chemistry events, including formation of ONOO2 and nitration of protein tyrosine residues [8], we analyzed the extent of protein nitration in Nox42/2 and wild-type mice subjected to tMCAO. In agreement with our findings on the generation of ROS, tissue nitration occurred to a lesser extent in ischemic brains from Nox42/2 mice than in those from wild-type controls (Figure 3B). Oxidative chemistry events such as the formation of ROS and peroxynitrite, as detected by According to the current experimental stroke guidelines [4,16,17], any protective effect also requires evaluation in models of both transient and permanent ischemia. We therefore subjected Nox42/2 mice to filament-induced permanent middle cerebral artery occlusion (pMCAO), a procedure in which no tissue reperfusion occurs (Figure 2E). In the absence of Nox4, infarct volumes (66.7628.6 mm3 versus 120.1615.6 mm3, p,0.05) and neurological deficits (Bederson score 2.361.7 versus 3.460.8, p,0.05) at day 1 after pMCAO were significantly reduced compared with those in wild-type controls, although to a lesser extent than they were in the tMCAO model (Figures 2E and S5). Brain infarctions following filament-induced pMCAO are large, and the infarct borders are often not very well defined, which limits the accuracy of any estimation on infarct volumes. We therefore used another model of permanent stroke, cortical photothrombosis, to further verify our findings. Here, the lesions According to the current experimental stroke guidelines [4,16,17], any protective effect also requires evaluation in models of both transient and permanent ischemia. We therefore subjected Nox42/2 mice to filament-induced permanent middle cerebral artery occlusion (pMCAO), a procedure in which no tissue reperfusion occurs (Figure 2E). NOX4 Is Induced during Ischemic Stroke in Mice and Humans To test this hypothesis, we generated constitutively NOX4- deficient (Nox42/2) mice and directly compared them to NOX1- deficient (Nox1y/2) and NOX2-deficient (Nox2y/2) mice. NOX4 September 2010 \| Volume 8 \| Issue 9 \| e1000479 2 PLoS Biology \| www.plosbiology.org Role of NOX4 in Stroke PLoS Biology \| www.plosbiology.org September 2010 \| Volume 8 \| Issue 9 \| e1000479 PLoS Biology \| www.plosbiology.org September 2010 \| Volume 8 \| Issue 9 \| e1000479 3 Role of NOX4 in Stroke Role of NOX4 in Stroke Figure 1. Induction of NOX4 expression after ischemic stroke in mice and humans. (A) Relative gene expression of Nox4 in the ischemic basal ganglia (left) and cortex (right) of wild-type mice after sham operation and 4 h, 12 h, and 24 h after tMCAO (n = 5). , p,0.05, one-way ANOVA, Bonferroni post-hoc test, compared with sham-treated controls. (B) Immunohistochemical detection of NOX4 protein in ischemic brains of wild-type mice (after sham operation or tMCAO, day 1) and humans (samples from stoke patients, after routine autopsy). We compared NOX4 immunolabeling in the ischemic forebrain cortex and the unaffected contralateral side. In ischemic samples, NOX4 was predominantly expressed in neurons (arrowheads) and endothelial cells (arrows). This distribution was confirmed by visualization of NOX4 and NeuN or NOX4 and von Willebrand Factor in the same structures. All scale bars represent 100 mm. doi:10.1371/journal.pbio.1000479.g001 (30.166.7 mm3 versus 89.5622.2 mm3, respectively) and less severe neurological deficits (p,0.001) than female controls (Figure 2A and 2B). Histological analysis revealed that all infarcts in Nox42/2 mice were restricted to the basal ganglia (arrow in Figure 2A and 2C), whereas in wild-type mice, the neocortex was also consistently affected. Serial magnetic resonance imaging of living mice up to 6 d after stroke showed that in Nox42/2 mice the infarct volume did not increase over time, thus indicating that deletion of the Nox4 gene provides sustained protection against stroke (Figure 2C). Moreover, infarcts always appeared hyperin- tense on blood-sensitive constructive interference in steady state (CISS) sequences. Hypointense areas, which typically indicate intracerebral hemorrhage, were absent from Nox42/2 mice and wild-type controls. This finding excludes the possibility of an increased rate of bleeding complications caused by Nox4 deficiency. To establish any potential specificity of this function for NOX4 compared to NOX1 and NOX2 in stroke, we carried out identical experiments in 6- to 8-wk-old Nox1y/2 and Nox2y/2 mice. No Apparent Vascular Phenotype of Nox42/2 Mice Other Than in Stroke Based on the physiological distribution of NOX4 in kidney [22], lung [23], and aorta [24], as well as cell biology data obtained using small interfering RNA approaches [23], one would predict basal phenotypes in a Nox42/2 mouse, such as arterial hypotension, reduced hypoxic pulmonary hypertension, and altered renal function. Importantly, these effects could potentially modulate or interfere with stroke outcome even in the absence of a specific neuronal or neurovascular mechanism. Surprisingly, systemic elimination of Nox4 did not result in any apparent abnormal vascular phenotype (Text S1; Figures S1 and S2; Table S1). In particular, blood pressure was normal, and hypoxic pulmonary hypertension still occurred despite a 20-fold induction of NOX4 in wild-type animals [23]. In contrast, Nox1- and p47phox-deficient mice (a Nox2 subunit) have a lower basal blood pressure, and their blood-pressure response to angiotensin II is reduced [25–27]. Our data suggest that any phenotype caused by deleting Nox4, unlike those caused by deleting Nox1 and Nox2, would indeed be brain-specific. To establish any potential specificity of this function for NOX4 compared to NOX1 and NOX2 in stroke, we carried out identical experiments in 6- to 8-wk-old Nox1y/2 and Nox2y/2 mice. However, in contrast to Nox42/2 mice, we observed no protection in these animals, neither in terms of infarct volumes nor on functional outcomes on day 1 after tMCAO, even with large subject sample sizes (n = 19 for Nox2y/2 mice, p.0.05; Figure 2A). Ischemic stroke is usually a disease of the elderly and, consequently, one should verify any stroke-protective effects observed in young adult laboratory animals also in an older cohort [4,16,17]. Indeed, 18- to 20-wk-old Nox42/2 mice also developed significantly smaller brain infarctions (27.8615.1 mm3 versus 81.8619.0 mm3, respectively) and less severe neurological deficits than age-matched controls, thereby confirming our results in young animals (Figure 2B). We also determined the functional outcome and mortality of 6- to 8-wk-old male Nox42/2 mice and matched wild-type controls over a longer time period after ischemic stroke (Figure 2D). Five days after 60 min of tMCAO, 15 of 15 wild-type mice (100%) had died, which is in line with previous reports [19]. In contrast, seven of ten Nox42/2 mice (70%) survived until day 5, and five of these were still alive after 1 wk (p = 0.0039) (Figure 2D). Protection from Ischemic Stroke in Nox42/2 Mice Is a Result of Reduced Oxidative Stress, Neuronal Apoptosis, and Blood-Brain-Barrier Leakage In the absence of Nox4, infarct volumes (66.7628.6 mm3 versus 120.1615.6 mm3, p,0.05) and neurological deficits (Bederson score 2.361.7 versus 3.460.8, p,0.05) at day 1 after pMCAO were significantly reduced compared with those in wild-type controls, although to a lesser extent than they were in the tMCAO model (Figures 2E and S5). September 2010 \| Volume 8 \| Issue 9 \| e1000479 4 Role of NOX4 in Stroke Role of NOX4 PLoS Biology \| www.plosbiology.org 5 September 2010 \| Volume 8 \| Issue 9 \| September 2010 \| Volume 8 \| Issue 9 \| e1000479 PLoS Biology \| www.plosbiology.org September 2010 \| Volume 8 \| Issue 9 \| e1000479 PLoS Biology \| www.plosbiology.org Role of NOX4 in Stroke Figure 2. Nox4 deficiency confers long-term neuroprotection and reduces mortality after acute ischemic stroke in young adult and aged mice of either sex. (A) Upper panel shows representative TTC staining of three corresponding coronal brain sections of 6- to 8-wk-old male and female wild-type (WT) mice, male Nox1y/2 mice, male Nox2y/2 mice, and male and female Nox42/2 mice, as well as 18- to 20-wk-old male wild- type and Nox42/2 mice on day 1 after tMCAO. The ischemic infarcts (white) appear smallest in the Nox42/2 mice of either age or sex (arrows), and this result was confirmed by infarct volumetry (lower panel). , p,0.0001, and , p,0.001, one-way ANOVA, Bonferroni post-hoc test compared with wild-type mice (n = 8–19 per group). (B) Neurological Bederson score (upper panel) and motor score (lower panel) on day 1 after tMCAO in the eight mouse groups indicated above. (C) Serial magnetic resonance images of cerebral infarcts 1 d and 6 d after tMCAO in wild-type and Nox42/2 mice (lower panel). The broken white lines show hyperintense ischemic lesions on day 1 after tMCAO in wild-type and Nox42/2 mice. Infarcts on day 1 are smaller in Nox42/2 mice than in wild-type mice and remain restricted to the basal ganglia on day 6. Hematoxylin and eosin staining confirmed neuronal damage in the cortex of wild-type mice 24 h after tMCAO (top panel, left), whereas cortical integrity was preserved in Nox42/2 mice (top panel, right). (D) Mortality (upper panel) and long-term functional outcome (Bederson score, lower panel) in 6- to 8-wk-old male Nox42/2 mice and wild-type controls. Survival curve (upper panel): , p = 0.0039, log-rank test compared with wild-type mice (n = 10–15 per group). Protection from Ischemic Stroke in Nox42/2 Mice Is a Result of Reduced Oxidative Stress, Neuronal Apoptosis, and Blood-Brain-Barrier Leakage Again, post-stroke application of VAS2870 to Nox42/2 mice had no additive neuroprotective or superoxide-lowering effect compared to the outcomes in wild-type animals treated with VAS2870 or untreated Nox42/2 mice (Figure 4B–4D). This observation is consistent with our ex vivo findings in ischemic brain slices and reaffirms that NOX4 rather than NOX1 or NOX2 is critically involved in the pathophysiology of ischemic stroke. Another, less specific inhibitor that also targets molecules other than NADPH oxidases [36,37], apocynin, had no effect on infarct size or functional outcome when given post-stroke and did not reduce the formation of ROS in vivo (Figure 4B and 4C). To further examine whether the neuroprotective effect observed in Nox42/2 mice is specifically related to reduced ROS formation and not due to other nonspecific or developmental defects, we performed a rescue experiment by restoring cerebral ROS levels in Nox42/2 mice during the course of ischemic stroke by applying exogenous H2O2 (Figure 4B–4D). Indeed, intrathecal administra- tion of H2O2 rescued the phenotype in Nox42/2 mice, and infarct volumes, functional deficits, and stroke-induced ROS formation returned to the levels observed in wild-type mice (Figure 4B–4D). To determine whether VAS2870 is also active when applied in vivo, we administered 2 mg of VAS2870 intrathecally to wild-type mice 2 h and 12 h after tMCAO. This experimental therapeutic approach significantly reduced brain infarct volumes (20.764.0 mm3 in VAS2870-treated mice versus 82.466.4 mm3 in vehicle-treated controls) and significantly improved neurological function, to the same extent as observed for the deletion of Nox4 in mice (Figure 4B and 4C). Moreover, less oxidative stress was detected in ischemic brains from VAS2870-treated animals than in those from vehicle- treated controls (Figure 4D). Again, post-stroke application of VAS2870 to Nox42/2 mice had no additive neuroprotective or superoxide-lowering effect compared to the outcomes in wild-type animals treated with VAS2870 or untreated Nox42/2 mice (Figure 4B–4D). This observation is consistent with our ex vivo findings in ischemic brain slices and reaffirms that NOX4 rather than NOX1 or NOX2 is critically involved in the pathophysiology of ischemic stroke. Another, less specific inhibitor that also targets molecules other than NADPH oxidases [36,37], apocynin, had no effect on infarct size or functional outcome when given post-stroke and did not reduce the formation of ROS in vivo (Figure 4B and 4C). g p (p ) ( g ) We also detected NOX4 in cerebral blood vessels (Figure 1B, white arrow indicates endothelial cells). Treatment with the NOX Inhibitor VAS2870 Effectively Protects Ischemic Brain Damage Even When Applied After Stroke Treatment with the NOX Inhibitor VAS2870 Effectively Protects Ischemic Brain Damage Even When Applied After Stroke Finally, we wanted to examine whether these genetic insights into the biology of oxidative stress in stroke and the role of NOX4 in general can be translated into a therapeutic intervention. Importantly, this intervention would have to be effective post- stroke and ideally it would be pharmacological. Therefore, we examined the efficacy of a validated, low-molecular-weight NADPH oxidase inhibitor, VAS2870 [15,32–34], in vital brain slices and in vivo. VAS2870 equally inhibits the ROS-generating activity of all NOX subunits, i.e., NOX1, NOX2, and NOX4. Vital brain slices [35] taken from wild-type mice 12 h after tMCAO produced significantly less ROS after pretreatment with 10 mM VAS2870, as did brain slices from untreated Nox42/2 mice (Figure 4A). Importantly, incubating ischemic slices from Nox42/2 mice with VAS2870 had no additional inhibitory effect on superoxide formation (Figure 4A). This finding further underlines the extraordinary role of NOX4 in generating oxidative stress Protection from Ischemic Stroke in Nox42/2 Mice Is a Result of Reduced Oxidative Stress, Neuronal Apoptosis, and Blood-Brain-Barrier Leakage Therefore, we hypothe- sized that Nox4 deficiency also influences the disruption of the blood–brain barrier and edema formation mediated by ROS [31]. Integrity of the blood–brain barrier was preserved in Nox42/2 mice on day 1 after tMCAO. This finding correlated with significantly less brain edema in Nox42/2 mice than in wild-type controls, as assessed by the extent of extravasation of Evans blue stain (8.065.9 mm3 in Nox42/2 mice versus 96.265.9 mm3 in wild-type mice). Importantly, almost no brain edema was seen in the brain regions where infarcts were regularly present in Nox42/2 mice (basal ganglia; Figure 3D, area delineated by the broken white line). This result indicates that the lesser edema seen in the Nox42/2 mice was a specific phenomenon and mechanistically relevant but was not due to smaller infarct volumes. To further examine whether the neuroprotective effect observed in Nox42/2 mice is specifically related to reduced ROS formation and not due to other nonspecific or developmental defects, we performed a rescue experiment by restoring cerebral ROS levels in Nox42/2 mice during the course of ischemic stroke by applying exogenous H2O2 (Figure 4B–4D). Indeed, intrathecal administra- tion of H2O2 rescued the phenotype in Nox42/2 mice, and infarct volumes, functional deficits, and stroke-induced ROS formation returned to the levels observed in wild-type mice (Figure 4B–4D). PLoS Biology \| www.plosbiology.org PLoS Biology \| www.plosbiology.org Protection from Ischemic Stroke in Nox42/2 Mice Is a Result of Reduced Oxidative Stress, Neuronal Apoptosis, and Blood-Brain-Barrier Leakage Long-term outcome (lower panel): , p,0.0001, and , p,0.05, one-way ANOVA, Bonferroni post-hoc test compared with wild-type mice (n = 10–15 per group). (E) Upper panel shows representative TTC staining of three corresponding coronal brain sections of 6- to 8-wk-old male wild-type mice (left) and matching Nox42/2 mice (right) on day 1 after pMCAO. Lower panel: Infarct volumes as measured by infarct volumetry (left) and Neurological Bederson score (right). Nox4 deficiency also protects the brain from permanent ischemia. *, p,0.001, and , p,0.05, two-tailed Student’s t-test compared with wild-type mice (n = 7–11 per group). (F) Representative coronal brain sections of wild-type and Nox42/2 mice stained with TTC on day 1 after permanent cortical photothrombosis (PT) (upper panel). Cortical infarctions are smaller in the absence of NOX4 (arrow). The lower panel shows infarct volumes in wild-type and Nox42/2 mice on day 1 after cortical photothrombosis. , p,0.001, two-tailed Student’s t-test compared with wild- type mice (n = 7 per group). All scale bars represent 100 mm. doi:10.1371/journal.pbio.1000479.g002 during the course of ischemic stroke, while other NOX isoforms such as NOX1 or NOX2 are obviously less relevant. dihydroethidium staining and nitrotyrosine immunolabeling, can induce neuronal apoptosis, which is a well-established mechanism of tissue damage in ischemic stroke [29,30]. Indeed, superimposed TUNEL and NeuN immunolabeling revealed widespread apop- tosis of neurons in wild-type mice 24 h after stroke onset (Figure 3C). In contrast, the number of apoptotic neurons in Nox42/2 mice subjected to tMCAO was significantly lower, and the basal apoptotic turnover rate in Nox42/2 mice fell within the range found in sham-operated mice (p.0.05) (Figure 3C). suc as NO o NO 2 a e obv ous y ess e eva t. To determine whether VAS2870 is also active when applied in vivo, we administered 2 mg of VAS2870 intrathecally to wild-type mice 2 h and 12 h after tMCAO. This experimental therapeutic approach significantly reduced brain infarct volumes (20.764.0 mm3 in VAS2870-treated mice versus 82.466.4 mm3 in vehicle-treated controls) and significantly improved neurological function, to the same extent as observed for the deletion of Nox4 in mice (Figure 4B and 4C). Moreover, less oxidative stress was detected in ischemic brains from VAS2870-treated animals than in those from vehicle- treated controls (Figure 4D). PLoS Biology \| www.plosbiology.org Discussion Finally, protein expression levels of NOX1 and NOX2 were almost unchanged in the brains of Nox42/2 mice (Figure S3C), underlining that the profound neuroprotection we observed is mediated by deficiency or blockade of NOX4 itself and not by secondary effects. The hypothesis that free radicals are involved in acute ischemic stroke and account for secondary infarct growth dates back to the 1970s [38] but has remained unproven [38,39]. The extent of neuroprotection that we observed is exceptional compared with that seen in many other pre-clinical stroke studies, in which the reduction of infarct size usually does not exceed 30%–40% [40]. Such moderate reductions of infarct volume have not translated into improvement of neurological status [3]. Most notably, continuous assessment of functional deficits until 7 d after stroke revealed that Nox4-null mice indeed showed a better amplitude rather than simply altered kinetics of recovery. This protection in Nox42/2 mice was further underlined by a significantly reduced post-stroke long-term mortality. Secondary infarct growth mediated for example by edema formation or hemorrhagic transformation is common during the course of brain ischemia and can lead to worsening of neurological symptoms [39]. Serial magnetic resonance imaging revealed that infarcts in Nox42/2 mice remain small, even at later stages of infarct development, and signs of intracerebral hemorrhage were consistently absent, thus indicating that NOX4 inhibition is likely to be safe and persistently effective. A plethora of compounds have provided neuroprotection in animal models of brain ischemia, but they all failed in human clinical trials [4]. This translational roadblock has been attributed mainly to inadequate pre-clinical study design and severe methodological shortcomings. Important confounding factors are a lack of randomization or rater-blinded evaluation of study results, and use of only one stroke model [16]. Strictly adhering to current expert recommendations for basic stroke trials, we here demonstrate that in the absence of NOX4, brain tissue can be salvaged after ischemia or reperfusion injury (as occurs in the tMCAO model). Most importantly, neuroprotection was preserved in old male and female Nox42/2 mice as well as in Nox42/2 mice subjected to permanent ischemia (i.e., cortical photothrombosis or pMCAO). Compared to in the tMCAO model, however, the reduction of infarct size in the pMCAO model was less pronounced though still significant. Discussion The right panel shows the extent of extravasation (i.e., edema volume) as determined by planimetry in the wild-type and Nox42/2 mice 24 h after tMCAO (n = 6 per group). For (A–C), ###, p,0.0001, and ##, p,0.001, compared with sham-treated mice; , p,0.0001, and , p,0.001, compared with wild-type mice by two-way ANOVA, Bonferroni post-hoc test. For (D), , p,0.001, Two-tailed Student9s t-test, compared with wild- type mice. All scale bars represent 100 mm. doi:10.1371/journal.pbio.1000479.g003 cerebral vessels of Nox42/2 mice subjected to tMCAO but not pMCAO (unpublished data). Clearly, elimination of NOX4 remains beneficial in the absence of arterial recanalization, a condition frequently observed in human stroke. finding for the wider concept of oxidative stress, which might also be of relevance for other disease states, such as neurotrauma and neuroinflammation, where oxidative stress, blood–brain barrier damage, and neurotoxicity are involved. Rather than focusing on antioxidants and the disappointing outcomes of their application, the identification of the relevant source of oxidative stress and preventing its formation may represent an approach with clinical potential. q y In our experiments, deficiency of NOX1 or NOX2 had no impact on infarct size or functional outcome after tMCAO. Although others have described protective effects of NOX2 deficiency after experimental stroke [42–44], we could not reproduce those findings. The exact reasons for this discrepancy are unclear at present. Differences in the experimental protocols and middle cerebral artery occlusion times, which varied between 30 min and 120 min in previous investigations, might play a role here [42–44]. In contrast to these previous studies, however, we used especially high numbers (n = 19) of Nox2y/2 mice to verify our findings. Moreover, type-II (beta) error of the differences between infarct volumes in Nox2y/2 mice and wild-type controls was only 7% in our study (93% power, respectively) (Tables S3–S5), which is a very powerful result compared to the positive reports on Nox2 deficiency in cerebral ischemia [42–44] as well as to many other experimental stroke studies in general [4,45]. Moreover, the fact that VAS2870, which specifically inhibits NADPH oxidases, could not further decrease infarct size and ROS formation in Nox42/2 mice ex vivo and in vivo (Figure 4) clearly argues against a major role of NOX1 or NOX2 in the pathophysiology of acute ischemic stroke. Discussion Here we identify NOX4 as a relevant molecular source of oxidative stress in cerebral ischemia, including some cases of human stroke. Our data suggest that NOX4-mediated oxidative stress leads to neuronal damage via leakage of the blood–brain barrier and neuronal apoptosis—two pathophysiological hallmarks of ischemic stroke. The extent of neuroprotection conferred by the absence of NOX4 in male and female Nox42/2 mice was exceptional and preserved in old animals. Importantly, the outcomes of these genetic experiments were mimicked when we pharmacologically inhibited NADPH oxidases within a clinically relevant time after induction of stroke. We consider this a key PLoS Biology \| www.plosbiology.org September 2010 \| Volume 8 \| Issue 9 \| e1000479 6 Role of NOX4 in Stroke September 2010 \| Volume 8 \| Issue 9 \| e1000479 September 2010 \| Volume 8 \| Issue 9 \| e1000479 PLoS Biology \| www.plosbiology.org 7 Role of NOX4 in Stroke Figure 3. Nox4 deficiency confers neuroprotection by reducing oxidative stress, neuronal apoptosis, and disruption of the blood– brain barrier. (A and B) Left panels show representative brain sections from sham-operated wild-type (WT) mice and wild-type and Nox42/2 mice 24 h after tMCAO. Sections were stained for ROS and oxidative chemistry using dihydroethidium (DHE) (A), or stained for reactive nitrogen species by using nitrotyrosine (B). Right panels show the number of cells per square millimeter that are positive for ROS or oxidative stress (A) or reactive nitrogen species (B) in the ischemic hemispheres of sham-operated wild-type mice and wild-type and Nox42/2 mice 12 h and 24 h after tMCAO (n = 4 per group). (C) Left panels show representative brain sections from sham-operated wild-type mice and wild-type and Nox42/2 mice 24 h after tMCAO, immunolabeled for the neuronal marker NeuN and subjected to TUNEL to show apoptosis. Right panel shows the number of TUNEL-positive neurons per square millimeter in the ischemic hemispheres of sham-operated wild-type mice and wild-type and Nox42/2 mice 24 h after tMCAO (n = 4 per group). (D) Left panels show corresponding coronal brain sections of wild-type and Nox42/2 mice on day 1 after tMCAO and injection of Evans blue. Extravasation of Evans blue was reduced in areas where infarcts were regularly present in Nox42/2 mice (basal ganglia, broken white line). September 2010 \| Volume 8 \| Issue 9 \| e1000479 PLoS Biology \| www.plosbiology.org Discussion Right panel shows number of ROS-positive cells per square millimeter in brain slices from mice in the different treatment groups (n = 5 per group). (B) Upper panel shows representative TTC staining of three corresponding coronal brain sections of wild-type mice treated with (left to right) carrier solution (10% DMSO; control), 100 mg of apocynin intravenously 1 h after tMCAO, or 2 mg of VAS2870 intrathecally 2 h and 12 h after tMCAO, untreated Nox42/2 mice, Nox42/2 mice treated with 2 mg of VAS2870 intrathecally 2 h and 12 h after tMCAO, and Nox42/2 mice treated with H2O2 intrathecally (15 mg/kg) immediately after the occlusion of the middle cerebral artery and then every hour until 6 h after stroke induction. Ischemic infarcts (white) appear smaller (arrows) in VAS2870-treated wild-type mice and Nox42/2 mice than in control mice, but those in apocynin-treated mice are similar to those in control mice. VAS2870 could not further decrease infarct volumes in Nox42/2 mice. Exogenous H2O2 reversed the stroke- protective phenotype in Nox42/2 mice. These results were confirmed by infarct volumetry (lower panel) (n = 7–10 per group). (C) Neurological Bederson score (upper panel) and motor score (lower panel) on day 1 after tMCAO in the different animal groups indicated in (B) (n = 7–10 per group). (D) Left panel shows representative brain sections from the different animal groups indicated in (B) stained for ROS by using dihydroethidium. Right panel shows corresponding number of ROS-positive cells per square millimeter in the ischemic hemispheres (n = 3–5 per group). For (A), ###, p,0.0001, compared with sham-operated mice, , p,0.0001 compared with control mice, ns, not significant, one-way ANOVA, Bonferroni post-hoc test. For (B–D), , p,0.0001, , p,0.001, , p,0.05, ns, not significant, one-way ANOVA, Bonferroni post-hoc test, compared with controls. All scale bars represent 100 mm. p m doi:10.1371/journal.pbio.1000479.g004 formation of ROS or of functional outcome after experimental stroke in vivo. formation of ROS or of functional outcome after experimental stroke in vivo. apocynin 1 h after the occlusion of the middle cerebral artery. In order to restore ROS levels in Nox42/2 mice, animals received repetitive intrathecal injections of H2O2 (15 mg/kg) immediately after the occlusion of the middle cerebral artery and then every hour until 6 h after stroke induction. Discussion Distinct pathomechanisms that can be positively influenced only in the presence of tissue reperfusion, i.e., after tMCAO but not pMCAO, such as progressive thrombus formation in the cerebral microvasculature [41], might account for this quantitative difference. Indeed, preliminary results suggest that clotting is attenuated in the Nevertheless, we cannot completely rule out contributions of other sources of ROS. Referring to this, Block et al. recently reported that a functional NOX4 is present and regulated in mitochondria, indicating the existence of a hitherto undescribed source of mitochondrial ROS [46]. An unprecedented need exists for more effective therapies for acute stroke, the second leading cause of death worldwide [1]. We have demonstrated that pharmacological inhibition of NADPH oxidases using the specific NADPH oxidase inhibitor VAS2870 [15,32–34] protects mice from brain ischemia within a clinically meaningful 2-h time window. In contrast, the commonly used organic compound apocynin may not be a NOX inhibitor in vascular cells but rather acts as a nonspecific antioxidant [36]. It also inhibits Rho kinase inhibitor [37], an activity that increases its nonspecific actions. If apocynin inhibits NADPH oxidases at all, it supposedly blocks the migration of the cellular NADPH oxidase complex subunit p47phox to the membrane, thus interfering with assembly of the functional NOX complex [47]. Therefore, it is unlikely to inhibit the NOX4-containing NADPH oxidase, which acts independently of any cytosolic subunits [12]. Indeed, in our experiments, application of apocynin had no effect on the PLoS Biology \| www.plosbiology.org September 2010 \| Volume 8 \| Issue 9 \| e1000479 PLoS Biology \| www.plosbiology.org 8 Role of NOX4 in Stroke PLoS Biology \| www.plosbiology.org 9 September 2010 \| Volume 8 \| Issue 9 \| e10004 September 2010 \| Volume 8 \| Issue 9 \| e1000479 September 2010 \| Volume 8 \| Issue 9 \| e1000479 PLoS Biology \| www.plosbiology.org PLoS Biology \| www.plosbiology.org Role of NOX4 in Stroke Figure 4. The NADPH oxidase inhibitor VAS2870 protects the brain from damage during acute ischemic stroke. (A) Left panel shows representative images of vital brain slices from sham-operated wild-type (WT) mice and wild-type mice and Nox42/2 mice 12 h after tMCAO. Slices were incubated ex vivo with VAS2870 (10 mM) or carrier solution (1% DMSO; control) for 30 min and stained with dihydroethidium (DHE) to detect ROS. Discussion In summary, we have demonstrated that NOX4-derived oxida- tive stress is a crucial player in the pathophysiology of acute ischemic stroke, while Nox4 deletion does not affect basal vascular or renal function. Nox4 gene reconstitution experiments in Nox42/2 mice and studies of the effects of different, structurally unrelated NOX inhibitors—should they become available—would be desirable to further substantiate the causality between NOX4 deficiency and protection from cerebral ischemia. Pharmacological inhibition of NADPH oxidases using specific compounds may also pave new avenues for the treatment of ischemic brain injury in humans. Because NADPH oxidase–mediated production of ROS may represent a general mechanism of neurotoxicity, our findings may extend to other ischemic disorders and neurodegenerative or inflammatory diseases. Further studies in relevant disease models are warranted. Cortical photothrombosis was induced in 6- to 8-wk-old wild- type or Nox42/2 mice as described previously [51,52]. Stroke Analysis Stroke analysis was performed as described previously [53,54]. To determine infarct size, mice were killed 24 h after tMCAO, pMCAO, or cortical photothrombosis. Brains were cut in 2-mm-thick coronal sections using a mouse brain slice matrix (Harvard Apparatus). The slices were stained with 2% TTC (Sigma-Aldrich) to visualize the infarcts. Planimetric measurements (ImageJ software, United States National Institutes of Health), calculating lesion volumes, were corrected for brain edema as described previously [55]. Determination of brain edema using Evans blue dye was performed as described previously [19]. Stroke Models If not otherwise mentioned, we performed 60 min of tMCAO in 6- to 8-wk-old male mice weighing 20–25 g, as described previously [48,49]. To exclude age- and gender-specific effects, 18- to 20-wk-old male and 6- to 8-wk-old female mice were used in some subgroups. For pMCAO the occluding filament was left in situ until sacrificing the animals [41]. Stroke Study Design Detailed study characteristics are provided in Table S2. We strictly followed the recent international expert recommendations for conducting research in mechanism-driven basic stroke studies [4,6,7,16,17,40]. Human Specimens Specimens from patients who had experienced a stroke were collected during routine autopsy at the Department of Neuropa- thology, University of Wu¨rzburg, Germany. Materials and Methods Magnetic resonance imaging was performed repeatedly at 24 h and 6 d after stroke on a 1.5-T magnetic resonance unit (Vision Siemens) as described previously [56]. We used a custom-made dual channel surface coil designed for examining mice (A063HACG; Rapid Biomedical). The imaging protocol com- prised a coronal T2-weighted sequence (slice thickness 2 mm) and a blood-sensitive coronal three-dimensional T2-weighted gradient echo CISS (slice thickness 1 mm) sequence. Magnetic resonance images were assessed with respect to infarct morphology and the occurrence of intracerebral bleeding. Refer to the Text S1 for more detailed methodology. The generation of the Nox4-null mice is described in Figure S3. Vital Brain Slices Vital brain slices from infarcted mouse brains (between –2 mm and –4 mm from bregma) were prepared as described previously [57]. Quantitative PCR Analysis After RNA isolation, we quantified NOX4 mRNA expression using real-time PCR and the TaqMan system (TaqMan Gene Expression Arrays for murine NOX4, assay ID Mm00479246_m1, Applied Biosystems), using 18s rRNA (TaqMan Predeveloped Assay Reagents, part number 4319413E, Applied Biosystems) to normal- ize the amount of sample RNA. At 2 h and12 h after the induction of tMCAO, subgroups of wild-type mice or Nox42/2 mice were randomly selected to receive either 2 mg of the NOX-specific inhibitor VAS2870 (Vasopharm GmbH [32,33]) or carrier solution (10% dimethyl sulfoxide, Sigma) intrathecally, as described previously [50]. In another group, wild-type mice were injected intravenously with 100 mg of PLoS Biology \| www.plosbiology.org Accession Numbers The GenBank (http://www.ncbi.nlm.nih.gov/Genbank) acces- sion numbers for the genes discussed in this paper are NOX1, NM_172203; NOX2, NM_007807; and NOX4, NM_015760. Quantification of Protein Expression Quantification of Protein Expression We quantified amounts of NOX1, NOX2, and NOX4 protein in the cortex and basal ganglia by Western blot analysis. ( p ) Found at: doi:10.1371/journal.pbio.1000479.s002 (1.64 MB TIF) Histology and Immunohistochemistry Histology was performed by using formalin-fixed mouse brains on day 1 after tMCAO. Samples were embedded in paraffin and PLoS Biology \| www.plosbiology.org September 2010 \| Volume 8 \| Issue 9 \| e1000479 10 Role of NOX4 in Stroke cut into 4-mm-thick sections (0.5 mm anterior from bregma). After deparaffinization and rehydration, tissues were stained with hematoxylin and eosin or Nissl staining solution (Sigma-Aldrich). Immunohistochemical detection of NOX4 was performed on formalin-fixed human brain slices or cryopreserved mouse brain slices. A NOX4-specific primary antibody [58] was applied at a dilution of 1:200 overnight at 4uC. To identify the cellular origin we performed double staining of NOX4 with the neuronal marker NeuN (1:1,000) and the endothelial marker von Willebrand Factor (1:25). arterial pressure (PAP) in isolated perfused lungs during normoxic (21% O2) ventilation. (E) Strength of hypoxic pulmonary vasoconstriction (HPV) as indicated by the maximum increase in PAP (DPAP) upon acute hypoxic ventilation (10 min, 1% O2) in isolated perfused lungs. No significant differences were observed between wild-type and Nox42/2 mice. Data are derived from six mice in each case. (F) Renal hypertrophy as assessed by kidney weight per body surface area (BSA) (g/m2). There was no significant difference in terms of renal mass between wild-type and Nox42/2 mice at 17 wk of age. (G) Albuminuria at 17 wk of age (mg/24 h). There was no significant difference in 24-h urinary albumin excretion between wild-type and Nox42/2 mice at 17 wk of age. Statistical Analysis Figure S3 Generation of Nox4 knockout mice and counter-regulation of NOX1 and NOX2. (A) Construct development for Nox4 knockout mice. Exons 14 and 15 are flanked by loxP sites and followed by a floxed neomycin resistance gene (neo) and a negative-selection cassette coding for diphtheria toxin A (dta) as described in the Text S1. Embryonic stem cell clones were generated by homologous recombination with the targeting vector. Transient expression of Cre recombinase results in three different recombination events. Type 1 results in deletion of the neo cassette and thus floxed exons 14 and 15. These cells can be used to generate conditional Nox4 knockout. Type 2 results in deletion of the floxed exons, and type 3 results in the deletion of exons 14 and 15 and the neo cassette. These cells were used to generate the Nox4 knockout mice. (B) Western blot demonstrating the absence of the 64-kDa NOX4 band in the aorta, lung, and kidney of Nox42/2 mice. (C) Expression of NOX1 and NOX2 is not upregulated in Nox42/2 mice. The uppermost left panel shows results of densitometric analysis of the NOX1 134-kDa band in brain samples of the cortex and basal ganglia from Nox42/2 (pale bar) and wild-type mice (black bar). Data are presented as the relative amount of the NOX1 band normalized to GAPDH and represent the mean 6 standard error of three samples. The right panel shows a Western blot comparison of brain and aorta samples from wild-type mice demonstrating the presence of the 134-kDa band in both samples. The center and lowest panels show results of densitometric analysis of the 91- and 53-kDa NOX2 bands seen in brain samples from the cortex and basal ganglia of Nox42/2 (pale bar) and wild-type mice (black bar). Data are presented as the Data are expressed as mean 6 standard deviation and were analyzed statistically using the PrismGraph 4.0 software package (GraphPad Software). In the case of multiple group comparisons, data were tested for Gaussian distribution with the D’Agostino and Pearson omnibus normality test and then analyzed by Bonferroni- corrected one-way ANOVA or two-way ANOVA. Otherwise, the two-tailed Student’s t-test was applied. For comparison of survival curves the log-rank test was used. P-values less than 0.05 were considered significant. Detailed power and type-II (beta) error calculations on infarct volumes are provided in Tables S3–S5. Oxidative Chemistry Biomarkers The presence of ROS and other oxidants such as ONOO2 was visualized on frozen mouse brain sections 12 h and 24 h after tMCAO or 24 h after pMCAO using dihydroethidium (Sigma; 2 mM stock) staining, as described previously [59], in coronal brain sections taken from identical regions (–0.5 mm from bregma) of sham-operated controls, wild-type and Nox42/2 mice that had undergone stroke, and wild-type mice and Nox42/2 mice treated with VAS2870 or H2O2. Found at: doi:10.1371/journal.pbio.1000479.s001 (1.23 MB TIF) Found at: doi:10.1371/journal.pbio.1000479.s001 (1.23 MB TIF) Figure S2 Cerebral blood flow, cerebral vasculature, and brain structure are normal in Nox42/2 mice. (A) Regional cerebral blood flow (rCBF) in the right territory of the middle cerebral artery as measured by laser Doppler flowmetry in wild-type (WT) mice and in Nox1y/2, Nox2y/2, and Nox42/2 mice (n = 4 per group) at baseline levels, after insertion of the thread (ischemia) and again 10 min after removal of the thread (reperfusion). No significant differences were observed between the groups at any time point. p.0.05, two-way ANOVA, Bonferroni post-hoc test, compared with baseline rCBF. (B) Assessment of the cerebral vasculature in wild-type and Nox42/2 mice. A complete circle of Willis (white arrows) was identified in all animals studied, and the distribution of the trunk and branch of the middle cerebral artery appeared to be anatomically identical among the genotypes. (C) Normal brain structure in Nox42/2 mice. Representative Nissl-stained 5-mm coronal paraffin-wax- embedded brain sections of 3-mo-old wild-type and Nox42/2 mice (n = 3 each), showing a macroscopic view (uppermost panel), formation of the hippocampus formation (center panel), and somatomotor areas of the neocortex (lowermost panel). Found at: doi:10.1371/journal.pbio.1000479.s002 (1.64 MB TIF) Immunohistochemical staining for nitrotyrosine to visualize additional reactive nitrogen species was conducted on cryopre- served brain sections taken from identical regions of the mouse brain (–0.5 mm from bregma) 12 h and 24 h after tMCAO, using a polyclonal nitrotyrosine antibody. Apoptotic neurons in the ischemic hemisphere 24 h after tMCAO were visualized by TUNEL on paraffin-wax-embedded slices, using the TUNEL in situ cell death detection kit, TMR red (Roche). NeuN/TUNEL double staining was performed on cryopreserved brain slices. Author Contributions Found at: doi:10.1371/journal.pbio.1000479.s006 (1.76 MB TIF) Found at: doi:10.1371/journal.pbio.1000479.s006 (1.76 MB TIF) Found at: doi:10.1371/journal.pbio.1000479.s006 (1.76 MB TIF) The author(s) have made the following declarations about their contributions: Conceived and designed the experiments: CK AS MHdA KJD NW HHS. Performed the experiments: CK HG MEA EJ MM DB TS CG PK KB MKS AMH SGM GS SM AS LB VGD HF TK. Analyzed the data: CK MEA EJ MM DB LB VGD HF TK MHdA KJD NW HHHWS. Contributed reagents/materials/analysis tools: CK HG KW AMS HHHWS. Wrote the paper: CK KW HHHWS. References 1. World Health Organization (2008) The top ten causes of death. Fact sheet number 310. Geneva: World Health Organization. Available: http://www.who. int/mediacentre/factsheets/fs310_2008.pdf. 5 p. 1. World Health Organization (2008) The top ten causes of death. Fact sheet number 310. Geneva: World Health Organization. Available: http://www.who. int/mediacentre/factsheets/fs310_2008.pdf. 5 p. 1. World Health Organization (2008) The top ten causes of death. Fact sheet number 310. Geneva: World Health Organization. Available: http://www.who. int/mediacentre/factsheets/fs310_2008.pdf. 5 p. 11. Omenn GS (2007) Chemoprevention of lung cancers: lessons from CARET, the beta-carotene and retinol efficacy trial, and prospects for the future. Eur J Cancer Prev 16: 184–191. 2. Elkins JS, Johnston SC (2003) Thirty-year projections for deaths from ischemic stroke in the United States. Stroke 34: 2109–2112. 12. Opitz N, Drummond GR, Selemidis S, Meurer S, Schmidt HH (2007) The ‘A’s and ‘O’s of NADPH oxidase regulation: a commentary on ‘‘Subcellular localization and function of alternatively spliced Noxo1 isoforms’’. Free Radic Biol Med 42: 175–179. 2. Elkins JS, Johnston SC (2003) Thirty-year projections for deaths from ischemic stroke in the United States. Stroke 34: 2109–2112. 3. O’Collins VE, Macleod MR, Donnan GA, Horky LL, van der Worp BH, et al. (2006) 1,026 experimental treatments in acute stroke. Ann Neurol 59: 467–477. 3. O’Collins VE, Macleod MR, Donnan GA, Horky LL, van der Worp BH, et al. (2006) 1,026 experimental treatments in acute stroke. Ann Neurol 59: 467–477. 13. Miller AA, Drummond GR, Schmidt HH, Sobey CG (2005) NADPH oxidase activity and function are profoundly greater in cerebral versus systemic arteries. Circ Res 97: 1055–1062. 4. Dirnagl U (2006) Bench to bedside: the quest for quality in experimental stroke research. J Cereb Blood Flow Metab 26: 1465–1478. 5. Whalley K (2006) Slicing into stroke therapeutics. Nat Rev Drug Discov 5: 632–632. 14. McCann SK, Dusting GJ, Roulston CL (2008) Early increase of Nox4 NADPH oxidase and superoxide generation following endothelin-1-induced stroke in conscious rats. J Neurosci Res 86: 2524–2534. 6. Sena ES, van der Worp HB, Bath PM, Howells DW, Macleod MR (2010) Publication bias in reports of animal stroke studies leads to major overstatement of efficacy. PLoS Biol 8: e1000344. doi:10.1371/journal.pbio.1000344. 15. Niethammer P, Grabher C, Look AT, Mitchison TJ (2009) A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish. Nature 459: 996–999. 7. van der Worp HB, Howells DW, Sena ES, Porritt MJ, Rewell S, et al. Text S1 Supplementary results, supplementary meth- ods, and supplementary references. Text S1 Supplementary results, supplementary meth- ods, and supplementary references. Found at: doi:10.1371/journal.pbio.1000479.s012 (0.32 MB DOC) Found at: doi:10.1371/journal.pbio.1000479.s012 (0.32 MB DOC) Figure S5 Motor function after pMCAO. Motor function was assessed by the grip test in 6- to 8-wk-old male Nox42/2 mice (n = 7) and wild-type (WT) controls (n = 11) 24 h after pMCAO. Two-tailed Student’s t-test compared with wild-type mice. ns, not significant. Found at: doi:10.1371/journal.pbio.1000479.s005 (0.18 MB TIF) Found at: doi:10.1371/journal.pbio.1000479.s007 (0.04 MB PDF) Found at: doi:10.1371/journal.pbio.1000479.s007 (0.04 MB PDF) Acknowledgments We thank Ba¨rbel Fu¨hler and Courtney Jackson for their help in breeding the Nox42/2 colonies in Gießen and Melbourne, respectively; Prof. Bettie Sue Masters, University of Texas Health Science Center San Antonio, United States, and Prof. Lutz Hein, University of Freiburg, Germany, for their advice in designing the knockout strategy; Prof. Karl-Heinz Krause, University of Geneva, Switzerland, for providing Nox1y/2 mice; Prof. Wolfgang Roggendorf, Department of Neuropathology and Brain Bank Center, University of Wu¨rzburg, Germany, and Andreas Reif, Department of Psychiatry, University of Wu¨rzburg, Germany, for providing human stroke specimens; Marissa Bowden and Melanie Glaser for technical assistance; Sandra Cox for carefully editing the manuscript, and Vasopharm GmbH, Wu¨rzburg, Germany, for providing VAS2870. We also thank the members of the German Mouse Clinic for comprehensive phenotyping of the mice and fruitful discussions. H.H.H.W.S. would like to dedicate this work to his mother, Renate Schmidt, who while this paper was in preparation on December 31, 2009, died of a severe stroke. Figure S6 Oxidative stress is reduced in brains from Nox42/2 mice after pMACO. Left panels show representative brain sections from wild-type (WT) and Nox42/2 mice 24 h after sham operation of pMCAO. Sections were stained for ROS and oxidative chemistry using dihydroethidium. Right panel shows the number of cells per square millimeter that are positive for ROS or oxidative stress in the ischemic hemisphere of wild-type and Nox42/2 mice 24 h after sham operation or pMCAO (n= 3–5 per group). ##, p,0.001 compared with sham-treated mice; , p,0.001 compared with wild-type mice by one-way ANOVA, Bonferroni post-hoc test. Supporting Information Figure S1 Systemic and pulmonary blood pressure as well as kidney function in Nox42/2 mice are unchanged. (A and B) Radiotelemetry recordings of basal mean arterial pressure (MAP) and heart rate (HR) of wild-type (WT) (open circles, n = 10) and Nox42/2 (filled squares, n = 14) mice. Data are represented as 1-h (A) and 24-h (B) averages of mean arterial pressure (left panels) and heart rate (right panels). Dark and light periods are denoted by black and white bars, respectively. (C) Right ventricular systolic pressure (RVSP) as assessed in vivo in anaesthetized Nox42/2 and wild-type mice. (D) Mean pulmonary PLoS Biology \| www.plosbiology.org September 2010 \| Volume 8 \| Issue 9 \| e1000479 11 Role of NOX4 in Stroke Found at: doi:10.1371/journal.pbio.1000479.s008 (0.09 MB PDF) Found at: doi:10.1371/journal.pbio.1000479.s008 (0.09 MB PDF) relative amount of either the 91-kDa band or 53 k-Da band normalized to GAPDH and represent the mean 6 standard error of three samples. The bottom right panel shows a Western blot comparison of NOX2 expression in the brain and aorta of wild- type mice, demonstrating the presence of the 91-kDa and 53-kDa bands in both tissues. Found at: doi:10.1371/journal.pbio.1000479.s003 (24.75 MB TIF) Table S3 Power and type-II (beta) error calculations on infarct volumes depicted in Figure 2A. Found at: doi:10.1371/journal.pbio.1000479.s009 (0.06 MB PDF) Table S4 Power and type-II (beta) error calculations on infarct volumes depicted in Figure 2E. Found at: doi:10.1371/journal.pbio.1000479.s010 (0.05 MB PDF) Table S4 Power and type-II (beta) error calculations on infarct volumes depicted in Figure 2E. Found at: doi:10.1371/journal.pbio.1000479.s003 (24.75 MB TIF) Found at: doi:10.1371/journal.pbio.1000479.s010 (0.05 MB PDF) Table S5 Power and type-II (beta) error calculations on infarct volumes depicted in Figure 4B. Figure S4 Long-term outcomes are improved in Nox42/2 mice after tMCAO. Long-term outcome of motor function (grip test) in 6- to 8-wk-old male Nox42/2 mice (n =10) and wild-type (WT) controls (n= 15) after tMCAO. Nox42/2 mice performed better over the whole observation period. , p,0.001 and *, p,0.05, one-way ANOVA, Bonferroni post-hoc test compared with wild-type mice. Found at: doi:10.1371/journal.pbio.1000479.s004 (0.27 MB TIF) Found at: doi:10.1371/journal.pbio.1000479.s011 (0.06 MB PDF) Table S1 Results of blood gas analysis and posterior communicating artery (PComA) score in wild-type and Nox42/2 mice. 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https://openalex.org/W4382699987	https://www.ijfmr.com/papers/2023/3/4112.pdf	English	null	The Scientific and Adventurous Elements in Margaret Atwood’s Oryx and Crake	International Journal For Multidisciplinary Research	2,023	cc-by-sa	2,033	Abstract: The present research paper examines the scientific and adventurous elements of Oryx and Crake (2003). These elements increase reader attention and add to the book's widespread appeal. The novel Oryx and Crake by Margaret Atwood describe the dystopia of the near future. Humans are destroying the universe due to anthropocentrism, which values humanity over non-humanity and is embedded in Western religions and philosophies. Margaret Eleanor Atwood is a Canadian poet, novelist, literary critic, essayist, creator, professor, and environmental activist. Her writing explores issues of gender, identity, religion, myth, global warming, and power politics. The most well-known book by Atwood, Oryx, and Crake, extrapolates present trends to envision possible futures. Atwood is well known for her innovative and thought-provoking writings. Margaret Atwood focused on how science impacts common people in her science fiction novel Oryx and Crake. This study article's main objective is to focus on important components of modern technology. Keywords: Environmental, technology, humanity, speculative fiction, science, adventurous, apocalyptic. Research Scholar, Smt. Radhabai Sarda Arts, Commerce and Science College Anjangaon (Surji), Dist. Amravati Research Scholar, Smt. Radhabai Sarda Arts, Commerce and Science College Anjangaon (Surji), Dist. Amravati Miss Shilpa Nareshrao More Miss Shilpa Nareshrao More Research Scholar, Smt. Radhabai Sarda Arts, Commerce and Science College Anjangaon (Surji), Dist. Amravati Main Text: Oryx and Crake's post-apocalyptic world was rich in science, technology, and businesses dedicated to transgenic research. The term "transgenic research" refers to genetic studies in which genetic material from one species is deliberately inserted into the DNA of a different species. Jimmy's father was a project manager for Transgenic Research. He was the main designer of the pigoon, a hybrid pig monster he intended to produce human kidneys and skin cells in. Other hybrid animals mentioned in the book include rakunks and wolrogs. Every advancement or discovery made by humans moves them closer to technology and materialism and farther from the natural world. The delayed rise of technology paved the way for knowledge paths because ancient people did not acquire the manners that modern people do. Humans can survive in the modern world without other humans, but not without technology. These are regarded as being given in exchange for human devastation, and they also improve the standard of human life. People support inventions for every task and need, including clothing, communication, transportation, food, and housing. In short, science has made it easier for humans to survive, but only humans can see the benefits of it. Snowman Said: “Strange to think of the endless labour, “Strange to think of the endless labour, The digging, the hammering, the carving, The digging, the hammering, the carving, The lifting, the drilling, day by day, Year by year, century by century, And now the endless crambling that Must be going on everywhere. Sandcastles in the wind.” Sandcastles in the wind.” Snowman considers how even things that seem the most substantial may be weak as he surveys the remnants of his extinct civilization. Human lifestyles and views are altered by science. International Journal for Multidisciplinary Research (IJFMR) compounds while the rest of the population is forced to live in unsafe conditions in urban areas. In this novel, Atwood uses a dual temporal frame to present the events. compounds while the rest of the population is forced to live in unsafe conditions in urban areas. In this novel, Atwood uses a dual temporal frame to present the events. Science fiction is one of the most inventive genres in literature, and it is a form of speculative fiction, which keeps up imagined aspects that don't exist in the real world. It examines the dystopian future and how it deals with the effects of science and technology to assist and treat those who are afflicted with ailments, genetic breakthroughs from business companies are being studied. These inventions eventually turn into wild creatures that pose a threat to humans. There does not appear to be any restriction on creativity in society, and there is no legal framework in place to control scientific discovery or creation. Today, these firms' true motivations are driving scientists to pursue profits at the expense of human welfare. Sustainability has emerged as one of humanity's most pressing issues because of scientific investigation. In the scientific community, where all living things are the subject of research and testing, the leveling of hierarchical divisions between animals and humans is even more clear. INTRODUCTION: Oryx and Crake by Margaret Atwood is a work of science fiction that also contains aspects of adventure. Atwood likened the post-apocalyptic world to being populated by a small band of innocent, primitive people. Whom he refers to as "Crackers;" The protagonist of the book, Snowman, is based on a group of characters in the novel. The novel is written from Snowman’s point of view. He's close to the "Crackers," a group of creators who have been altered to resemble humans. The book was first published by McClelland and Stewart in 2003 and was named to the 2003 and 2004 Orange Prize for Fiction shortlists. The story of centered on the scientific and technological fields. A New England is the setting for Oryx and Crake. This essay discusses the cataclysmic tragedy that the human race's negligence has caused to emerge in the twenty-first century. Science and technological advancements enable us to live in peace, but they also bring us famine, death, and worldwide catastrophe, as well as climate change and nuclear war. The dystopian novel Oryx and Crake by Margaret Atwood serves as a warning against environmental degradation by illustrating potential human consequences. It takes place in a dystopian future when numerous transhumanist and unethical companies rule and manufacture various biotechnology items. There are class differences; the wealthy and intelligent reside in company Volume 5, Issue 3, May-June 2023 IJFMR23034112 1 ational Journal for Multidisciplinary Research (IJFMR) the day as soon as the sun rises. Before the advent of science, people, and animals coexisted on every continent. To support their families, men in families often work in agriculture and animal care. They don't use pesticides, they start their days in the fields, and they grow good harvests. Women finish their household chores and help their spouses with family duties early in the morning. Children are safe from chemicals and in a nice atmosphere. They take classes and learn, which is advantageous. Following his routine afternoon stroll, Snowman commutes It might also be defined as having a systematic understanding of the physical or material world. Modern society uses mobile phones to start the day from the moment the sun rises. People and animals coexisted throughout the globe before scientific advancement changed things. Men in families typically work in agriculture and animal husbandry to make a living off natural resources. Their day begins in the fields, they don't use pesticides, and they produce good crops. Early in the morning, women complete their household chores and assist their husbands with family responsibilities. Children are in a pleasant environment without being under the chemicals. They attend classes and receive education, which is beneficial. After his daily afternoon stroll, Snowman travels to the collapsed bridge so he can sip water from the beer bottles and take a shower in the runoff. When he sees a sign that reads "Men at Work," he is reminded of the extraordinary amount of human labor that went into creating such an engineering marvel. For many people, the bridges represent civilization, a vast and intricate human creation that took centuries to create but only one generation to destroy. Snowman may celebrate the fall of civilization, but his view also has a spiritual undertone People protect nature and animals out of respect for religion because they understand that these resources ensure the safety of human life. Numerous organisms, including flora and wildlife, can be found in this globe. These are the priceless resources that nature has bestowed onto humans. Most forest regions have been converted Researchers are viewed as playthings, and there are no values for animal life. There is a dearth of food for other forest organisms since people have grabbed some of the resources. Impact of Science in Margaret Atwood’s Oryx and Crake: Our perspectives are changed by science. It could also be described as having a methodical comprehension of the material or physical universe. Mobile phones are used by modern culture to begin IJFMR23034112 Volume 5, Issue 3, May-June 2023 2 International Journal for Multidisciplinary Research (IJFMR) E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com Essence. Jimmy met a female who was Crake's assistant while working for that company. She resembled he young woman that appeared on Jimmy's childhood pornographic website. Essence. Jimmy met a female who was Crake's assistant while working for that company. She resembled the young woman that appeared on Jimmy's childhood pornographic website. Essence. Jimmy met a female who was Crake's assistant while working for that company. She resembled the young woman that appeared on Jimmy's childhood pornographic website. Conclusion: Oryx was brave mind girl she faced all crisis in her life, and she learn there was profit for everything. Crake also used Oryx physically, but she never questioned him, because Crake takes her from the muddy place to scientific labs. But atlas she was killed by Crake by cutting her throat. Here it is evident due to seeing violent videos of murders it becomes easy for him to kill the person who is close to him. But Jimmy cannot be able to bear the circumstances in front of him. Sheer in anger he killed Crake and after the death of Crake the hybrid animals he creates become violent. Most of the humans are dead and now Jimmy become Snowman who lived in trees to escape from hybrid animals. There is a lack of food and water. Snowman remembers his past along with crackers. The world in which Snowman lives is destroyed due to science which has no ethics. e e e ces: 1. Atwood Margaret Oryx and Crake. Great Britain: Virago press, 2003 2. https://en.m.wikipedia.org/wiki/oryx_and_crake 3. http://larsschmeink.de/?p=2865lang=en 4. http://compulsivereader.com/2004/03/30/a-review-oforyx-and-crake-by-Margaret-atwood ational Journal for Multidisciplinary Research (IJFMR) Hen and cattle slaughter is increasing daily, and some animals are also sent to research facilities where experiments are conducted on them until they pass away. However, for Jimmy's father, these kinds of behaviours are commonplace. Jimmy had some memories of his father, but he never really understood his mother, who was a mystery figure to him. His mother Sharon could not live in the materialistic society and was disturbed by scientific practises that injure animals. She gave up. Technology forbids social interaction, and under compound culture, people are chained. Most of them are abducted for sexual torture and organ harvesting. Massive technological advancement has never provided a solution to this issue. Most of the cases are unsolved, and their parents continue to live in the hope that they will one day visit them. Technology advancement makes crime more prevalent in society. Children of working parents may experience issues with loneliness. Children suffer from psychological issues because of these events, and technology has developed video games that teach players how to harm others. Technology disturbs human nature and transforms human behaviour into that of an animal. Violence is increasing along with science and technology, and Crake lacks appropriate social connection and attachment to anyone. Jimmy was hired by a library, where one of his responsibilities was to burn old books. Even though books are now regarded as valuable, human nature is changing. Most of them don't consult books or libraries to gather materials. Internet sources were used, and many favour nature over materialism. He left his work after a few days since he could not survive there as an art lover. After a protracted absence, Crake paid him a visit and showed him around his scientific corporation, Rewoven Volume 5, Issue 3, May-June 2023 IJFMR23034112 IJFMR23034112 3 3 International Journal for Multidisciplinary Research (IJFMR) E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com Essence. Jimmy met a female who was Crake's assistant while working for that company. She resembled the young woman that appeared on Jimmy's childhood pornographic website. References: 1. Atwood Margaret Oryx and Crake. Great Britain: Virago press, 2003 2. https://en.m.wikipedia.org/wiki/oryx_and_crake 3. http://larsschmeink.de/?p=2865lang=en 4. http://compulsivereader.com/2004/03/30/a-review-oforyx-and-crake-by-Margaret-atwood IJFMR23034112 Volume 5, Issue 3, May-June 2023 4
https://openalex.org/W4308451201	https://www.zora.uzh.ch/id/eprint/224376/1/ZORA_Soto_20et_20al__20_282022a_29_20Diversity_20and_20Distributions.pdf	English	null	Tracking a killer shrimp: <i>Dikerogammarus villosus</i> invasion dynamics across Europe	Diversity and distributions	2,022	cc-by	17,079	g y p Soto, Ismael; Cuthbert, Ross N; Ahmed, Danish A; et al; Altermatt, Florian (2023). Tracking a killer shrimp: Dikerogammarus villosus invasion dynamics across Europe. Diversity and Distributions, 29(1):157-172. DOI: https://doi.org/10.1111/ddi.13649 Zurich Open Repository and Archive University of Zurich University Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2023 Tracking a killer shrimp: Dikerogammarus villosus invasion dynamics across Europe DOI: https://doi.org/10.1111/ddi.13649 DOI: https://doi.org/10.1111/ddi.13649 To cite this version: Ismael Soto, Ross N Cuthbert, Danish A Ahmed, Antonín Kouba, Sami Domisch, et al.. Tracking a killer shrimp: Dikerogammarus villosus invasion dynamics across Europe. Diversity and Distributions, Wiley, 2022, ฀10.1111/ddi.13649฀. ฀hal-03843377฀ DOI: https://doi.org/10.1111/ddi.13649 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-224376 Journal Article Published Version The following work is licensed under a Creative Commons: Attribution 4. Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-224376 Journal Article Published Version Originally published at: Soto, Ismael; Cuthbert, Ross N; Ahmed, Danish A; et al; Altermatt, Florian (2023). Tracking a killer shrimp: Dikerogammarus villosus invasion dynamics across Europe. Diversity and Distributions, 29(1):157-172. DOI: https://doi.org/10.1111/ddi.13649 invasion dynamics across Europe Ismael Soto, Ross N Cuthbert, Danish A Ahmed, Antonín Kouba, Sami Domisch, Jaime R G Marquez, Ayah Beidas, Giuseppe Amatulli, Jens Kiesel, Longzhu Q Shen, et al. Diversity and Distributions. 2022;00:1–16. Tracking a killer shrimp: Dikerogammarus villosus invasion dynamics across Europe Ahmed4 \| Antonín Kouba1 \| Sami Domisch5 \| Jaime R. G. Marquez5 \| Ayah Beidas4 \| Giuseppe Amatulli6 \| Jens Kiesel7,8 \| Longzhu Q. Shen5,9 \| Margarita Florencio10,11 \| Herlander Lima12 \| Elizabeta Briski2 \| Florian Altermatt13,14 \| Gaït Archambaud- Suard15 \| Peter Borza16 \| Zoltan Csabai17,18 \| Thibault Datry19 \| Mathieu Floury20 \| Maxence Forcellini19 \| Jean- François Fruget21 \| Patrick Leitner22 \| Marie- Hélène Lizée15 \| Anthony Maire23 \| Anthony Ricciardi24 \| Ralf B. Schäfer25 \| Rachel Stubbington26 \| Gea H. Van der Lee27 \| Gábor Várbíró28 \| Ralf C. M. Verdonschot27 \| Peter Haase8,29 \| Phillip J. Tracking a killer shrimp: Dikerogammarus villosus invasion dynamics across Europe Haubrock1,29 1Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, University of South Bohemia in České Budějovice, Vodňany, Czech Republic 1Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, University of South Bohemia in České Budějovice, Vodňany, Czech Republic 2GEOMAR Helmholtz- Zentrum für Ozeanforschung Kiel, Kiel, Germany 2GEOMAR Helmholtz- Zentrum für Ozeanforschung Kiel, Kiel, Germany 2GEOMAR Helmholtz- Zentrum für Ozeanforschung Kiel, Kiel, Germany 3School of Biological Sciences, Queen's University Belfast, Belfast, UK 3School of Biological Sciences, Queen's University Belfast, Belfast, UK Applied Mathematics and Bioinformatics (CAMB), Department of Mathematics and Natural Sciences, Gulf University for Sci wait 4Center for Applied Mathematics and Bioinformatics (CAMB), Department of Mathematics and Natural Sciences, Gulf University for Science and Technology, Hawally, Kuwait epartment of Community and Ecosystem Ecology, Leibniz Institute of Freshwater Ecology and Inland Fisheries (IGB), Berlin, G 6School of the Environment, Yale University, New Haven, Connecticut, USA 6School of the Environment, Yale University, New Haven, Connecticut, USA 7Department of Hydrology and Water Resources Management, Institute for Natural Resource Conservation, Christian- Albre 7Department of Hydrology and Water Resources Management, Institute for Natural Resource Conservation, Christian- Albrechts- University Kiel, Kiel, Germany 8Faculty of Biology, University of Duisburg– Essen, Essen, Germany culty of Biology, University of Duisburg– Essen, Essen, Germany 9Institute for Green Science, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA 9Institute for Green Science, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA 10Inland- Water Ecosystems Team (I- WET), Departamento de Ecología, Edificio de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid, Spain Spain 11Centro de Investigación en Biodiversidad y Cambio Global (CIBC- UAM), Universidad Autónoma de Madrid, Madrid, Spain 12GloCEE – Global Change Ecology & Evolution Group, Department of Life Sciences, University of Alcalá, Alcalá de Henares, Spain 13Department of Aquatic Ecology, Eawag: Swiss Federal Institute of Aquatic Science and Technology, Dübendorf, Switzerland 14Department of Evolutionary Biology and Environmental Studies, University of Zurich, Zürich, Switzerland 15INRAE, UMR RECOVER, Aix Marseille Univ., Centre d'Aix- en- Provence, Aix- en- Provence Cedex 5, France 16Centre for Ecological Research, Institute of Aquatic Ecology, Budapest, Hungary 17Department of Hydrobiology, University of Pécs, Pécs, Hungary 18Department of Botany and Zoology, Faculty of Science, Masaryk University, Brno, Czech Republic 19RiverLY Research Unit, National Research Institute for Agriculture, Food and Environment (INRAE), Villeurbanne, France 20UMR 5023 LEHNA, Univ Lyon, Université Claude Bernard Lyon 1, CNRS, ENTPE, Villeurbanne, France 21ARALEP, Ecologie des Eaux Douces, Villeurbanne Cedex, France 22Institute of Hydrobiology and Aquatic Ecosystem Management, University of Natural Resources and Life Sciences, Vienna, Austria 23EDF R&D, Laboratoire National d'Hydraulique et Environnement (LNHE), Chatou Cedex, France 24Redpath Museum and Bieler School of Environment, McGill University, Montreal, Quebec, Canada 25Institute for Environmental Sciences, University of Koblenz Landau, Landau, Germany 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. Tracking a killer shrimp: Dikerogammarus villosus invasion dynamics across Europe Haubrock1,29 1Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, University of South Bohemia in České Budějovice, Vodňany, Czech Republic 2GEOMAR Helmholtz- Zentrum für Ozeanforschung Kiel, Kiel, Germany 3School of Biological Sciences, Queen's University Belfast, Belfast, UK 4Center for Applied Mathematics and Bioinformatics (CAMB), Department of Mathematics and Natural Sciences, Gulf University for Science and Technology, Hawally, Kuwait 5Department of Community and Ecosystem Ecology, Leibniz Institute of Freshwater Ecology and Inland Fisheries (IGB), Berlin, Germany 6School of the Environment, Yale University, New Haven, Connecticut, USA 7Department of Hydrology and Water Resources Management, Institute for Natural Resource Conservation, Christian- Albrechts- University Kiel, Kiel, Germany 8Faculty of Biology, University of Duisburg– Essen, Essen, Germany 9Institute for Green Science, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA 10Inland- Water Ecosystems Team (I- WET), Departamento de Ecología, Edificio de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid, Spain 11Centro de Investigación en Biodiversidad y Cambio Global (CIBC- UAM), Universidad Autónoma de Madrid, Madrid, Spain 12GloCEE – Global Change Ecology & Evolution Group, Department of Life Sciences, University of Alcalá, Alcalá de Henares, Spain 13Department of Aquatic Ecology, Eawag: Swiss Federal Institute of Aquatic Science and Technology, Dübendorf, Switzerland 14Department of Evolutionary Biology and Environmental Studies, University of Zurich, Zürich, Switzerland 15INRAE, UMR RECOVER, Aix Marseille Univ., Centre d'Aix- en- Provence, Aix- en- Provence Cedex 5, France 16Centre for Ecological Research, Institute of Aquatic Ecology, Budapest, Hungary 17Department of Hydrobiology, University of Pécs, Pécs, Hungary 18Department of Botany and Zoology, Faculty of Science, Masaryk University, Brno, Czech Republic 19RiverLY Research Unit, National Research Institute for Agriculture, Food and Environment (INRAE), Villeurbanne, France 20UMR 5023 LEHNA, Univ Lyon, Université Claude Bernard Lyon 1, CNRS, ENTPE, Villeurbanne, France 21ARALEP, Ecologie des Eaux Douces, Villeurbanne Cedex, France 22Institute of Hydrobiology and Aquatic Ecosystem Management, University of Natural Resources and Life Sciences, Vienna, Austria 23EDF R&D, Laboratoire National d'Hydraulique et Environnement (LNHE), Chatou Cedex, France 24Redpath Museum and Bieler School of Environment, McGill University, Montreal, Quebec, Canada 25Institute for Environmental Sciences, University of Koblenz Landau, Landau, Germany 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. © 2022 The Authors. Diversity and Distributions published by John Wiley & Sons Ltd. Ismael Soto1 \| Ross N. Cuthbert2,3 \| Danish A. HAL Id: hal-03843377 https://hal.archives-ouvertes.fr/hal-03843377 Submitted on 8 Nov 2022 Haubrock1,29 aculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, University of South ohemia in České Budějovice, Vodňany, Czech Republic GEOMAR Helmholtz- Zentrum für Ozeanforschung Kiel, Kiel, Germany chool of Biological Sciences, Queen's University Belfast, Belfast, UK Center for Applied Mathematics and Bioinformatics (CAMB), Department of Mathematics and Natural Sciences, Gulf University for Science and Technology, awally, Kuwait Department of Community and Ecosystem Ecology, Leibniz Institute of Freshwater Ecology and Inland Fisheries (IGB), Berlin, Germany chool of the Environment, Yale University, New Haven, Connecticut, USA Department of Hydrology and Water Resources Management, Institute for Natural Resource Conservation, Christian- Albrechts- University Kiel, Kiel, Germany aculty of Biology, University of Duisburg– Essen, Essen, Germany nstitute for Green Science, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA nland- Water Ecosystems Team (I- WET), Departamento de Ecología, Edificio de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid, pain Centro de Investigación en Biodiversidad y Cambio Global (CIBC- UAM), Universidad Autónoma de Madrid, Madrid, Spain GloCEE – Global Change Ecology & Evolution Group, Department of Life Sciences, University of Alcalá, Alcalá de Henares, Spain Department of Aquatic Ecology, Eawag: Swiss Federal Institute of Aquatic Science and Technology, Dübendorf, Switzerland Department of Evolutionary Biology and Environmental Studies, University of Zurich, Zürich, Switzerland NRAE, UMR RECOVER, Aix Marseille Univ., Centre d'Aix- en- Provence, Aix- en- Provence Cedex 5, France Centre for Ecological Research, Institute of Aquatic Ecology, Budapest, Hungary Department of Hydrobiology, University of Pécs, Pécs, Hungary Department of Botany and Zoology, Faculty of Science, Masaryk University, Brno, Czech Republic RiverLY Research Unit, National Research Institute for Agriculture, Food and Environment (INRAE), Villeurbanne, France 14724642, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ddi.13649 by EDF Lab Paris Saclay, Wiley Online Library on [06/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA Received: 1 June 2022 \| Revised: 1 October 2022 \| Accepted: 3 October 2022 Received: 1 June 2022 \| Revised: 1 October 2022 \| Accepted: 3 October 2022 DOI: 10.1111/ddi.13649 DOI: 10.1111/ddi.13649 \| 1 wileyonlinelibrary.com/journal/ddi HAL Id: hal-03843377 https://hal.archives-ouvertes.fr/hal-03843377 Submitted on 8 Nov 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 Distributed under a Creative Commons Attribution\| 4.0 International License eceived: 1 June 2022 \| Revised: 1 October 2022 \| Accepted: 3 October 2022 OI: 10.1111/ddi.13649 E S E A R C H A R T I C L E Tracking a killer shrimp: Dikerogammarus villosus invasion dynamics across Europe smael Soto1 \| Ross N. Cuthbert2,3 \| Danish A. Ahmed4 \| Antonín Kouba1 \| ami Domisch5 \| Jaime R. G. Marquez5 \| Ayah Beidas4 \| Giuseppe Amatulli6 \| ens Kiesel7,8 \| Longzhu Q. Shen5,9 \| Margarita Florencio10,11 \| Herlander Lima12 \| lizabeta Briski2 \| Florian Altermatt13,14 \| Gaït Archambaud- Suard15 \| eter Borza16 \| Zoltan Csabai17,18 \| Thibault Datry19 \| Mathieu Floury20 \| Maxence Forcellini19 \| Jean- François Fruget21 \| Patrick Leitner22 \| Marie- Hélène Lizée15 \| Anthony Maire23 \| Anthony Ricciardi24 \| Ralf B. Schäfer25 \| Rachel Stubbington26 \| Gea H. Van der Lee27 \| Gábor Várbíró28 \| Ralf C. M. Verdonschot27 \| Peter Haase8,29 \| Phillip J. Tracking a killer shrimp: Dikerogammarus villosus invasion dynamics across Europe Diversity and Distributions. 2022;00:1–16. \| 1 wileyonlinelibrary.com/journal/ddi Tracking a killer shrimp: Dikerogammarus villosus invasion dynamics across Europe Ismael Soto1 \| Ross N. Cuthbert2,3 \| Danish A. Ahmed4 \| Antonín Kouba1 \| Sami Domisch5 \| Jaime R. G. Marquez5 \| Ayah Beidas4 \| Giuseppe Amatulli6 \| Jens Kiesel7,8 \| Longzhu Q. Shen5,9 \| Margarita Florencio10,11 \| Herlander Lima12 \| Elizabeta Briski2 \| Florian Altermatt13,14 \| Gaït Archambaud- Suard15 \| Peter Borza16 \| Zoltan Csabai17,18 \| Thibault Datry19 \| Mathieu Floury20 \| Maxence Forcellini19 \| Jean- François Fruget21 \| Patrick Leitner22 \| Marie- Hélène Lizée15 \| Anthony Maire23 \| Anthony Ricciardi24 \| Ralf B. Schäfer25 \| Rachel Stubbington26 \| Gea H. Van der Lee27 \| Gábor Várbíró28 \| Ralf C. M. Verdonschot27 \| Peter Haase8,29 \| Phillip J. 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 Location: Europe. Methods: We analysed 96 European time series between 1994 and 2019 and identi- fied trends in the relative abundance (i.e. dominance %) of D. villosus in invaded time series, as well as a set of site- specific characteristics to identify drivers and determi- nants of population changes and invasion dynamics using meta- regression modelling. We also looked at the spread over space and time to estimate the invasion speed (km/year) of D. villosus in Europe. We investigated the impact of D. villosus abundance on recipient community metrics (i.e. abundance, taxa richness, temporal turnover, Shannon diversity and Pielou evenness) using generalized linear models. Results: Population trends varied across the time series. Nevertheless, community dominance of D. villosus increased over time across all time series. The frequency of occurrences (used as a proxy for invader spread) was well described by a Pareto dis- tribution, whereby we estimated a lag phase (i.e. the time between introduction and spatial expansion) of approximately 28 years, followed by a gradual increase before new occurrences declined rapidly in the long term. D. villosus population change was associated with decreased taxa richness, community turnover and Shannon diversity. Main Conclusion: Our results show that D. villosus is well- established in European waters and its abundance significantly alters ecological communities. However, the multidecadal lag phase prior to observed spatial expansion suggests that initial intro- ductions by D. villosus are cryptic, thus signalling the need for more effective early detection methods. m/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License K E Y W O R D S biological invasion, crustacean, freshwater ecosystem, invasive alien species, long- term monitoring, time- series Correspondence Correspondence Ismael Soto, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, University of South Bohemia in České Budějovice, Vodňany, Czech Republic. Email: isma-sa@hotmail com Abstract Aim: Invasive alien species are a growing problem worldwide due to their ecological, economic and human health impacts. The “killer shrimp” Dikerogammarus villosus is a notorious invasive alien amphipod from the Ponto- Caspian region that has invaded many fresh and brackish waters across Europe. Understandings of large- scale popu- lation dynamics of highly impactful invaders such as D. villosus are lacking, inhibiting predictions of impact and efficient timing of management strategies. Hence, our aim was to assess trends and dynamics of D. villosus as well as its impacts in freshwater rivers and streams. Tracking a killer shrimp: Dikerogammarus villosus invasion dynamics across Europe © 2022 The Authors. Diversity and Distributions published by John Wiley & Sons Ltd. d-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commo 16Centre for Ecological Research, Institute of Aquatic Ecology, Budapest, Hungary epartment of Hydrobiology, University of Pécs, Pécs, Hungary \| 1 wileyonlinelibrary.com/journal/ddi 2 \| SOTO et al. 26School of Science & Technology, Nottingham Trent University, Nottingham, UK 27Wageningen Environmental Research, Wageningen University and Research, Wageningen, The Netherlands 28Department of Tisza River Research, Centre for Ecological Research, Institute of Aquatic Ecology, Debrecen, Hungary 29Department of River Ecology and Conservation, Senckenberg Research Institute and Natural History Museum Frankfurt, Gelnhausen, Germany SOTO et al. Funding information Funding information Kuwait Foundation for the Advancement of Sciences (KFAS), Grant/Award Number: PR1914SM- 01; Gulf University for Science and Technology (GUST); Leverhulme Trust Early Career Fellowship, Grant/Award Number: ECF- 2021- 001; EU Horizon 2020, Grant/Award Number: 871128; German Federal Ministry of Education and Research, Grant/Award Number: 033W034A Accordingly, this invader's potential to successfully es- tablish after introduction – from even a single gravid female – into a novel environment is substantial (Devin et al., 2004; Lockwood et al., 2005). Reflecting its impacts on recipient ecosystems (e.g. extirpation of native species and changes in biotic indices; Kouba et al., 2021; MacNeil et al., 2013), D. villosus has been the focus of various management actions, although measures for manage- ment post- establishment are undeveloped, and prevention of ini- tial introduction has been advocated (Bradbeer et al., 2020; Wood et al., 2021). Moreover, understanding the population dynamics of this species remains limited at large spatial scales regarding tem- poral and climatic gradients (e.g. time since invasion; variability in temperature and precipitation), hindering quantification of impact and population trends that could inform management strategies. For example, earlier invasions, such as those detected in Hungary (Bij de Vaate et al., 2002: in 1926; Huber et al., 2015 in 1970; Figure 1), might already be regressing from their peak abundance to a sta- ble, intermediate level or exhibiting “boom- bust” dynamics (Rolla, Consuegra, Hall, & Garcia de Leaniz, 2020; Strayer et al., 2017) and therefore be expanding to new areas more slowly than populations with a more recent invasion history (Seebens et al., 2018). More re- cent invasions may be at an early stage of spread, potentially pre- ceding future exponential population increases. In addition, invasion success and exerted impacts are intertwined with anthropogenic stressors such as hydromorphological alterations and dam construc- tion, which can influence spread rates (Colautti et al., 2006; MacNeil & Platvoet, 2013). Invaders may take considerable time before becoming estab- lished, detected and disruptive (Crooks, 2005; Spear et al., 2021), and such time lags are very difficult to predict (Coutts et al., 2018). Furthermore, the invaders may not remain disruptive or could be- come less so, owing to population declines (e.g. reflecting boom- and- bust cycles and/or community adjustment; Strayer et al., 2017). Finally, the type and magnitude of ecological impacts an invader causes are dependent, in part, on its abundance (Sofaer et al., 2018; Yokomizo et al., 2009), and thus can change through time accord- ing to the invader's population dynamics. 3 The form of population dynamics may differ among invaders, invasion pathways, biogeographical regions, abiotic or biotic gra- dients and spatio- temporal scales (Arim et al., 2006; Haubrock et al., 2020). Characterization of population dynamics of invasive alien species at a broad spatio- temporal scale has been limited by insufficient long- term data. However, advances in availability in long- term biodiversity monitoring (Dornelas et al., 2018; Mirtl et al., 2018) and data analysis approaches for biodiversity time se- ries (Bowler et al., 2017; Dornelas et al., 2014; Pilotto et al., 2020) have been made. These and other initiatives have collated large datasets for diverse taxonomic groups and examined biodiver- sity and ecosystem function trends at regional and global scales (Seebens et al., 2019, 2020). Large- scale, community- level data can also be used to examine distribution and abundance patterns of invasive alien species, as well as their potential effects on eco- logical communities over time (Dornelas et al., 2014; Haubrock et al., 2020). et al., 2021), causing disruptions across multiple trophic levels (i.e. trophic cascades; Van Riel, Van der Velde, & Bij de Vaate, 2006). Its displacement of native species (MacNeil et al., 2011; MacNeil & Platvoet, 2013) also facilitates the establishment of other invasive alien species (Bollache et al., 2004; Leuven et al., 2009). Notably, it is the only invasive amphipod with documented monetary costs (Kouba et al., 2021). Yet, information on this invader's effects on native species assemblages over time is still scarce. et al., 2021), causing disruptions across multiple trophic levels (i.e. trophic cascades; Van Riel, Van der Velde, & Bij de Vaate, 2006). Its displacement of native species (MacNeil et al., 2011; MacNeil & Platvoet, 2013) also facilitates the establishment of other invasive alien species (Bollache et al., 2004; Leuven et al., 2009). Notably, it is the only invasive amphipod with documented monetary costs (Kouba et al., 2021). Yet, information on this invader's effects on native species assemblages over time is still scarce. Dikerogammarus villosus invasion success has been promoted by its suitability for transport by anthropogenic vectors (e.g. ships; Anderson et al., 2014), wide thermal and salinity tolerances (Cuthbert et al., 2020), aggressive competitive behaviours (Kobak et al., 2016), effective anti- predator strategies (Rolla, Consuegra, & de Leaniz, 2020) and high growth rates and fecundity (Holdich & Pöckl, 2007; Pöckl, 2009), as one female can carry nearly 200 eggs (Pöckl, 2009). 1 \| INTRODUCTION 1 invaders (i.e. introduced species that become invasive; Fournier et al., 2019; Pyšek et al., 2020) and assess their impacts (Essl et al., 2020). With the ongoing global increase in invasion rates (Seebens et al., 2021) and a growing threat to ecosystems and econ- omies (Pyšek et al., 2020), there is an urgent need to characterize population dynamics at large scales to inform effective detection, management actions and monitoring (Cuthbert, Kotronaki, Carlton, et al., 2022; Seebens et al., 2021). Humans have translocated thousands of invasive alien species be- yond their native ranges (Seebens et al., 2021). Their establishment and spread have been recognized as a leading cause of biodiver- sity loss and a growing socio- economic burden worldwide (Bellard et al., 2016; Diagne et al., 2021). It is therefore imperative to un- derstand and predict large- scale invasion patterns, identify future SOTO et al. SOTO et al. \| 3 2.1 \| Data compilation To investigate the population dynamics of D. villosus and the re- sponse of recipient communities across Europe, we considered 1816 time series (Peter Haase, unpublished data) reporting the abundance of macroinvertebrate taxa in streams and rivers across 22 European countries. Macroinvertebrates were sampled using dif- ferent methods and protocols (see Table S1) among time series, but were consistent within each time series. Each time series comprised macroinvertebrate assemblages collected at a single site in multiple years. We initially selected all 132 time series with D. villosus popula- tions and then excluded 36 time series that contained ≤2 sampled years, retaining 96 time series (Figure 1), of which most are from large European rivers (e.g. Danube or Rhine) across six European countries (i.e. Austria, France, Germany, Hungary, Netherlands and Switzerland). For time series in which samples were not collected in all years, we coded missing years as “not available” (NA). Time series spanned a mean ± SD of 10.1 ± 3.4 years and contained 7.7 ± 3.6 sam- pling years between 1994 and 2019. Although raw abundances are not comparable directly across time series due to differences in sampling methods, we approx- imated the temporal trend in D. villosus relative abundance (%) by averaging abundance records across all samples from a given year, as a proxy of dominance of the species to avoid introducing a bias from the comparison of different sampling methods. We considered only those years in which at least 15 assemblages were sampled and thus omitted 1994, 2018 and 2019 (5– 7 sampled assemblages). Once removed, the mean number of assemblages sampled was 27.3 ± 6.8, 4 \| SOTO et al. individual regressions (as in our case from different time series). For this, we used the “rma.mv” function of the R package metafor (Viechtbauer, 2010), using the time- series Mann– Kendall trend test (S- statistics) and respective variance as the effect size. The Mann– Kendall trend test is a non- parametric test to evaluate a monotonic increase or decrease in trends. In particular, we used the trends calculated by modified Mann– Kendall trend tests with variance correction to account for temporal auto- correlation (Hamed & Rao, 1998; Maire et al., 2019). To correct the spatial autocorrelation between time series, we used a random effect model, specifying the geographic coordinates as a random effect according to a Gaussian correlation structure (Cressie, 1993; Maire et al., 2019). This approach uses a regression model and enables comparability of time series by analysing the individual time series abundance trends (i.e. its slope) rather than the raw abundances. These models account for the variance of each individual temporal trend and treat each population as an individual spatial unit associ- ated with scale and sampling protocol (Viechtbauer et al., 2015). Lastly, based on two data clusters, we classified the time series according to their first year of sampling into two groups and visu- ally inspect if there are differences in population growth among groups. For this, we selected 2003 as a middle point between ear- lier (i.e. the first year of the time series before 2003) and later (i.e. the first year of the time series after 2003) invasions. hypothesized (iv) changes in recipient communities to be associated with D. villosus population dynamics. Given that the long- term population dynamics of even widespread conspicuous invaders are often poorly characterized (Strayer et al., 2006), this gap challenges our ability to (i) predict in which situations the invader's impacts on invaded ecosystems will be maximal and (ii) decide if/when manage- ment should intervene. However, species having well- documented invasion histories within large contiguous regions offer temporally highly resolved data whose collation and analysis could reveal es- sential information to guide risk assessment and management prioritization. wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licen One such species is the “killer shrimp” Dikerogammarus vil- losus, an invasive alien freshwater amphipod listed among the “100 worst” invaders in Europe (Nentwig et al., 2018). The con- firmed native range of D. villosus is the northern margins of the Ponto- Caspian region (i.e. Black Sea, Caspian Sea and Azov Sea; Dedyu, 1980; Mordukhai- Boltovskoi, 1960). This voracious pred- ator has spread rapidly through European inland waterways and the Baltic Sea, aided by canalization and anthropogenic vectors (Cuthbert et al., 2020; De Ventura et al., 2017), and also poses an invasion risk to the North American Great Lakes (Kramer et al., 2017). Facilitated by the known influence of streams on the spread of D. villosus, it has substantial impacts on biodiver- sity in invaded regions, causing marked declines in native mac- roinvertebrates via predation and competition (Dick et al., 2002; Dick & Platvoet, 2000). It also potentially impacts egg and em- bryonic stages of large, ecologically important crustaceans, fish and amphibians (Roje et al., 2021; Taylor & Dunn, 2017; Warren To characterize the population dynamics of D. villosus across time series, we collated European riverine macroinvertebrate bio- monitoring data containing this species. We hypothesized that the (i) dominance of D. villosus populations is increasing within time series, whereas the number of new occurrences is declining in the long- term, indicating a deceleration in the invasion; (ii) recent inva- sions exhibit more rapid population growth, whereas populations originating from earlier invasions are stable or declining; (iii) popu- lation dynamics of D. villosus are mediated by site- specific climatic and spatial characteristics, with populations affected by warming and the presence of anthropogenic barriers such as dams. Lastly, we 2.3 \| Effects of site- specific characteristics on D. villosus To investigate the spatial and climatic drivers of D. villosus trends, we used site- specific characteristics of each time series. Climatic re- gions (i.e. Köppen- Geiger climate zone) were extracted from Beck et al. (2018). Biogeographical regions were defined following the European Environment Agency classification map and estimated visually using the site- specific coordinates (EEA, 2021). We further classified the type of ecosystem of each time series based on Strahler order (i.e. stream <8 or large rivers ≥8) to evaluate differences in the degree of invasions between both. We obtained site- specific runoff data, expressed as the annual Q (mm), from the TerraClimate dataset at 4- km spatial resolution (Abatzoglou et al., 2018). We extracted the elevation of each site from the MERIT Hydro digital elevation model (Yamazaki et al., 2019) at 90- m spatial resolution and used the Hydrography90m (Amatulli et al., 2022) stream network, catch- ments and sub- catchments (catchments between network nodes) as underlying spatial units. For each site, we computed the stream slope using the r.stream.slope function. We extracted land cover data from the European Space Agency Climate Change Initiative (ESA CCI) Land Cover time series v2.0.7 dataset, at 300- m spatial resolution (ESA, 2017), as the percentage cover of a given land cover category within the sub- catchment. We used the Global Reservoir and Dam Database (GRanD) v1.3 to identify dams along the river network. We then measured the distance to the nearest dam to investigate the effect of instream barriers. We extracted mean daily temperature and total daily precipitation data from a gridded European scale observation- based dataset (spatial resolution: 0.1°; Cornes et al., 2018) and calculated the average annual temperature and precipitation for each sampled assemblage in each sampling year (Pilotto et al., 2020), as well as their respective S- statistics (i.e. the Mann– Kendall trend test statistic, see above), as indicators of climatic changes. Precipitation in particular can be used as a proxy for river discharge (Higashino & Stefan, 2019) and was included as it affects the availability of water and nutrients as well as the habitat suitability for many species (Gallardo et al., 2012; Shi et al., 2010). We then calculated the mean maximum and minimum temperature and precipitation and each respective S- statistic. Dikerogammarus villosus slopes (to investigate factors determining D. villosus' rate of change over time) and relative abundances (as a proxy of D. 2.3 \| Effects of site- specific characteristics on D. villosus villo- sus' dominance in invaded ecosystems over time) were analysed as a function of these spatial, temporal, and site- specific characteris- tics, to identify significant drivers of temporal trends (see Table S2). We used generalized linear models (GLMs) via the MASS R package (Ripley et al., 2013). We used a Gaussian distribution for continuous data and quasibinomial distribution (to account for high variance of 2.2 \| Trend identification and meta- regression modelling To synthesize and describe the directionality and the trends in the number of sampled D. villosus individuals, we used a meta- regression modelling approach, which synthesizes the slopes of FI G U R E 1 Map summarizing the native and invaded range of Dikerogammarus villosus in Europe, showing populations reported by the Global Biodiversity Information Facility (GBIF, 2021; grey triangles) and our time series (white triangles). Years within countries indicate the year of the first introduction according to the sTWIST database of first records (Seebens et al., 2018). The invasion pathways by which D. villosus has spread (i.e. Rhine, Danube, Volga and Dnieper rivers) are inferred from Bij de Vaate et al. (2002). FI G U R E 1 Map summarizing the native and invaded range of Dikerogammarus villosus in Europe, showing populations reported by the Global Biodiversity Information Facility (GBIF, 2021; grey triangles) and our time series (white triangles). Years within countries indicate the year of the first introduction according to the sTWIST database of first records (Seebens et al., 2018). The invasion pathways by which D. villosus has spread (i.e. Rhine, Danube, Volga and Dnieper rivers) are inferred from Bij de Vaate et al. (2002). SOTO et al. \| 5 5 and maximum 42 in the year 2007. We tested a linear model for the dominance of D. villosus over time using least squares regression and thus estimated the proportion of abundance (%) at the time the as- semblage was sampled. the dataset) for proportion data (i.e. relative abundances) after visu- ally inspecting their respective residual distributions for normality through histograms. To identify the best model, we first tested for collinearity among the numerical variables using the variance inflation factor (VIF) for continuous predictors using the “corvif” function (Zuur et al., 2009). We selected VIF >5 as the threshold, and those variables with high VIF values for each model were assessed for their ecological rel- evance based on expert knowledge (Table S3; Dorman et al., 2013; Zuur et al., 2009). Regarding the four categorical variables (country, biogeographical region, Köppen- Geiger classification and ecosystem type), we used chi- square tests to investigate the collinearity, and retained only the biogeographical region (Table S4). We considered each model and the respective predictors, using expert- based opin- ion to determine if the inclusion predictors would make sense from an ecological perspective (Table S5). Hence, the model consisted of a single response variable (i.e. the Mann– Kendall trend test slopes of D. villosus abundances or the relative proportion of D. villosus) and site- specific characteristics (see above; Table S2). Following the alpha- betical order, the Alpine region was used as a reference factor (i.e. as intercept), and therefore, we do not infer any results about this region. 6 \| SOTO et al. All models used restricted maximum likelihood estimation (REML). We also quantified the proportion of variance in the model not attributed to sampling error by using the I2 statistic. In addition, we evaluated the results of the meta- regression by a graphical rep- resentation (i.e. forest plots) using the “forest” function of the R package metafor (Figure S2; Viechtbauer, 2010). To inspect poten- tial biases that may alter the results, we checked the symmetry of the data using funnel plots and statistically evaluated this symmetry using the Egger's test (Egger et al., 1997; see Figure S3; Table S6). As a complementary analysis, we computed the distance between the locations of every sampled site from the first invaded site using site location data (GPS coordinates recorded as latitude and longitude) over the years 1994– 2021. An estimate for the invasion speed (km/ year) was obtained by computing the mean distance (i.e. total distance averaged over the number of occurrences per year) over time and modelled using a linear equation (Bagnara et al., 2022). We also calcu- lated the differences between the first record of D. villosus in our data and sTWIST database (the most comprehensive source of first records of alien species, integrating several databases and merging them into a single database; Seebens et al., 2018). All analyses were carried out in R v.4.1.3. (R Core Team, 2022). The reproducible R script is available with the manuscript and lists all R packages that were used. 3 \| RESULTS To assess the effect of D. villosus abundance on recipient communi- ties (proxied by the S- statistic of D. villosus trends), we computed five common metrics for each community and year within each time series: total abundance (i.e. individuals), taxon richness (i.e. the total num- ber of taxa), temporal turnover (i.e. the proportion of species either gained or lost over time relative to the total number of species ob- served; Carvalho et al., 2012), the Shannon diversity index (Shannon & Weaver, 1949) and Pielou's evenness (Pielou, 1966). The metrics were calculated considering all species in the community except D. villosus, potentially including both native and other non- native species. For evenness, we followed the formula: H/ln(S) (where H is the Shannon Index and S is the taxon richness of a community). Metrics were cal- culated using the “diversity” function in the R package vegan (Oksanen et al., 2013) and the “turnover” function in the R package codyn (Hallett et al., 2016). In analysing these metrics as response variables in meta- regression models, we included the middle point of each time series (see above) in addition to the rate change of D. villosus to infer the effects of temporal variability in changing temporal trends (i.e. slopes) over time and evaluate the change of sampled individuals of D. villosus individuals to test its associated effect on community metrics. 2.4 \| Modelling occurrence frequency and invasion speed We combined the first occurrence of D. villosus in each time series in our data with those occurrences (as coordinates and year of records) in the Global Biodiversity Information Facility database (GBIF, 2021). Eight GBIF occurrences were removed due to insufficient informa- tion (e.g. no recorded year), resulting in 400 records. We excluded those sample years from the dataset of 400 re- cords that reported relatively high occurrence frequencies, that is, any number of occurrences that was greater than Q3 + 3 × IQR, where Q3 is the upper quartile and IQR is the interquartile range of the dataset. A single outlier was found with 120 occurrences in the year 2009, and thus, it was removed. We modelled the remain- ing 280 occurrences to represent invader spread, using a logistic distribution and a two- tailed Pareto distribution. A key difference between these distributions is the decay rate at the end tails: the logistic distribution decays exponentially fast (thin tails), and for the Pareto distribution, the decay is much slower according to an inverse power law (fat tails; Nolan, 2020). Moreover, Pareto distributions with distinct parameters were considered (i.e. two- tailed) for the early and late phases of the invasion. Both distributions were fit- ted against the occurrence data using the non- linear regression tool lsqcurvefit in Matlab. The better- fitting model was determined based on lower number of parameters and higher R2 value (see Note S2). Further, we estimated the frequency of occurrences f0 at the time of first sampling (t = 0) the time of introduction tintro, and the duration of the lag phase tlag that is, the period before D. villosus was observed in additional assemblages, evaluated at 10% of the largest recorded occurrence frequency f. FI G U R E 2 Changes in trends (slopes) of Dikerogammarus villosus in individual time series (S- statistics ± confidence intervals): red represents negative trends, blue positive trends, and grey indicates no significant change over time (a). Relative abundance of D. villosus in sampled sites at the European level. Proportions were averaged over the number of sampled assemblages each year from 1995 (t = 0) to 2017 (t = 22; black dots) (b). 3.1 \| Trends of D. villosus across Europe Across the sampling sites in Europe included in this study, the abun- dance trend of D. villosus increased in 49 locations, decreased in 44 locations and has not changed in 3 locations (Figure 2a). Our analysis therefore suggests that the total number of D. villosus individuals in the study region experienced no overall significant change in its rising trajectory between 1994 and 2019s (S- statistics = 4.74; CI: −9.32, 18.82, p = .50; Table 1; Figure 2a), albeit expressing low het- erogeneity (I2 = 3.62%). In addition, we did not find differences in the population growth between earlier (for which t = 0 was before 2003) and later time series (i.e. t = 0 after 2003; Figure S1). Averaged across all time series, the overall proportion of D. villosus was well described by a linear model (r = .45), suggesting a steep rise in relative abundance over time. An average of 8.66% of D. villosus was recorded per sampled assemblage at the first time point. On average across all time series, the rate of increase in relative abundance (i.e. dominance) increased by 0.31% per year for each sampled assemblage (Figure 2b). In addition, our first records for D. villosus were on average FI G U R E 2 Changes in trends (slopes) of Dikerogammarus villosus in individual time series (S- statistics ± confidence intervals): red represents negative trends, blue positive trends, and grey indicates no significant change over time (a). Relative abundance of D. villosus in sampled sites at the European level. Proportions were averaged over the number of sampled assemblages each year from 1995 (t = 0) to 2017 (t = 22; black dots) (b). 3.2 \| Effect of site- specific characteristics on D. villosus trend We did not identify a change in individuals of D. villosus sampled over time, despite a positive tendency (i.e. change in the S- statistics; GLM: 0.59 ± 0.45; p = .19; Figure 3a; Table S7). The rate of change in the trend of D. villosus increased significantly across the Mediterranean biogeographic region (p < .05). The average minimum temperature and the rate change of the maximum temperature had positive effects on the rate of change of D. villosus individuals (p < .05), while the distance to the next barrier had negative effects (p < .05; Figure 3b; Table S7). Regarding the relative abundance of D. villosus over time, we identified a significant increase (p < .05; Figure 3c; Table S7). This increase was shown in all biogeographic regions (relative to Alpine), as well as with increasing distance to the next barrier and the elevation of the stream (p < .01; Figure 3d; Table S7). The relative abundance decreased in streams relative to large rivers (p < .01; Figure 3d; Table S7). We estimated the invasion speed of D. villosus at the time of first sampling as 80.27 km/year, with deceleration at a rate of 2.83 km/ year2, eventually reaching minimum speed at time t = 28.34 years (see Figure 4b). The estimation of null speed corresponds to a cessa- tion in the frequency of occurrences at approximately the same time (49.03 years after tintro, Figure 4a). 3.4 \| Impact of D. villosus on community metrics trends We did not find a significant trend over time for community metrics (Table 1). The rate of change in the number of D. villosus individuals sampled over time (i.e. its slope) had a significant negative effect on trends in taxon richness, temporal turnover and Shannon diversity (Figure 5; Table 1). We did not find a significant effect of the number of sampled D. villosus individuals on total community abundance and Pielou's evenness trends (Table 1; Figure 5). \| 7 10.8 years later than those referenced in the sTWIST database. This difference was reduced to 2.8 years after excluding time series from Austria and Switzerland (n = 2), for which the difference between both databases was 14 years (Seebens et al., 2018). f = 78 in the year 2008 (t* = 14). The occurrence frequency of D. vil- losus was best described by a two- tailed Pareto distribution (R2 = .90 for t ≤ t* and R2 = .98 for t ≥ t* see Figure 4a), which fits better than the logistic distribution (R2 = .86; see Note S2). Moreover, the Pareto distribution depends on fewer parameters. On considering an occur- rence frequency of 1 (i.e. first invaded site), we predicted the time of introduction tintro = −20.67 years prior to the first sampling event. The estimated number of occurrences at the time of first sampling was f0 = 3.37. The duration of the lag phase was 27.64 years after tintro. Beyond t, occurrence frequency rapidly declined, reaching low levels 45– 50 years after tintro, indicating low levels of spread for D. villosus. 3.1 \| Trends of D. villosus across Europe (b) (a) Slope 0 5 10 15 20 25 30 5 0 10 15 20 25 30 time, t (years) ) % ( n e c n a d n u b a e v it a l e R n(t) = 0.31t + 8.66, r = 0.45 n = at + n0 -100 -50 0 50 100 150 200 (b) 0 5 10 15 20 25 30 5 0 10 15 20 25 30 time, t (years) ) % ( n e c n a d n u b a e v it a l e R n(t) = 0.31t + 8.66, r = 0.45 n = at + n0 (a) (a) Slope -100 -50 0 50 100 150 200 ) % ( n e c n a d n u b a e v it a l e R FI G U R E 2 Changes in trends (slopes) of Dikerogammarus villosus in individual time series (S- statistics ± confidence intervals): red represents negative trends, blue positive trends, and grey indicates no significant change over time (a). Relative abundance of D. villosus in sampled sites at the European level. Proportions were averaged over the number of sampled assemblages each year from 1995 (t = 0) to 2017 (t = 22; black dots) (b). FI G U R E 2 Changes in trends (slopes) of Dikerogammarus villosus in individual time series (S- statistics ± confidence intervals): red represents negative trends, blue positive trends, and grey indicates no significant change over time (a). Relative abundance of D. villosus in sampled sites at the European level. Proportions were averaged over the number of sampled assemblages each year from 1995 (t = 0) to 2017 (t = 22; black dots) (b). \| 7 SOTO et al. 3.3 \| Modelling occurrence frequency and invasion speed at the European level The mean ± SD for the number of D. villosus occurrences was 13.33 ± 19.07, with the maximum recorded occurrence frequency TA B LE 1 Meta- regression results according to time and Dikerogammarus villosus abundance for the following response variables: D. villosus abundance (a), community abundance (b), richness (c), turnover (d), diversity (e) and evenness (f) of recipient community. TA B LE 1 Meta- regression results according to time and Dikerogammarus villosus abundance for the following response variables: D. villosus abundance (a), community abundance (b), richness (c), turnover (d), diversity (e) and evenness (f) of recipient community. Response variable Predictor Estimate Standard error p- Value Confidence interval (lower) Confidence interval (upper) (a) D. villosus abundance I2 = 3.62% Intercept 4.74 7.18 .50 −9.32 18.82 (b) Abundance I2 = 21.86% Intercept −15.70 196.46 .93 −400.76 369.34 Middle point of time series 0.13 0.13 .32 −0.13 0.39 Change in D. villosus abundance 0.14 0.08 .10 −0.03 0.31 (c) Richness I2 = 68.96% Intercept −327.49 237.33 .16 −792.66 137.67 Middle point of time series 0.11 0.20 .57 −0.28 0.52 Change in D. villosus abundance −0.24 0.09 .01 −0.43 −0.05 (d) Turnover I2 = 4.55% Intercept 47.70 187.37 .75 −319.53 414.94 Middle point of time series −0.03 0.09 .75 −0.21 0.15 Change in D. villosus abundance −0.17 0.08 .03 −0.32 −0.01 (e) Diversity Shannon I2 = 21.80% Intercept 74.45 188.24 .69 −294.50 443.41 Middle point of time series < 0.01 0.15 .95 −0.30 0.32 Change in D. villosus abundance −0.22 0.08 .01 −0.39 −0.04 (f) Evenness Pielou I2 = 14.42% Intercept 148.23 190.31 .43 −224.78 521.25 Middle point of time series −0.03 0.12 .78 −0.27 0.20 Change in D. villosus abundance −0.15 0.08 .08 −0.33 0.02 14724642, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ddi.13649 by EDF Lab Paris Saclay, Wiley Online Library on [06 14724642, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ddi.13649 by EDF Lab Paris Saclay, Wiley Online Library on [06/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for 14724642, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ddi.13649 by EDF Lab Paris Saclay, Wiley Online Library on [06/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; SOTO et al. 8 \| 1995 2000 2005 2010 2015 Middle point of time series 20 10 0 -10 Trend of D. villosus (a) D 8 \| SOTO et al. 3.3 \| Modelling occurrence frequency and invasion speed at the European level FI G U R E 3 Trend of Dikerogammarus villosus abundance over time ± standard error (SE, blue shaded area) (a). Effect ± SE of the predictors included in the D. villosus model (b). Relative abundance of D. villosus over time ± standard error (SE, blue shaded area) (c). Effect ± SE of the predictors included in the model (d). Solid trend lines are significant and dashed lines not significant. Blue dot: positive effect; red dot: negative effect; filled dots: significant effects; empty dots: non- significant effects. 1995 2000 2005 2010 2015 2000 2010 2020 Middle point of time series 20 10 0 -10 Trend of D. villosus Relative abundance of D. villosus Year 15% 10% 0% 20% (c) (d) (a) (b) Mediterranean Continental Atlantic Average Tmin Stream Average Precipitation Trend of Tmax Middle point of time series Trend of Precipitation Average Tmax Elevation Average Temperature Distance to the next barrier Trend of Temperature Pannonian Slope of stream Mediterranean Continental Slope Atlantic Pannonian Year Elevation Average Temperature Distance to the next barrier Tmax Average Tmax Temperature Tmin Precipitation Streams -150 -100 -50 0 50 100 Estimates Estimates -4 -2 0 2 4 6 24642, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ddi.13649 by EDF Lab Paris Saclay, Wiley Online Library on [06/11/2022]. See the Terms and Condition 1995 2000 2005 2010 2015 Middle point of time series 20 10 0 -10 Trend of D. villosus (a) (b) Mediterranean Continental Atlantic Average Tmin Stream Average Precipitation Trend of Tmax Middle point of time series Trend of Precipitation Average Tmax Elevation Average Temperature Distance to the next barrier Trend of Temperature Pannonian Slope of stream -150 -100 -50 0 50 100 Estimates (b) Mediterranean Continental Atlantic Average Tmin Stream Average Precipitation Trend of Tmax Middle point of time series Trend of Precipitation Average Tmax Elevation Average Temperature Distance to the next barrier Trend of Temperature Pannonian Slope of stream -150 -100 -50 0 50 100 Estimates (b) 10 0 Trend of D. villosus nlinelibrary.wiley.com/doi/10.1111/ddi.13649 by EDF Lab Paris Saclay, Wiley Online Library on [06/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/te (d) Mediterranean Continental Slope Atlantic Pannonian Year Elevation Average Temperature ance to the next barrier Tmax Average Tmax Temperature Tmin Precipitation Streams Estimates -4 -2 0 2 4 6 (d) FI G U R E 3 Trend of Dikerogammarus villosus abundance over time ± standard error (SE, blue shaded area) (a). 4 \| DISCUSSION Contrary to our first hypothesis, our meta- regression models identified no trend in the D. villosus population across time series. This lack of identifiable patterns could (i) reflect the complex pop- ulation dynamics of invasive alien species at large spatial scales, (ii) climatic variability across European countries and biogeographical regions or (iii) genetic differentiation across invaded sites, ultimately leading to differing trends (Arim et al., 2006; Haubrock et al., 2022). For example, in France, all temporal trends (i.e. S- Statistics) were positive, whereas most trends were negative in Hungary. The sus- tained dominance of D. villosus in recipient ecosystems increased over time, which could reflect its ability to rapidly reach high pop- ulation densities in combination with its capacity to predate, elim- inate and replace native and alien species (Dick & Platvoet, 2000; Nentwig et al., 2018; Warren et al., 2021). In addition, the difference between the first record of D. villosus in sTWIST and our database was ~10.8 years. Yet, after excluding Switzerland and Austria— which had a difference of ~14 years between both databases likely due to the scarce time series from that country (n = 2)— the difference among both databases shrank to ~2.8 years only, underlining the ac- curacy of our data. 3.3 \| Modelling occurrence frequency and invasion speed at the European level Effect ± SE of the predictors included in the D. villosus model (b). Relative abundance of D. villosus over time ± standard error (SE, blue shaded area) (c). Effect ± SE of the predictors included in the model (d). Solid trend lines are significant and dashed lines not significant. Blue dot: positive effect; red dot: negative effect; filled dots: significant effects; empty dots: non- significant effects. om/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 4.1 \| Overview We characterized the population dynamics of one of the most noto- rious invasive alien species in Europe, D. villosus, and its effects on freshwater macroinvertebrate community metrics across available European time series. Contrary to our first hypothesis, we detected no significant trend in the number of D. villosus individuals sampled, although its dominance in invaded ecosystems increased over time. Contrary to our second hypothesis, the growth of earlier and more recently invading populations was comparable. Supporting our third hypothesis, D. villosus populations were influenced by site- specific climatic and spatial characteristics. Finally, supporting our fourth hypothesis, D. villosus was negatively associated with trends in mac- roinvertebrate community taxon richness, temporal turnover and Shannon diversity. These results highlight the need towards proac- tive management actions to contain D. villosus as well as to better understand the potential synergistic effects of stressors (Ricciardi et al., 2021). 14724642, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ddi.13649 by EDF Lab Paris Saclay, Wiley Online Library on [06/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of \| 9 SOTO et al. FI G U R E 4 Occurrence frequency of D. villosus between 1994 (t = 0) and 2021 (t = 27) (circle markers) (a). Pareto distributions were fitted with R2 = .90 for t ≤t∗, RMSE = 6.7 and R2 = .98 for t > t∗, RMSE = 3.6, where the maximum occurrence frequency f = 78 was recorded at t* = 14 years. Estimated model parameters for the left- hand tail are s1 = 1.81, 휇1 = 1.45, which were used to predict the: time of introduction tintro = −20.67 years, number of occurrences at the time of first sampling (t = 0) f0 = 3.37, duration of the lag phase tlag = 27.64 years (after tintro) with occurrence frequency flag = 7.8 (fixed at 10% of f). Model parameters for the right- hand tail are s2 = 1.08 × 104, 휇2 = 7.51 × 103, see Note S2 for mathematical details related to the Pareto distributions, and how these key points were determined from estimated model parameters. Linear model v = −2.83t + 80.27 fitted using least squares regression against estimated values of annual invasion speed with R = −0.70. The estimated speed at the time the first site was sampled is v0 = 80.27 km/year, and acceleration v1 = −2.83 km/year2 (b). 4.1 \| Overview time, t (years) time, t (years) , d e e p s n o i s a v n I v ) r a e y / m k ( 70 60 50 40 30 20 10 0 -25 -20 -15 -10 -5 0 5 10 15 20 25 30 , y c n e u c e r f e c n e r r u c O f ) t ( 0 5 10 15 20 25 30 120 100 80 60 40 20 0 (a) (b) v = -2.83t + 80.27, R=-0.70 (tintro,1) (0,f0) (tlag,flag) (t,f) \| 9 SOTO et al. time, t (years) time, t (years) , d e e p s n o i s a v n I v ) r a e y / m k ( 70 60 50 40 30 20 10 0 -25 -20 -15 -10 -5 0 5 10 15 20 25 30 , y c n e u c e r f e c n e r r u c O f ) t ( 0 5 10 15 20 25 30 120 100 80 60 40 20 0 (a) (b) v = -2.83t + 80.27, R=-0.70 (tintro,1) (0,f0) (tlag,flag) (t,f) \| 9 SOTO et al. (a) FI G U R E 4 Occurrence frequency of D. villosus between 1994 (t = 0) and 2021 (t = 27) (circle markers) (a). Pareto distributions were fitted with R2 = .90 for t ≤t∗, RMSE = 6.7 and R2 = .98 for t > t∗, RMSE = 3.6, where the maximum occurrence frequency f = 78 was recorded at t* = 14 years. Estimated model parameters for the left- hand tail are s1 = 1.81, 휇1 = 1.45, which were used to predict the: time of introduction tintro = −20.67 years, number of occurrences at the time of first sampling (t = 0) f0 = 3.37, duration of the lag phase tlag = 27.64 years (after tintro) with occurrence frequency flag = 7.8 (fixed at 10% of f*). Model parameters for the right- hand tail are s2 = 1.08 × 104, 휇2 = 7.51 × 103, see Note S2 for mathematical details related to the Pareto distributions, and how these key points were determined from estimated model parameters. 4.1 \| Overview Linear model v = −2.83t + 80.27 fitted using least squares regression against estimated values of annual invasion speed with R = −0.70. The estimated speed at the time the first site was sampled is v0 = 80.27 km/year, and acceleration v1 = −2.83 km/year2 (b). In addition, contrary to our fourth hypothesis, we observed no difference in the growth rates of earlier and more recently invading populations, suggesting that the time since invasion does not influ- ence D. villosus population dynamics. In addition, time series repre- senting earlier and more recent invasions originated from different biogeographical regions: earlier ones were mostly from Atlantic and Continental regions, and more recent ones from Pannonian and Alpine regions. Crooks, 2005; Sakai et al., 2001; Spear et al., 2021). Although report- ing efforts may be increased over time, this lag phase may explain the rapid decrease in new occurrences 35 years after an assemblage was first invaded (Ricciardi, 2013; Rouget et al., 2016). Nevertheless, records extracted from GBIF have to be taken with caution, as the taxonomic validity cannot always be ensured, simultaneously sug- gesting that many observations may be missing (Nekola et al., 2019: Shirey et al., 2019). Predicting future trends in the abundance of D. villosus is hampered by context dependencies, which may cause sud- den shifts in population dynamics at different temporal scales, for example reflecting boom- bust dynamics (Strayer et al., 2017). Impact of D. villosus on community metrics Understandings of how invasive alien species impact the recipient ecosystems and the potential synergistic effects of anthropogenic stressors (abiotic and biotic factors) have become priorities in in- vasion science (Ricciardi et al., 2021). Supporting our fourth hy- pothesis, we identified a negative relationship between temporal changes in the number of sampled D. villosus individuals and in three metrics representing macroinvertebrate communities: taxon richness, temporal turnover and Shannon diversity. The negative impacts of D. villosus on invaded ecosystems are well- documented and include the depredation of a wide range of macroinverte- brates (e.g. chironomids, leeches, isopods and juvenile crayfish; Buřič et al., 2009; Dick et al., 2002; Platvoet et al., 2009), including via “wasteful” killing (Dick et al., 2002). Invasive amphipods have also been shown to display lower levels of omnivory than native species (Cuthbert, Kotronaki, Hütt, et al., 2022). This predatory capacity can reduce or replace functionally equivalent species via intraguild predation (e.g. native Gammarus duebeni by alien G. tigri- nus; Rewicz et al., 2014). Dikerogammarus villosus also has negative effects on ecosystem functioning, including alteration of habitat structure, leaf litter decomposition and energy flows through food webs, potentially causing large- scale trophic cascades (Koester et al., 2016; MacNeil et al., 2011; Piscart et al., 2011; Van Riel et al., 2006). These impacts can create vacant niches that increase community susceptibility to other invasions and exacerbate the collective impacts of invasive alien species (Boets et al., 2010, 2011). 0.00 Response FI G U R E 5 Effect of general temporal trend (circles) and the number of sampled individuals of Dikerogammarus villosus (D. villosus shape) on the rate at which trends in community metrics (total abundance, richness, temporal turnover, Shannon diversity and Pielou's evenness) changed across all time series. Empty dots represent no significant effects ± standard error (bars). Hollow shapes represent no significant effects of D. villosus, while the filled shapes represent significant effects. Red represents negative effects of D. villosus and negative trends in community metrics, while blue represents positive effects and positive trends of community metrics. FI G U R E 5 Effect of general temporal trend (circles) and the number of sampled individuals of Dikerogammarus villosus (D. (Bacela- Spychalska et al., 2013). Furthermore, dams can be used as refuges and “stepping stones” for further spread, but also limit the spread upstream. Therefore, invasive alien species can accumulate near these barriers (Rahel, 2013). FI G U R E 5 Effect of general temporal trend (circles) and the number of sampled individuals of Dikerogammarus villosus (D. villosus shape) on the rate at which trends in community metrics (total abundance, richness, temporal turnover, Shannon diversity and Pielou's evenness) changed across all time series. Empty dots represent no significant effects ± standard error (bars). Hollow shapes represent no significant effects of D. villosus, while the filled shapes represent significant effects. Red represents negative effects of D. villosus and negative trends in community metrics, while blue represents positive effects and positive trends of community metrics. Total abundance Richness Turnover Diversity (Shannon) Evenness (Pielou) -0.25 0.00 0.25 Response D. villosus effect Community metrics Total abundance Richness Turnover Diversity (Shannon) Evenness (Pielou) -0.25 0.00 0.25 Response D. villosus effect Community metrics villosus was lower in streams in comparison to the major European rivers. This result may be partially explained by fast water flow or lower temperature in streams (Allan & Castillo, 2007; Grabowski et al., 2009), but also larger rivers having a greater level of conflu- ence with smaller streams, functioning as shipping canals and there- fore being prone to higher invasion rates. These results could be also affected by differences in sampling effort and the sampling methods among time series, which could have created biases and delays in detecting D. villosus at local and regional scales. 4.3 \| The influence of site- specific characteristics on D. villosus We estimated that the introduction of D. villosus occurred on average 21 years before the first sampling event suggesting current monitor- ing of European streams is insufficient for early detection of invasive alien species. Following their introduction, such species often have low abundance during an initial establishment phase before increas- ing or becoming detected (Crooks et al., 1999), although lag phases are rarely measured in freshwater systems (but see Karatayev et al., 2011). Here, after a considerably long lag phase of 28 years from the time of introduction, D. villosus then only took another seven years to reach peak abundance. A lengthy lag period could reflect non- mutually exclusive phenomena including, inter alia, limits on the or- ganism's reproductive rate in the early phase of exponential growth (e.g. Allee effects); (ii) multiple failed introductions prior to coloniza- tion success; (iii) genotypic selection of locally adapted organisms; and (iv) sudden population growth triggered by disturbance, environ- mental stochasticity or interspecific interactions (Crooks et al., 1999; Understanding how site- specific characteristics influence invasive populations can enable the identification of factors facilitating and limiting their spread. Supporting our third hypothesis, D. villosus populations were influenced by site- specific abiotic characteris- tics, in particular, elevation, distance to the next barrier and climatic variables (average minimum temperature and the trend of maxi- mum temperature). Focusing first on the rate of change of D. villo- sus trends, the distance to the next barrier had a negative effect on D. villosus populations. The reservoirs created by the construction of barriers such as dams are a hotspot for the introduction of inva- sive alien species, due to, for example, recreational fishing activity (Anderson et al., 2014), with D. villosus able to survive for up to three and a half days out of water attached to ropes and other equipment 14724642, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ddi.13649 by EDF Lab Paris Saclay, Wiley Online Library on [06/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are gov 10 SOTO et al. 10 ACKNOWLEDGEMENTS We acknowledge the contribution of Dr. rer. nat. Hanno Seebens, who helped with the extrapolation model of the occurrences of Dikerogammarus villosus. We also thank the Yale Centre for Research Computing for guidance and use of the research computing in- frastructure. D.A.A. is funded by the Kuwait Foundation for the Advancement of Sciences (KFAS) (PR1914SM- 01) and the Gulf University for Science and Technology (GUST) internal seed funds (Case no. 234597 & 253536). R.N.C. acknowledges funding from a Leverhulme Trust Early Career Fellowship (ECF- 2021- 001). P.J.H. and P.H. received funding from the EU Horizon 2020 project eLTER PLUS (Grant Agreement No. 871128). S.D. acknowledges funding by the Leibniz Competition (J45/2018) and support by the German Federal Ministry of Education and Research (BMBF; 033W034A). Open Access funding enabled and organized by Projekt DEAL. The scope of our study was limited by the time series represented in our database. Invasive alien species, such as D. villosus, can affect food webs through either bottom- up or top- down regulation, po- tentially triggering trophic cascades that cause major disturbances in invaded ecosystems (Van Riel et al., 2006). The greater effects of D. villosus following steeper increases in its abundance suggest that the main effects on invaded communities are driven by high population densities, which overwhelm the ecological resistance of the recipient community. Surprisingly, the data from GBIF and our time series do not overlap in some cases, highlighting areas for future abundance survey efforts. Dikerogammarus villosus was first recorded in Italy in 1992 (Seebens et al., 2018), Belgium in 1998 (Seebens et al., 2018), France at the beginning of the 2000s (Devin et al., 2001) and the United Kingdom in 2010 (Bacela- Spychalska et al., 2013; Seebens et al., 2018), but D. villosus was not present in any time series from these countries, perhaps because our data are restricted to lotic systems. Our study was therefore limited by our focus on rivers and streams. Dikerogammarus villosus also occurs in lentic freshwaters including lakes, ponds, and brackish waters, and has marked impacts on these ecosystems (Bacela- Spychalska et al., 2013; Bollache et al., 2004; Minchin et al., 2019). Its oc- currence in other countries could also be underestimated, result- ing in relatively few time series, for example the Netherlands or Switzerland (Altermatt et al., 2014; Bij de Vaate & Klink, 1995). As such, further research would be needed to comprehensively characterize D. Impact of D. villosus on community metrics For invasive alien species, climatic variables – in particular temperature – can be the most important environmental variables determining the survival, reproduction and establishment in recipient ecosystems (Müller & Baur, 2011). Temperature is also well- known for its influence on life cycle char- acteristics such as fecundity in D. villosus (Pöckl et al., 2003). In con- gruence with Kobak et al. (2017), D. villosus preferred warm water and exhibited a stronger tendency to select extreme temperatures. ry.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License However, we stress that changes in community trends cannot be attributed exclusively to the effect of D. villosus, as correlation does not indicate causation, and were likely also altered by other anthropogenic and natural stressors not included in our models, in- cluding changes in water quality, disturbance events or even other invasive alien species (Didham et al., 2005; Haubrock et al., 2020; Pilotto et al., 2020). The combination of invasive alien species and other human impacts can promote the local extirpation of native species, reducing community diversity and driving biotic homogeni- zation (Dormann et al., 2007; Ekroos et al., 2010; McKinney, 2004; McKinney & Lockwood, 1999), but can also promote or prevent in- vasions and/or increases in invader populations (Simberloff & Von Holle, 1999; Beaury et al., 2020). The dominance of D. villosus increased over time. This increase can be explained by propagule/colonization pressures, such as by the species exploiting increasing anthropogenic invasion corridors such as canals (MacIsaac et al., 2001; Lockwood et al., 2005). Reduced abiotic and biotic resistance resulting from degradation of ecosys- tems directly or indirectly by humans could also promote invasion (Hufbauer et al., 2012). The more rapid increase in D. villosus pop- ulation growth at higher elevations nevertheless contradicts other studies that show lower elevation as high bioclimatic suitability for D. villosus (Gallardo et al., 2012), but suggests the species is invad- ing higher elevated regions as a potential response to the ongoing climate change (Pauchard et al., 2016). Lastly, the dominance of D. SOTO et al. 11 11 CONFLICT OF INTEREST The authors declare no competing interests. The authors declare no competing interests. DATA AVAILABILITY STATEMENT The data that support the findings of this study are openly available in a GitHub reposit at https://github.com/IsmaS A/Diker ogamm arus- villo sus- popul ation - dynam ics.git. PEER REVIEW The peer review history for this article is available at https://publo ns.com/publo n/10.1111/ddi.13649. ACKNOWLEDGEMENTS villosus impacts across all European freshwaters. 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Canadian Journal of Fisheries and Aquatic Sciences, 59(7), 1159– 1174. https:// doi.org/10.1139/f02- 098 ORCID ORCID Ismael Soto https://orcid.org/0000-0002-7288-6336 Ross N. Cuthbert https://orcid.org/0000-0003-2770-254X Danish A. Ahmed https://orcid.org/0000-0002-2490-1546 Antonín Kouba https://orcid.org/0000-0001-8118-8612 Sami Domisch https://orcid.org/0000-0002-8127-9335 Giuseppe Amatulli https://orcid.org/0000-0002-8341-2830 Jens Kiesel https://orcid.org/0000-0002-4371-6434 Longzhu Q. Shen https://orcid.org/0000-0001-5629-3007 Margarita Florencio https://orcid.org/0000-0002-6688-7770 Elizabeta Briski https://orcid.org/0000-0003-1896-3860 Florian Altermatt https://orcid.org/0000-0002-4831-6958 Gaït Archambaud- Suard https://orcid.org/0000-0001-9493-2279 Zoltan Csabai https://orcid.org/0000-0003-1700-2574 Mathieu Floury https://orcid.org/0000-0002-4952-5807 Maxence Forcellini https://orcid.org/0000-0003-4921-2189 Patrick Leitner https://orcid.org/0000-0001-8122-4265 Anthony Maire https://orcid.org/0000-0003-0920-773X Anthony Ricciardi https://orcid.org/0000-0003-1492-0054 Ralf B. Schäfer https://orcid.org/0000-0003-3510-1701 Rachel Stubbington https://orcid.org/0000-0001-8475-5109 Gábor Várbíró https://orcid.org/0000-0001-5907-3472 Ralf C. M. Verdonschot https://orcid.org/0000-0002-0977-5975 Peter Haase https://orcid.org/0000-0002-9340-0438 Phillip J. Haubrock https://orcid.org/0000-0003-2154-4341 Overall, our results show that D. villosus is well- established across the vast majority of Europe. Considering that D. villosus has invaded many freshwater and brackish ecosystems beyond those sites covered by the time series we considered in our analyses, it remains impossible to conclude anything about the capacity of D. villosus to expand further in Europe. Nonetheless, our documen- tation of D. villosus highlights the need for greater effort to reduce delays in the detection of invasive alien species to implement man- agement techniques in an early stage of invasion, when such meth- ods can be more effective (Ahmed et al., 2022; Lodge et al., 2016) and less expensive (Cuthbert, Diagne, et al., 2022; Fantle- Lepczyk et al., 2022; Hulme et al., 2009). These measures are especially im- portant in those regions currently uninvaded, such as the North American Great Lakes that can act as “stepping stones” to assist further spread across the continent. Our use of long- term, large- scale time series also emphasizes the importance of long- term data in ecology (Crooks et al., 1999). Further long- term studies are nec- essary to increase our understanding of the population dynamics of D. villosus and other aquatic invaders across the breadth of ecosys- tems, and the context- dependencies that differentiate such dynam- ics, to provide better management information for stakeholders and governments. 14724642, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ddi.13649 by EDF Lab Paris Saclay, Wiley Online Library on [06/11/2022]. See the Terms SOTO et al. 12 REFERENCES Abatzoglou, J. T., Dobrowski, S. Z., Parks, S. A., & Hegewisch, K. C. (2018). 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Water Resources Research, 55(6), 5053– 5073. https://doi.org/10.1029/2019W R024873 Yokomizo, H., Possingham, H. P., Thomas, M. B., & Buckley, Y. M. (2009). Managing the impact of invasive species: The value of knowing the density– impact curve. Ecological Applications, 19(2), 376– 386. https://doi.org/10.1890/08- 0442.1 Zuur, A. F., Ieno, E. N., Walker, N. J., Saveliev, A. A., & Smith, G. M. (2009). Mixed effects models and extensions in ecology with R (Vol. 574). Springer. BIOSKETCH Ismael Soto is a PhD student at the faculty of fisheries and pro- tection of waters at the University of South Bohemia (Czech Republic). His works is focused on the long- term trends and im- pacts of invasive alien species in European inland waters. Author contributions: IS: Methodology, Formal analysis, Data Curation, Writing – Original Draft, Writing – review & editing. RC, DA: Conceptualization, Writing – Original Draft, Writing – review & editing. BIOSKETCH Ismael Soto is a PhD student at the faculty of fisheries and pro- tection of waters at the University of South Bohemia (Czech Republic). His works is focused on the long- term trends and im- pacts of invasive alien species in European inland waters. Author contributions: IS: Methodology, Formal analysis, Data Curation, Writing – Original Draft, Writing – review & editing. RC, DA: Conceptualization, Writing – Original Draft, Writing – review & editing. AK, SD, JM, AB, GA, JK, LS, MF, HL, EB, FA, GA, PB, ZC, TD, MF, MF, JF, PL, ML, AM, AR, RS, RS, GV, GV, RV: Resources, Writing – Review & Editing. PH: Funding acquisi- tion and Project, Writing – Review & Editing Administration. PJH: Conceptualization, Supervision, Writing – original draft; Writing – review & editing.
https://openalex.org/W2014539578	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0004279&type=printable	English	null	On Population Growth Near Protected Areas	PloS one	2,009	cc-by	4,294	Abstract Background: Protected areas are the first, and often only, line of defense in efforts to conserve biodiversity. They might be detrimental or beneficial to rural communities depending on how they alter economic opportunities and access to natural resources. As such, protected areas may attract or repel human settlement. Disproportionate increases in population growth near protected area boundaries may threaten their ability to conserve biodiversity. Methodology/Principal Findings: Using decadal population datasets, we analyze population growth across 45 countries and 304 protected areas. We find no evidence for population growth near protected areas to be greater than growth of rural areas in the same country. Furthermore, we argue that what growth does occur near protected areas likely results from a general expansion of nearby population centers. Conclusions/Significance: Our results contradict those from a recent study by Wittemyer et al., who claim overwhelming evidence for increased human population growth near protected areas. To understand the disagreement, we re-analyzed the protected areas in Wittemyer et al.’s paper. Their results are simply artifacts of mixing two incompatible datasets. Protected areas may experience unusual population pressures near their edges; indeed, individual case studies provide examples. There is no evidence, however, of a general pattern of disproportionate population growth near protected areas. Citation: Joppa LN, Loarie SR, Pimm SL (2009) On Population Growth Near Protected Areas. PLoS ONE 4(1): e4279. doi:10.1371/journal.pone.0004279 Received September 24, 2008; Accepted December 11, 2008; Published January 26, 2009 Received September 24, 2008; Accepted December 11, 2008; Published January 26, 2009 Copyright: 2009 Joppa 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. pa 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. Funding: LNJ is supported by a National Science Foundation Graduate Research Fellowship. SRL is supported by a NASA Earth Systems Sciences Graduate Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: StuartPimm@me.com Competing Interests: The authors have declared that no competing interests exist. Abstract * E-mail: StuartPimm@me.com Whether used sustainably or not, protected areas are often the last remnants of natural resources available to rural communi- ties. Theoretically, the combination of infrastructure, employ- ment, and necessary goods and services could cause protected areas to serve as surrogate urban centers, attracting human settlement and population growth. Such a trend would be a testament to the value associated with ecosystem services, ecotourism, and natural resources for rural economies. This optimism comes at a cost. By encouraging population growth and accelerating the isolation of the protected area from natural landscapes, the net impact of protected areas on conserving biodiversity may be negligible. Lucas N. Joppa1, Scott R. Loarie2, Stuart L. Pimm1* 1 Nicholas School of the Environment, Duke University, Durham, North Carolina, United States of America, 2 Department of Global Ecology, Carnegie Institute, Stanford, California, United States of America PLoS ONE \| www.plosone.org On Population Growth Near Protected Areas Lucas N. Joppa1, Scott R. Loarie2, Stuart L. Pimm1* Citation: Joppa LN, Loarie SR, Pimm SL (2009) On Population Growth Near Protected Areas. PLoS ONE 4(1): e4279. doi:10.1371/jou Results Using methods we employ elsewhere [8], we create a series of 10-km wide buffers inside and outside of our sample of 304 protected areas (the same as those analyzed by Wittemyer et al.) and calculate the population densities within these annuli. This technique is necessary to avoid inherent problems with creating a single buffer, as doing so ignores events immediately outside the buffer. Figures 1a and 1b show these annuli around Kafue National Park (NP) in Zambia. We use Kafue NP as an example because Wittemyer et al. highlighted the area in their study and we have extensive experience working there. It is difficult to convey results for all 304 protected areas in the same manner as Figure 1c. Nevertheless, Figures 2a and 2b provide an adequate summary for all the protected areas in our sample. If people were immigrating to protected area boundaries, population growth 0–10 km away from the boundary would be higher than growth 10–20 km away. (And, by extension 10–20 should be larger than 20–30, 0–20 larger than 20–40, and so on.) Figure 1c plots the population densities against distance from 20 km inside to 50 km outside the boundary of Kafue NP. From these densities, we derive the corresponding annual growth rates Figure 1. Changes in population density in and around Kafue National Park, Zambia, from 1970 to 2000. A, B) Population density around Kafue National Park (established 1971) in 1970 (A), and 2000 (B) expressed as people per 25 km2, the unit of analysis. Parks are outlined in heavy black, and lighter black lines radiating from them represent increasing 10 km intervals. From 1970 to 2000, growth in the buffer zone around Kafue increases because of increasing and multi-directional growth from pre-existing populations outside the 10 km buffer. C) Population densities at 10 km intervals inside and outside of Kafue National Park (left-hand y axis) and annual growth rates (right-hand y axis). We show densities for 1970, 1980, 1990, and 2000. There is no tendency for population densities to increase near the boundary. doi:10.1371/journal.pone.0004279.g001 Figure 1. Changes in population density in and around Kafue National Park, Zambia, from 1970 to 2000. A, B) Population density around Kafue National Park (established 1971) in 1970 (A), and 2000 (B) expressed as people per 25 km2, the unit of analysis. Population and Protected Areas Population and Protected Areas directly. Plots similar to Fig. 1c for all 304 protected areas are available on request. As is clear in Figure 1c, annual growth rates remain virtually unchanged with increasing distance outside of Kafue National Park. High growth rates are sometimes found inside park boundaries (as in Kafue), but here data are very sparse and prone to measurement error. Our results for Kafue NP contradict those of Wittemyer et al.’s. We provide an explanation for this disagreement presently. Introduction Protected areas are often the primary defense against species extinctions and habitat loss [1]. The global network of protected areas now covers more than 12% of the terrestrial earth surface [2]. With rapid population growth, human activity increasingly dominates landscapes surrounding this network [3]. Do protected areas influence human activity near their borders [4]? The answer is critical to assessing the effectiveness of protected areas towards conserving biodiversity as well as the contribution of biodiversity towards rural development. Many argue that protected areas are detrimental to rural development by excluding people from traditional lands and further marginalizing them by denying access to natural resources [5,6]. In that regard, there are concerns as to whether allocating resources away from rural economies, and towards biodiversity, is justified. In contrast, increasing numbers of rural people are moving to cities and towns in search of economic opportunities [7]. This argument would suggest that human activity and population growth near rural protected areas would be below the country average. Such a trend would benefit biodiversity. A recent study across African and Neotropical moist forests [8] found no evidence of increased deforestation near protected area boundaries, lending support for this argument. Wittemyer et al. [12] claim to provide the first consistent evidence supporting this latter argument. The title of their paper, ‘‘Accelerated Human Population Growth at Protected Area Edges’’, is a succinct summary of their results, claiming population growth rates near park boundaries are higher than national rural growth rates. The authors compared growth rates in a single 10 km buffer around each of 306 protected areas [2] in 45 African and South and Central American countries from geographically explicit data [13,14] with a UN-supplied single estimate of the country’s rural growth rate [15]. Using a more spatially explicit approach, we re-analyzed population growth around protected areas. There is no evidence to support disproportionate population growth near protected areas. There are systematic differences between the two indepen- dent datasets Wittemyer et al. used to generate the study’s results and this discrepancy is sufficient to explain their results. Others suggest that protected areas provide benefits for rural communities [9]. Protected areas require infrastructure, such as roads leading to their entrance, and people to work in them. Natural areas also provide many ecosystem services [10,11]. 1 January 2009 \| Volume 4 \| Issue 1 \| e4279 January 2009 \| Volume 4 \| Issue 1 \| e4279 Results Parks are outlined in heavy black, and lighter black lines radiating from them represent increasing 10 km intervals. From 1970 to 2000, growth in the buffer zone around Kafue increases because of increasing and multi-directional growth from pre-existing populations outside the 10 km buffer. C) Population densities at 10 km intervals inside and outside of Kafue National Park (left-hand y axis) and annual growth rates (right-hand y axis). We show densities for 1970, 1980, 1990, and 2000. There is no tendency for population densities to increase near the boundary. doi:10.1371/journal.pone.0004279.g001 January 2009 \| Volume 4 \| Issue 1 \| e4279 PLoS ONE \| www.plosone.org 2 Figure 2. Differences in annual population growth at increasing distances from all 304 parks. A) Annual population growth in 10 km buffer zones minus the annual population growth in 20 km buffers on the x-axis, number of protected areas on the y-axis (304 parks). If 10 km buffers were experiencing accelerated growth, most values would be greater than zero. This is not the case. B) Same as for (A), but comparing 0–20 km buffer zones with 20–40 km buffer zones. Again, there is no evidence for disproportionate population growth near protected area boundaries. doi:10.1371/journal.pone.0004279.g002 Population and Protected Areas Population and Protected Areas Figure 2. Differences in annual population growth at increasing distances from all 304 parks. A) Annual population growth in 10 km buffer zones minus the annual population growth in 20 km buffers on the x-axis, number of protected areas on the y-axis (304 parks). If 10 km buffers were experiencing accelerated growth, most values would be greater than zero. This is not the case. B) Same as for (A), but comparing 0–20 km buffer zones with 20–40 km buffer zones. Again, there is no evidence for disproportionate population growth near protected area boundaries. doi:10.1371/journal.pone.0004279.g002 matters, and again, Kafue NP provides an example (Figures 1a and 1b). If rural protected areas attract human settlement, one might expect isolated population centers to spring up, unassociated with preexisting population centers. This should be obvious through visual inspection. If one assumes that protected areas draw people to them, then population growth of the 0–10 km buffer minus that in the 10– 20 km buffer should be greater than zero. PLoS ONE \| www.plosone.org Results As found for Kafue, and shown for all 304 protected areas in Figures 2a and 2b, there is no tendency for growth rates to be higher adjacent to park boundaries (0–10 km) than further away (10–20 km) in either Africa (mean = 20.0013) or South America (mean = 20.0007). We repeat this analysis for the comparison of 0–20 km and 20– 40 km buffers and find the same result (Figure 2b). Indeed, we have made many such comparisons and always find no differences. Such comparisons refute Wittemyer et al., but match our previous results on land use changes near parks [8]. Instead, what one sees around Kafue NP follows well- understood features of human demography. What growth does occur in buffers is often from the growth of existing population centers incidentally expanding towards protected areas. Figures 1a and 1b show this clearly for Kafue NP, where we map human density in the decade of Kafue NP’s establishment (Figure 1a), and human density in the current decade (Figure 1b). When Kafue NP was established, there were few people living immediately outside the boundary, but several population centers existed at distances greater than 30 km to the northwest and east. Over time, these population centers grew multi-directionally. Kafue NP, which Although we find no evidence that population growth is disproportionate near protected area boundaries, populations are indeed growing. This is inevitable as human population continues to expand worldwide. Here it is the mechanism of the growth that PLoS ONE \| www.plosone.org January 2009 \| Volume 4 \| Issue 1 \| e4279 January 2009 \| Volume 4 \| Issue 1 \| e4279 3 Population and Protected Areas relationship between the two datasets ensures Wittemyer et al.’s results. remained stationary on the landscape, was simply in the way of population expansion. Inspection of many other parks shows this to be a common trend. We then repeated Wittemyer et al.’s analysis, calculating both rural and buffer growth rates from [13,14,16]. Using these derived rural growth rates, we calculated dramatic differences from Wittemyer et al.’s main results. Using incompatible datasets, they found buffer growth to be higher than rural growth for 245 of 306 (80%) parks and 38 of 45 (84%) countries. In Figures 3b and 3c, we show that by using a single dataset those results reduce to 155 of 304 (51%) parks and 24 of 45 countries (53%). Results There are no more parks with higher growth rates near them than parks with lower growth rates (p = 0.774, p = 0.766, respectively; binomial test). Discussion Do protected areas, and their perceived benefits, attract people to them? The question is of utmost importance. Conservation efforts can draw both positive and negative actors to the scene. In a manner similar to locating the last remaining population of an endangered species, the creation and funding of a protected area could potentially cause more harm than ignoring the area altogether [4]. We find no evidence that human population growth near protected area boundaries is higher than in rural areas and show that Wittemyer et al.’s counter-result is methodologically flawed. Using a global map of urban and rural extents, it is possible to mask out areas identified as ‘‘urban’’ in the geographically explicit data [16]. This is ideal, as doing so allows one to calculate rural growth rates from the same dataset used to calculate growth rates near protected areas. A comparison between UN-supplied rural growth rates and those independently derived from consistent datasets shows why Wittemyer et al.’s results could have been no other way. The geographically explicit data are not raw population counts, but predictions from a complex model that, in perhaps indirect and complex ways may include the proximity of a park boundary as a factor. Original population data comes from the level of census unit. The average resolution for all African countries (excluding South Africa) is 82 km2/census unit. Some countries, such as Chad and Angola, have much lower resolutions (303 and 263 km2/census unit, respectively). While individual maps appear highly resolved, much of this cancels out when one divides the two datasets to calculate growth rates. Maps of growth collapse to the coarse level of census unit or lower, calling into question the suitability of these modeled population datasets for fine spatial- scale analyses. As these numbers show, the data are likely too sparse to draw fine-scale conclusions about population growth. In As UN-supplied rural growth rates increase, so too did those from the country’s synoptic data on rural growth [15] (Spearman’s rank correlation: r = 0.501, n = 45, p,0.001). Wittemyer et al. found a similar correspondence (Spearman’s rank correlation: r = 0.501, n = 45, p,0.001), and used this highly significant correlation to justify mixing the two datasets. Unfortunately, what is needed here is not simply a strong correlation but a one-to-one correspondence. Wittemyer et al.’s results are artifacts of comparing incompatible data To understand why Wittemyer et al. found spurious results, we repeated their analysis as best we could. Using their methodology, we closely matched their results and found 253 of 304 (83%) parks with higher growth in the buffers (obtained from one data set) compared to rural growth (from the other). This compares well with Wittemyer et al.’s 245 of 306 (80%) parks. These results are artifacts of mixing two incompatible datasets. Wittemyer et al. calculated population growth rates near protected areas using geographically explicit data [13,14], and compared these rates to a UN-supplied single estimate of the country’s rural growth rate [15]. Unfortunately, the geographically explicit dataset provides consistently higher rural growth rates than does the UN-supplied one. We show this is true by deriving an alternative calculation of rural growth, one originally presented in Wittemyer et al.’s supplemental materials [12]. Discussion Figure 3 shows the strong correlation but also that the geographically explicit data are, with just one exception, higher than the UN-estimates. This Figure 3. Replication and re-analysis of Wittemyer et al.’s methods and results. A) Evidence for the incompatibility of the two datasets used to calculate growth rates in Wittemyer et al.’s study. We plot rural growth rates from [15] on the x-axis, and rural growth rates derived from [13,14,16] on the y-axis. Red points represent South American countries, while black indicates African countries. Almost all (44 of 45) countries are above the 1:1 line, indicating that the datasets used to calculate buffer growth rates provide consistently higher rural growth rates than does the UN dataset. The point below the 1:1 line is Belize, one of only two countries in South and Central America where Wittemyer et al. failed to find a positive result. B) The difference between average growth rates in park buffer zones and rural growth rates for all 45 countries, when we calculate rural growth from the same dataset as buffer growth. C) The same as ‘‘B’’, but this time displaying the results for 304 individual parks. Neither ‘‘B’’ nor ‘‘C’’ is statistically significant (p = 0.766, p = 0.774, respectively). doi:10.1371/journal.pone.0004279.g003 Figure 3. Replication and re-analysis of Wittemyer et al.’s methods and results. A) Evidence for the incompatibility of the two datasets used to calculate growth rates in Wittemyer et al.’s study. We plot rural growth rates from [15] on the x-axis, and rural growth rates derived from [13,14,16] on the y-axis. Red points represent South American countries, while black indicates African countries. Almost all (44 of 45) countries are above the 1:1 line, indicating that the datasets used to calculate buffer growth rates provide consistently higher rural growth rates than does the UN dataset. The point below the 1:1 line is Belize, one of only two countries in South and Central America where Wittemyer et al. failed to find a positive result. B) The difference between average growth rates in park buffer zones and rural growth rates for all 45 countries, when we calculate rural growth from the same dataset as buffer growth. C) The same as ‘‘B’’, but this time displaying the results for 304 individual parks. Neither ‘‘B’’ nor ‘‘C’’ is statistically significant (p = 0.766, p = 0.774, respectively). Discussion doi:10.1371/journal.pone.0004279.g003 January 2009 \| Volume 4 \| Issue 1 \| e4279 PLoS ONE \| www.plosone.org 4 Population and Protected Areas contrast, our analyses of land-use changes are consistently of 1 km2 resolution [8]. areas with no people in the 10 km buffer zone at the time of protected areas establishment, or with urban settlements greater than 1000 people. More generally, the scarcity of suitable datasets poses a challenge for conservation [17] and we note the need for more high-resolution biological and social data. We calculated human population growth rates using decadal modeled population datasets for Africa [13] and South and Central America [14]. To replicate Wittemyer et al.’s results, we obtained country-specific rural growth rates from [15]. To calculate rural growth rates from the decadal population datasets, we masked out all areas identified as ‘‘urban’’ by [16]. Wittemyer et al. provide further details of the analysis in their supplemental materials [12]. When assessed using much finer-scale metrics such as land- cover, we find that protected areas perform admirably [8]. However, efforts to keep protected areas protected must increase as the global network becomes increasingly isolated [3] and ever more in contact with growing human populations. As both the protected area network and human population grow, collisions between these areas and people struggling to find land on which to survive will continue. Kafue NP (Figures 1a and 1b) provides an example of this. Connecting existing protected areas through corridors [18], creating future protected areas in places they can be most effective [1], and effectively managing all protected lands will be essential to ensure the future of biodiversity. We then created 10 km wide annuli in and around each protected area, from 20 km inside the protected area to 50 km outside. Using the decadal datasets, we were able to calculate growth rates at ten-year intervals for each of the annuli. We obtained the annual growth rate by dividing the total growth rate by the number of years the analysis encompassed. In order to summarize these results, for each protected area we then divided the growth rate in the 0–10 km buffer by the growth rate in the 10–20 km buffer. Values greater than one indicate higher population growth near protected areas than away. Methods All datasets are global in scale, in raster (grid) format, and projected into Albers Equal Area projection at a resolution of 5km grid square (25 km2). We used ArcGIS 9.1 to harmonize projections, cell size, and extent across datasets. We carried out all further analyses in R 2.6. We obtained information on park location from the 2007 World Database on Protected Areas [2]. The 304 protected areas in our analysis are a sample of the 306 protected areas included in the analysis by Wittemyer et al. A full description of the criteria used to choose the sample of protected areas can be found in their supplemental materials [12], but in brief Wittemyer et al. only chose areas greater than 10 km2, established before 1995, not on oceanic islands, and IUCN category I or II (non-consumptive use categories) or World Heritage Sites. They also excluded protected References 11. Daily G (1997) Nature’s services: Societal dependence on natural ecosystems. Chicago: University of Chicago Press: 412 p. 1. Pimm SL, Ayres M, Balmford A, Branch G, Brandon K, et al. (2001) Can we defy Nature’s end? Science 233: 2207–2208. 2. UNEP World Conservation and Monitoring Center & IUCN. World database on protected areas (IUCN-WCPA/UNEP-WCMC, Washington DC, 2003). Available at http://www.unep-wcmc.org/wdpa/ (Sept. 2006). 12. Wittemyer G, Elsen P, Bean W, Burton ACO, Brashares J (2008) Accelerated human population growth at protected area edges. Science 321: 123–126. 13. UNEP & Center for International Earth Science Information Network (CIESIN). Africa Population Distribution Database (UN Environment Pro- gramme GRID Sioux Falls, 2004). Available at http://www.na.unep.net/ datasets/datalist.php (July 2008). p p g p ( p ) 3. DeFries R, Hansen H, Turner BL, Reid R, Liu J. (2007) Land use change around protected areas: Management to balance human needs and ecological function. Ecol Appl 17: 974–988. 4. Liu J, Linderman M, Ouyang Z, An L, Yang J, et al. (2001) Ecological degradation in protected areas: The case of Wolong Nature Reserve for Giant Pandas. Science 292: 98–101. 14. UNEP International Center for Tropical Agriculture & World Resource Institute. Latin America Population Distribution Database (UN Environment Programme GRID Sioux Falls, 2004). Available at http://www.na.unep.net/ datasets/datalist.php (July 2008). 5. Adams WM, Aveling R, Brockington D, Dickson B, Elliot J, et al. (2004) Biodiversity conservation and the eradication of poverty. Science 306: 1146. 15. UN Population Division. World Urbanization Prospects: The 2007 revision population database. (UNPD, New York, NY, 2007). Available at http://esa.un. org/unup (July 2008). 6. Peluso NL (1993) Coercing Conservation? The politics of state resource control. Glob Environ Change 3: 199–217. 7. Myers N, Kent J (2004) The new consumers: The influence of affluence on the environment. Chicago: Island Press: 199 p. 16. Center for International Earth Science Information Network (CIESIN) et al. Global Rural-Urban Mapping Project (GRUMP), Alpha Version: Urban Extent. Palisades, NY: Socioeconomic Data and Applications Center (SEDAC), Columbia University. Available at http://sedac.ciesin.columbia.edu/gpw. (July 2008). 8. Joppa LN, Loarie SR, Pimm SL (2008) On Proc Natl Acad Sci U S A 105: 6673–6678 8. Joppa LN, Loarie SR, Pimm SL (2008) On the protection of ‘‘protected areas’’. Proc Natl Acad Sci U S A 105: 6673–6678. Proc Natl Acad Sci U S A 105: 6673–6678. 9. Scherl LM, Wilson A, Wild R, Blockhus J, Franks P, et al. Author Contributions Conceived and designed the experiments: LNJ SRL SLP. Performed the experiments: LNJ. Analyzed the data: LNJ SRL. Wrote the paper: LNJ SRL SLP. Conceived and designed the experiments: LNJ SRL SLP. Performed the experiments: LNJ. Analyzed the data: LNJ SRL. Wrote the paper: LNJ SRL SLP. Discussion When repeating Wittemyer et al.’s analysis, we followed their methodol- ogy of calculating population growth inside the 0–10 km buffer using the decadal datasets [13,14] and subtracting from that the UN-supplied rural growth rate of the country [15]. Positive values indicate protected areas with higher human population growth than rural areas of the same country. 7. Myers N, Kent J (2004) The new consumers: The influence of affluence on the environment. Chicago: Island Press: 199 p. References (2004) Can protected areas contribute to poverty reduction? Opportunities and limitations. Gland, Switzerland: IUCN: 72 p. 17. Loarie SR, Joppa LN, Pimm SL (2007) Satellites miss environmental priorities. Trends Ecol Evol 22: 630–632. 18. Anderson A, Jenkins C (2006) Applying nature’s design: Corridors as a Strategy for biodiversity conservation. New York: Columbia University Press: 231 p. 10. Constanza R, d’Arge R, Groot R, Farber S, Grasso M (1997) The value of the world’s ecosystem services and natural capital. Nature 387: 253–260. PLoS ONE \| www.plosone.org January 2009 \| Volume 4 \| Issue 1 \| e4279 5 5
https://openalex.org/W2112842296	https://hal.archives-ouvertes.fr/hal-01454159/document	English	null	Staphylococcus aureus seroproteomes discriminate ruminant isolates causing mild or severe mastitis	Veterinary research	2,011	cc-by	17,026	Staphylococcus aureus seroproteomes discriminat ruminant isolates causing mild or severe mastitis Caroline Le Maréchal, Julien Jardin, Gwénaël Jan, Sergine Even, Coralie Pulido, Jean-Michel Guibert, David Hernandez, Patrice François, Jacques Schrenzel, Dieter Demon, et al. To cite this version: Caroline Le Maréchal, Julien Jardin, Gwénaël Jan, Sergine Even, Coralie Pulido, et al.. Staphylococ- cus aureus seroproteomes discriminate ruminant isolates causing mild or severe mastitis. Veterinary Research, 2011, 42 (35), pp.1-20. 10.1186/1297-9716-42-35. hal-01454159 Staphylococcus aureus seroproteomes discriminate ruminant isolates causing mild or severe mastitis Distributed under a Creative Commons Attribution 4.0 International License © 2011 Le Maréchal et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Staphylococcus aureus is a major cause of mastitis in ruminants. In ewe mastitis, symptoms range from subclinical to gangrenous mastitis. S. aureus factors or host-factors contributing to the different outcomes are not completely elucidated. In this study, experimental mastitis was induced on primiparous ewes using two S. aureus strains, isolated from gangrenous (strain O11) or subclinical (strain O46) mastitis. Strains induced drastically distinct clinical symptoms when tested in ewe and mice experimental mastitis. Notably, they reproduced mild (O46) or severe (O11) mastitis in ewes. Ewe sera were used to identify staphylococcal immunoreactive proteins commonly or differentially produced during infections of variable severity and to define core and accessory seroproteomes. Such SERological Proteome Analysis (SERPA) allowed the identification of 89 immunoreactive proteins, of which only 52 (58.4%) were previously identified as immunogenic proteins in other staphylococcal infections. Among the 89 proteins identified, 74 appear to constitute the core seroproteome. Among the 15 remaining proteins defining the accessory seroproteome, 12 were specific for strain O11, 3 were specific for O46. Distribution of one protein specific for each mastitis severity was investigated in ten other strains isolated from subclinical or clinical mastitis. We report here for the first time the identification of staphylococcal immunogenic proteins common or specific to S. aureus strains responsible for mild or severe mastitis. These findings open avenues in S. aureus mastitis studies as some of these proteins, expressed in vivo, are likely to account for the success of S. aureus as a pathogen of the ruminant mammary gland. S. aureus mastitis in dairy sheep ranges from subclinical mastitis to lethal gangrenous mastitis. Such variability relies on staphylococcal virulence factors as well as host factors. Until now, no study has been performed to identify the transcripts and proteins commonly or specifically produced in vivo by S. aureus strains during mastitis. To obtain such information using direct tran- scriptomic or proteomic approaches upon S. aureus samples collected within the infection site stumbles on technical bottlenecks such as the low amounts of S. aureus cells and the difficulty to localize the infection site within the udder. Staphylococcus aureus seroproteomes discriminate ruminant isolates causing mild or severe mastitis Caroline Le Maréchal1,2,3, Julien Jardin1,2, Gwenaël Jan1,2, Sergine Even1,2, Coralie Pulido3, Jean-Michel Guibert3, David Hernandez4, Patrice François4, Jacques Schrenzel4, Dieter Demon5, Evelyne Meyer5, Nadia Berkova1,2, Richard Thiéry3, Eric Vautor3,6†, Yves Le Loir1,2† Correspondence: Yves.LeLoir@rennes.inra.fr † Contributed equally 1INRA, UMR1253 Science et Technologie du Lait et de l’Œuf, F-35042 Rennes, France Full list of author information is available at the end of the article RESEARCH Open Access Staphylococcus aureus seroproteomes discriminate ruminant isolates causing mild or severe mastitis HAL Id: hal-01454159 https://hal.science/hal-01454159v1 Submitted on 29 May 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Le Maréchal et al. Veterinary Research 2011, 42:35 http://www.veterinaryresearch.org/content/42/1/35 Introduction S. aureus mastitis in dairy sheep ranges from subclinical mastitis to lethal gangrenous mastitis. Such variability relies on staphylococcal virulence factors as well as host factors. Until now, no study has been performed to identify the transcripts and proteins commonly or specifically produced in vivo by S. aureus strains during mastitis. To obtain such information using direct tran- scriptomic or proteomic approaches upon S. aureus samples collected within the infection site stumbles on technical bottlenecks such as the low amounts of S. aureus cells and the difficulty to localize the infection site within the udder. Mastitis is the first cause of economical loss in milk production worldwide [1] and is a major concern in milk transformation [2]. The problem is however cur- rently hard to tackle for mastitis in dairy cows, sheep and goats. Especially, S. aureus mastitis is typically refractory to antibiotic treatment. Prophylactic mea- sures, including the development of an effective vaccine, have so far proven unsuccessful for the control of the disease. S. aureus is well-known to produce a large vari- ety of virulence factors (including numerous proteins like toxins or adhesins). Consequently, it induces a large panel of infections, and the clinical acuteness of each infection type may also be variable. For example, Serological proteome analysis (SERPA) is a promising technique that can be used to shed light on the host’s immune response to staphylococcal infection. This tech- nique was used to mine new antigen candidates for vac- cine development in human infections [3]. SERPA has also been used to identify proteins produced in vivo, * Correspondence: Yves.LeLoir@rennes.inra.fr † Contributed equally 1INRA, UMR1253 Science et Technologie du Lait et de l’Œuf, F-35042 Rennes, France Full list of author information is available at the end of the article ll list of author information is available at the end of the article Le Maréchal et al. Veterinary Research 2011, 42:35 http://www.veterinaryresearch.org/content/42/1/35 Page 2 of 20 lineage found associated to ewe mastitis in south east of France [6,7]. Growth conditions and preparation of pro- tein extracts were as described in Le Maréchal et al. 2009 [11]. Briefly, overnight cultures in BHI were diluted 1:1000 in fresh RPMI 1640 medium (Sigma, Saint Quen- tin Fallavier, France). RPMI was extemporaneously depleted of iron (and hereafter referred to as iron- depleted RPMI) by adding deferoxamine (0.15 mM) (Sigma). Genome sequencing of O11 and O46 strains q g To facilitate the analysis of SERPA results, the genome of the two strains used in experimental infection was fully sequenced using the Solexa technology (P Mayer, L Fari- nelli, and E Kawashima, 1997. Patent application WO98/ 44151) according to the manufacturer’s protocol (Illu- mina, San Diego, CA, USA). Briefly, genomic DNA was physically fragmented by nebulization into 50- to 500-bp fragments. After end repair and ligation of the bar-coded paired-end adaptors, the products were purified on agar- ose gel to recover products with inserts of ~200 bp. Quality control was performed by cloning an aliquot of the library into a TOPO plasmid and capillary sequen- cing eight clones per library. The samples were then used to generate DNA colonies using one channel of a paired- end flow cell at dilutions of 4 pM. The flow cell was then submitted to 2 × 74 cycles of sequencing on the genome analyzer. Base calling was performed using the GAPipe- line 1.4.0 software; a total of 27.6 million reads (pass fil- ter) were obtained. After bar code selection, 13.9 and 11.8 million reads of 71 bases in length were obtained for the strains O11 and O46 respectively. The pool of sequences obtained was analyzed and assembled using the Edena assembler [13], which resulted in a set of 87 and 96 contigs for O11 and O46, respectively. The gene content of each strain (2787 and 2822 Coding Sequences -CDSs- for O11 and O46, respectively) was thus estab- lished and used in protein identification after SERPA. Detailed genome analysis of these strains is described elsewhere (Le Maréchal et al., submitted). Introduction Growth conditions in which there is restriction in the bioavailability of iron can indeed lead to an increase of the expression of virulence factors which are normally expressed in vivo [12]. S. aureus strains were grown in 500 mL flasks under agitation (150 rpm) at 37 °C (a flask-to-broth volumetric ratio of 5), for aerobic conditions, or in falcon tubes (50 mL) completely filled with medium and incubated at 37 °C without agitation for anaerobic conditions. Protein samples for superna- tant, cell wall or total fraction were prepared exactly as previously described [11]. during infection [4]. In combination with whole genome shotgun sequencing, SERPA is a powerful tool to iden- tify immunoreactive proteins produced by S. aureus dur- ing the infection [5]. Genotyping studies indicated that S. aureus strains isolated from dairy sheep farms in the south east of France were clonally related and are predominantly represented by a single pulse-field gel electrophoresis (PFGE) type OV/OV’ [6,7]. Such close phylogenetic rela- tionship was recently confirmed at the global scale using Multi Locus Sequence Typing and comparative genome hybridization [8,9]. Among strains in this OV/OV’ PFGE profile, one was isolated from subclinical mastitis (O46) and another one from gangrenous mastitis (O11). Few genetic differences were identified [10] and their role in the development of mastitis with such different severity has not yet been determined. Moreover identification of the proteins produced by S. aureus in mastitis with dif- ferent severity is an important step to better understand the host-pathogen interactions and to provide targets for the development of efficient prevention or treatment strategies against mastitis. Therefore, we applied SERPA to identify proteins that are produced by both S. aureus strains O11 and O46 (core seroproteome) and the ones specifically produced by each strain (“accessory” seroproteome). : O11 and O46 were used for experimental infections and their genome was fully sequenced (see Materials and Methods). Bacterial strains, growth conditions and preparation of protein samples S. aureus strains used in this study are presented in Table 1. S. aureus O46 was isolated from a case of ovine subcli- nical mastitis and O11 from a gangrenous lethal mastitis [10]. Genetic and genotypic background of S. aureus O46 and O11 are well-documented. They share the same pul- sotype (OV/OV’) and are representative of the major Table 1 Staphylococcus aureus strains used in this study Strain Type of mastitis Origin Isolated in: O11 gangrenous south east France 2002 1628 gangrenous south east France 2010 1624 clinical south east France 2003 1625 clinical south east France 2008 1626 clinical south east France 2008 1536 clinical south west France 1998 O46* subclinical south east France 2002 1627 subclinical south east France 2008 O55 subclinical south east France 2003 O117 subclinical Corsica. France 2001 1535 subclinical south west France 1998 O82 subclinical south east France 2003 *: O11 and O46 were used for experimental infections and their genome was fully sequenced (see Materials and Methods). Table 1 Staphylococcus aureus strains used in this study Strain Type of mastitis Origin Isolated in: 2-Dimensional Electrophoresis (2-DE) Samples (200 μg of proteins for Coomassie blue staining and 50 μg for Western blotting) were precipitated with 2D Le Maréchal et al. Veterinary Research 2011, 42:35 http://www.veterinaryresearch.org/content/42/1/35 Page 3 of 20 clean up kit (GE Healthcare, Orsay, France) according to the manufacturer’s instructions. Pellets were solubilised in sample solution containing 7 M urea, 2 M thio-urea, 25 mM dithiothreitol (DTT), 4% (w/v) 3-[(3-Cholamido- propyl)dimethylammonio]-1-propane-sulfonate (CHAPS) and 2% (w/v) ampholyte containing buffer (IPG-Buffer 4-7 or 3-10 NL, GE Healthcare). Isoelectric focusing was car- ried out using pH 4 to 7 (Cell wall and total proteins) or 3 to 10 NL (exoproteins) 13 cm Immobiline Dry Strips on a Multiphor II electrophoresis system (Amersham Bios- ciences; GE Healthcare, Orsay, France) for a total of 60 kVh using a standard procedure described previously [14]. The second dimensional separation was performed on the Ettan™DALTtwelve electrophoresis system (GE Health- care) using 14% acrylamide separating gels without a stacking gel at a voltage of 50 V for 1 h and 180 V for about 7 h. Kaleidoscope Prestained Standards (Biorad) were used as standard. Gels were transferred onto mem- brane or stained with R250 Coomassie blue (Serva, Heil- delberg, Germany) or MS-compatible silver nitrate (Sigma) [15]. D0, D7, D14, D21 and D28 post inoculation (pi). Briefly, blood samples were kept for 2 h at room temperature before centrifugation. Sera were then stored at -20°C. Milk samples were taken 24 and 36 h pi for bacteriolo- gical examination and determination of SCC. Milk was 1/10 diluted and 100 μL of this dilution was plated on selective Rabbit Plasma Fibrinogen Baird-Parker medium to confirm S. aureus presence in the mammary gland. SCC was measured with the Fossomatic method [16] to follow the onset of the infection. clean up kit (GE Healthcare, Orsay, France) according to the manufacturer’s instructions. Pellets were solubilised in sample solution containing 7 M urea, 2 M thio-urea, 25 mM dithiothreitol (DTT), 4% (w/v) 3-[(3-Cholamido- propyl)dimethylammonio]-1-propane-sulfonate (CHAPS) and 2% (w/v) ampholyte containing buffer (IPG-Buffer 4-7 or 3-10 NL, GE Healthcare). Isoelectric focusing was car- ried out using pH 4 to 7 (Cell wall and total proteins) or 3 to 10 NL (exoproteins) 13 cm Immobiline Dry Strips on a Multiphor II electrophoresis system (Amersham Bios- ciences; GE Healthcare, Orsay, France) for a total of 60 kVh using a standard procedure described previously [14]. 2-Dimensional Electrophoresis (2-DE) The second dimensional separation was performed on the Ettan™DALTtwelve electrophoresis system (GE Health- care) using 14% acrylamide separating gels without a stacking gel at a voltage of 50 V for 1 h and 180 V for about 7 h. Kaleidoscope Prestained Standards (Biorad) were used as standard. Gels were transferred onto mem- brane or stained with R250 Coomassie blue (Serva, Heil- delberg, Germany) or MS-compatible silver nitrate (Sigma) [15]. D0, D7, D14, D21 and D28 post inoculation (pi). Briefly, blood samples were kept for 2 h at room temperature before centrifugation. Sera were then stored at -20°C. Milk samples were taken 24 and 36 h pi for bacteriolo- gical examination and determination of SCC. Milk was 1/10 diluted and 100 μL of this dilution was plated on selective Rabbit Plasma Fibrinogen Baird-Parker medium to confirm S. aureus presence in the mammary gland. SCC was measured with the Fossomatic method [16] to follow the onset of the infection. Western blot analysis Total cell lysates were prepared as previously described [11]. Total protein extracts of S. aureus strains O11 and O46 were separated by SDS-PAGE on 12% acrylamide separating slab gels (70 × 100 × 0.5 mm), with a 4% acry- lamide stacking gel on a mini-protean III gel system (BioRad, Ivry sur Seine, France) according to Laemmli [17]. Protein migration was performed for 2 h at room temperature at constant 80 V voltage. Samples were diluted in sample buffer and denatured at 100 °C for 3 min. Gels were transferred onto a PVDF membrane (GE Healthcare) at constant 250 mA amperage in Tow- bin transfer buffer [18] using a Trans-Blot cell (Biorad) for 1.25 h. Membranes were washed three times with Tris Buffered Saline (TBS) at pH 7.5 and saturated in blocking solution (3% non-fat dry milk in TBS with 0.3% Tween 20 (TBS-T)) at 4 °C overnight. After saturation in blocking solution, membranes were washed 3 × 10 min with TBS-T and exposed to the different ewe sera used as primary antibody for 4 h at room temperature. After washing, membranes were incubated with alkaline phos- phatase conjugated anti-sheep IgG (Sigma) diluted 1:15,000 in 25 mL blocking solution for 1 h and finally BCIP/NBT (Sigma) was used to visualize immunoreactive proteins, according to the manufacturer’s instructions. Selection of the hyper-immune sera Se ect o o t e ype u e se a Sera samples were analysed by western blotting as described above. Sera sampled on D0, D7, D14, D21 and D28 pi were compared using the mini-protean II Multi- screen apparatus (Biorad) (600 μL of serum diluted 1:10,000 in blocking solution). Immunostained Western blots were scanned using an Image Scanner II (Amersham biosciences) and further analyzed using Image- Quant 1D software. The number, volume and area of bands were taken into account for the analysis. Optimal dilution for the selected sera was determined as described above. Sera and dilutions yielding the best ratio signal/background were selected. Identification of immunoreactive proteins Intramammary challenge with S. aureus in ewes Experimental mastitis was performed according to the Regional Committee for Animal Use and Care (Côte d’Azur, France) and is recorded under reference NCA/ 2008-14/12-09. Healthy lactating primiparous ewes of Lacaune breeds were selected based on the absence of intramammary infections and milk somatic cell counts below 100 000 cells/mL. Repeated full bacteriological analysis of milk from the two quarters that were going to be infected showed that the ewes were negative for Staphylococcus sp. and Mycoplasma sp. Absence of nasal carriage for staphylococci was also checked after enrichment and culturing of swab samples of the nares of ewes on selective media, as described previously [6]. At D0, 12 ewes were divided into 2 groups and urethral catheters (Portex® Jackson Cat Catheter, Coveto, France) were inserted into the teat canal after a thor- ough disinfection of the teat orifice with 70% ethanol. 1 mL PBS containing 20 CFU of S. aureus (O11 or O46) was injected through the catheter, which was removed afterwards. Six ewes were thus infected by strain O11 (group O11) and six by strain O46 (group O46). Severity of the mastitis induced in ewes was esti- mated according to criteria presented in Additional file 1, Table S1. Mastitis was classified as subclinical, clini- cal, pyogenic and gangrenous. Classification was based on clinical symptoms, presence of S. aureus cells and Somatic Cell Count (SCC) in milk. Sample processing Bacterial proteins separated by 2-DE were transferred onto a PVDF membrane (GE Healthcare) as described Sera from ewes were prepared from blood samples col- lected aseptically from the jugular vein of the animals at Le Maréchal et al. Veterinary Research 2011, 42:35 http://www.veterinaryresearch.org/content/42/1/35 Page 4 of 20 gradient from 10 to 50% of solvent B in 40 min was applied for the elution at a flow rate of 0.3 μL/min. Eluted peptides were directly electrosprayed into the mass spectrometer operated in positive mode. A full continuous MS scan was carried out followed by three data-dependent MS/MS scans. Spectra were collected in the selected mass range 400 to 2 000 m/z for MS and 60 to 2 000 m/z for MS/MS spectra. The three most intense ions from the MS scan were selected individually for collision-induced dissociation (1+ to 4+ charged ions were considered for the MS/MS analysis). The mass spectrometer was operated in data-dependent mode automatically switching between MS and MS/MS acqui- sition using Analyst QS 1.1 software. The instrument was calibrated by multipoint calibration using fragment ions that resulted from the collision-induced decomposi- tion of a peptide from b-casein, b-CN (193-209). The proteins present in the samples were identified from MS and MS/MS data by using MASCOT v.2.2 software for search into two concatenated databases: (i) a homemade database containing all the predicted proteins of the S. aureus strains O11 and O46 used in this study and (ii) a portion of the UniProtKB database corresponding to the S. aureus taxonomic group [20]. Search parameters were set as follows. A trypsin enzyme cleavage was used, the peptide mass tolerance was set to 0.2 Da for both MS and MS/MS spectra, and two variable modifications (oxidation of methionine and deamidation of asparagine and glutamine residues) were selected. For each protein identified in NanoLC-ESI-MS/MS, a minimum of four peptides with MASCOT score corresponding to a P value below 0.05 or an Exponentially Modified Protein Abundance Index [21] greater than 0.4 were necessary for validation with a high degree of confidence. For automatic validation of the peptides from MASCOT search results, the 1.19.2 version of the IRMa software was used [22]. above. Series of four gels were migrated and treated in parallel. Three gels were used for immunoblotting, and the fourth one was Coomassie blue-stained for spot matching and further identification. Sample processing After saturation in blocking solution the membranes were treated with selected sera in blocking solution during 4 h. Then, the membranes were washed with TBS-T and incubated with alkaline phosphatase conjugated anti-sheep IgG (Sigma) diluted 1:15 000 in 25 mL blocking solution for 1 h. Finally BCIP/NBT (Sigma) was used to visualize immunoreactive proteins, according to the manufac- turer’s instructions. Membranes were scanned using an Image Scanner II (Amersham biosciences) and further analyzed using Image- Master 2D software. Immunoblot profiles for 2-DE-separated proteins were reproducible in at least two individual experiments. Images of the 2D electrophoresis gels and the BCIP-NBT treated mem- branes were compared to detect immunoreactive pro- teins. Spots that were absent or had a significantly different intensity in one strain were considered as pro- teins that differed between O11 and O46. Spots corre- sponding to proteins of interest were excised and identified using Nano-Liquid Chromatography (Nano- LC) MS/MS analysis. Nano-LC MS/MS analysis y Proteins were identified by tandem mass spectrometry (MS/MS) after an in-gel trypsin digestion adapted from Shevchenko [19]. Briefly, gel pieces were excised from the gel, washed with acetonitrile and ammonium bicar- bonate solution, and then dried under vacuum in a SpeedVac concentrator (SVC100H-200; Savant, Thermo Fisher Scientific, Waltham, MA, USA). In-gel trypsin digestion was performed overnight at 37 °C and stopped with spectrophotometric-grade trifluoroacetic acid (TFA) (Sigma-Aldrich). The supernatants containing peptides were then vacuum dried in a Speed-Vac con- centrator and stored at -20 °C until mass spectrometry analysis. Nano-LC experiments were performed using an on-line liquid chromatography tandem mass spectrome- try (MS/MS) setup using a Dionex U3000-RSLC nano- LC system fitted to a QSTAR XL (MDS SCIEX, Ontario, Canada) equipped with a nano-electrospray ion source (ESI) (Proxeon Biosystems A/S, Odense, Denmark). Samples were first concentrated on a PepMap 100 reverse-phase column (C18, 5 μm, 300-μm inner dia- meter (i.d.) by 5 mm length) (Dionex, Amsterdam, The Netherlands). Peptides were separated on a reverse- phase PepMap column (C18, 3 μm, 75 μm i.d. by 150 mm length) (Dionex) at 35 °C, using solvent A (2% (vol/ vol) acetonitrile, 0.08% (vol/vol) formic acid, and 0.01% (vol/vol) TFA in deionized water) and solvent B (95% (vol/vol) acetonitrile, 0.08% (vol/vol) formic acid, and 0.01% (vol/vol) TFA in deionized water). A linear Intramammary infection with S. aureus in mice Th i l d d d di Intramammary infection with S. aureus in mice The animal study was conducted according to current Good Scientific Practice-principles (2000) and approved by the Ethical Committee of the Faculty of Veterinary Medicine, Ghent University (Belgium). Sixteen CD-1 lactating female mice (Harlan Laboratories Inc., Horst, The Netherlands) were used 12-14 days after birth of the offspring. The pups were removed 1 to 2 h before bacterial inoculation of mammary glands and a mixture of ketamine/xylazine was used for anesthesia of the lac- tating mice. The orifice of both L4 (on the left) and R4 (on the right) abdominal mammary glands was exposed by a small cut at the near end of the teat. 100 μL PBS without (n = 2) or with 150 CFU of S. aureus strain O11 (n = 7) or O46 (n = 7) was injected slowly with a 32-gauge blunt needle through the teat canal. Rectal Le Maréchal et al. Veterinary Research 2011, 42:35 http://www.veterinaryresearch.org/content/42/1/35 Page 5 of 20 Le Maréchal et al. Veterinary Research 2011, 42:35 http://www.veterinaryresearch.org/content/42/1/35 mastitis (n = 2), mild clinical mastitis (n = 2) and gang- renous mastitis (n = 1). Subclinical mastitis was deter- mined with the presence of bacteria (250 CFU/mL of milk 36 h pi), a raise in SCC (> 200 000 cells/mL in each milk sample after 24 h) and absence of fever or symptoms. Except for the ewe with subclinical mastitis, bacteria were detected in all milk samples and reached more than 106 CFU/mL 36 h pi. All animals had fever (above 40 °C 36 h pi) and SCC increased quickly and was above 106 cells/mL at 36 h pi. The proportion of the gangrenous mastitis was significantly higher in the group of ewes infected with O11 strain compared to the group infected with O46 (p = 0.08) (Additional file 2, Figure S1). body temperature of the mice was measured at 0 h and 18 h pi. At 18 h pi mice were anesthetized with keta- mine/xylazine to collect blood by cardiac puncture and serum was obtained after clotting at 37 °C and cold cen- trifugation. After cardiac puncture, mice were eutha- nized by cervical dislocation and mammary glands were isolated. Glands of six mice of each group were homo- genated and bacterial CFU was quantified by plating serial logarithmic dilutions in PBS. Lysates from the homogenates were prepared in 1% NP40-based buffer. Ewes infected with O11 or O46 S. aureus strains developed mastitis with different severities Ewes infected with O11 or O46 S. aureus strains developed mastitis with different severities p Although O11 and O46 strains share the same genotype and are highly genetically similar [10], they were isolated from dramatically different ewe mastitis episodes. One can thus wonder whether the clinical signs associated with O11 and O46 infection were or not related to strains characteristics or a fortuitous matter of sampling time (mastitis can indeed evolve from subclinical to severe clinical or even gangrenous within a few days). To check this, two groups of ewes were infected either with O11 or O46 S. aureus strains, as described in the previous section. Onset of the symptoms was followed up during the course of the experiment. All animals became infected and signs of mastitis were evident in most ewes as soon as 24 h pi. The animals shed the S. aureus strains over the sampling period and remained infected for the duration of the experiment. Shedding from the infected glands varied and S. aureus load in milk ranged from 10 CFU/mL to 3.16 × 108 CFU/mL, depending on the individual ewe, and on the day pi (not shown). Symptoms evoked by intramammary inocula- tion varied among ewes. In group O11, five out of six ewes developed a gangrenous mastitis, the last one developed a pyogenic mastitis according the criteria defined in Additional file 1, Table S1. In group O46, symptoms were more heterogeneous and mastitis cases were classified in subclinical mastitis (n = 1), pyogenic Statistical analyses A Fisher test was used with a risk a = 10% to determine the difference between the ewes infected with S. aureus O11 and the ewes infected with strain O46. Differences in rectal body temperature and cytokine levels in the mouse mastitis model were analyzed with the unpaired T-test. P < 0.05 was considered statistically significant. Intramammary infection with S. aureus in mice Th i l d d d di Serum and mammary gland lysates were quantified for IL-1b, IL-6, TNF, KC and MCP-1 using BD™Cyto- metric Bead Array technology. Mammary glands (inocu- lated and PBS control glands) of 4 mice (2 from each group) were embedded and used for further histopatho- logical analysis (Vetopath, Antibes, France). g S. aureus O11 and O46 induce dramatically different clinical features in infected mice. To confirm our obser- vation that S. aureus strains O11 or O46 induce differ- ent types of mastitis, the mouse mastitis model was employed in the current study. Strains O11 and O46 grew equally well in the infected mouse mammary glands and induced mastitis, as determined by tempera- ture measurement (hypothermia in O11 group and hyperthermia in O46 group, 24 h pi; Additional file 3, Figure S2) and histopathological analysis: polymorpho- nuclear neutrophils (PMN) infiltration was observed only in infected (either with O11 or O46 strains) mam- mary gland tissue (not shown). To analyse the role of each strain in the development of mastitis, cytokine pro- file of the serum and mammary gland tissue lysates of mice infected with O11 strain was compared to those infected with O46 strain. The results of cytokine quanti- fication showed that mice infected with S. aureus O46 had significantly higher IL-1b and TNF levels in the mammary gland lysates and significantly higher systemic (serum) levels of IL-1b and MCP-1 (Additional files 4 and 5, Figures S3 and S4). Altogether, these results demonstrate that despite their close genetic relation- ships, S. aureus O11 and O46 reproducibly induced mastitis with significantly different clinical signs. Detection and identification of immunogenic staphylococcal proteins by SERPA Protein samples were prepared from O11 and O46 strains grown in conditions that best mimic the mastitis context [11]. Each fraction (total, cell wall and superna- tant) of O11 and O46 culture was immunoblotted using either group O11 or group O46 sera. Altogether, 89 pro- teins were identified as immunoreactive (Table 2). Com- parison of SERPA results (see Figure 2, and Additional files 6 and 7, Figures S5 and S6) on the three fractions analyzed showed that immunoreactive proteins were mainly identified in supernatant samples prepared from aerobic and anaerobic cultures (Figure 2 and Additional file 7, Figure S6) and cell wall samples (Additional file 6, Figure S5, upper panels) whereas total protein samples were poorly recognized (Additional file 6, Figure S5, lower panels). A vast majority of the immunoreactive Composition of the core and accessory seroproteomes SERPA revealed 74 proteins as being recognised by both group O11 and group O46 sera (Table 2). These proteins defined the core seroproteome, i.e. a pool of staphylococ- cal proteins that are recognized by the host immune sys- tem in both S. aureus strains (Figure 3C). Moreover, 15 proteins were differentially recognized by group O11 sera or by group O46 sera. These proteins defined the acces- sory seroproteome, i.e. a pool of strain-specific proteins that are recognized by the host immune system. Among these 15 proteins, 12 appeared to be immunogenic only in infections with O11 (Table 2). These proteins include 7 virulence-associated proteins (Sbi, SspB, SspA, Aur, IsdH, Opp1A, and VWbp), 2 stress response proteins (AhpF, TrxB), 1 hypothetical protein (product of CDS O11_0736 or O46_1969 in O11 or O46), Ldh and a cysteine synthase. Of note, when considering the corresponding genes, all of these 12 genes are present and highly similar in O11 and O46 strains. Only ahpF and vwbp present 2 and 1 non- synonymous single nucleotide polymorphisms (NS-SNPs), respectively. These results suggest that O11 produce these 12 proteins in vivo in a mastitis context whereas O46 does not, or to a much lesser extent. Three proteins appeared to be immunoreactive with group O46 sera only. Two of them are hypothetical proteins corresponding to SAOV0649 (CDS 011_2290 and 046_0078, in O11 and O46, respectively) encoding a probable esterase or lipase, and a gene 011_0490/046_2740 with no homology in ED133 genome sequence. The third protein is IsaA, a viru- lence-associated immunodominant antigen A. Antibody production in response to infection of ewes with S. aureus strains O11 or O46 To compare the relative level of antibodies developed in response to S. aureus presence in the mammary gland, serum sampled on D0, D7, D14, D21 and D28 pi were analysed by Western blotting using either O46 or O11 total bacterial extracts as described in the previous sec- tion. The number of bacterial proteins recognised by sera and signal intensity increased from D0 to D28 for both O11 and O46 samples (Figure 1). The intensity of the signal revealed with sera collected on D0 was low and much weaker compared to those obtained with sera collected on D21 or D28. Western blots membranes were analysed as described in the previous section. Sera Le Maréchal et al. Veterinary Research 2011, 42:35 http://www.veterinaryresearch.org/content/42/1/35 Page 6 of 20 proteins are thus found in supernatant and cell wall frac- tions (88.7%), and only 11.3% are found in the total frac- tions (Figure 3A). Of note, secreted and surface proteins are expected to be exposed to the host immune system. The predicted location of the proteins (according to the SurfG+ analysis of O11 and O46 genome sequences) [23], showed that the immunoreactive proteins were mainly predicted cytoplasmic (52.8%) (Figure 3A). Pro- teins were classified into categories based on functional annotation (Figure 3B). Most immunogenic proteins identified here are found in various functional categories involved in cellular machinery and metabolism; while 20% were virulence factors and virulence associated proteins. collected either on D21 or D28 were selected for further analysis. Sera yielding the best signals in each group (one sample for each of the six ewes) were pooled to be used in SERPA experiments. Two pools were thus obtained: sera from ewes infected with O11 and sera from ewes infected by O46, hereafter referred to as group O11 sera and group O46 sera, respectively. Detection and identification of immunogenic staphylococcal proteins by SERPA The genes encoding IsaA and the probable esterase or lipase are highly homologous in O11 and O46 with only 1 synon- ymous SNP found when comparing O11_2290 and O46_0078. Interestingly, the CDS O11-0490 in O11 gen- ome shares homology with O46_2740 in O46. However, sequence analysis reveals that O11-0490 corresponds to a truncated form of O46_2740. This gene encodes a pre- dicted protein that presents 59% identity and 76% homol- ogy with exfoliative toxin D [24]. D0 D7 D14 D21D28 O11 D0 D7 D14 D21D28 O46 Figure 1 Western blot analysis of total lysates of S. aureus O11 (left panel) or S. aureus O46 (right panel) using D0, D7, D14, D21, D28 diluted 1:10,000 sera of ewes infected by O11 and O46. Bacteria were grown in iron-depleted RPMI without aeration to late exponential phase. Protein samples were prepared from total cell, submitted to 1-DE, western blotted on PVDF membrane and revealed using each of the 6 serum samples collected on ewes infected in each group. One representative example is given for each group of infected ewes (with O11 or O46). D0 D7 D14 D21D28 O11 D0 D7 D14 D21D28 O46 O11 O46 Figure 1 Western blot analysis of total lysates of S. aureus O11 (left panel) or S. aureus O46 (right panel) using D0, D7, D14, D21, D28 diluted 1:10,000 sera of ewes infected by O11 and O46. Bacteria were grown in iron-depleted RPMI without aeration to late exponential phase. Protein samples were prepared from total cell, submitted to 1-DE, western blotted on PVDF membrane and revealed using each of the 6 serum samples collected on ewes infected in each group. One representative example is given for each group of infected ewes (with O11 or O46). Figure 1 Western blot analysis of total lysates of S. aureus O11 (left panel) or S. aureus O46 (right panel) using D0, D7, D14, D21, D28 diluted 1:10,000 sera of ewes infected by O11 and O46. Bacteria were grown in iron-depleted RPMI without aeration to late exponential phase. Protein samples were prepared from total cell, submitted to 1-DE, western blotted on PVDF membrane and revealed using each of the 6 serum samples collected on ewes infected in each group. One representative example is given for each group of infected ewes (with O11 or O46). Le Maréchal et al. Detection and identification of immunogenic staphylococcal proteins by SERPA Veterinary Research 2011, 42:35 http://www.veterinaryresearch.org/content/42/1/35 Table 2 Proteins identified in this study Table 2 Proteins identified in this study Description1 Spots2 O11 CDS3 O46 CDS3 ED133 CDS3 pI4 Mass (Da)5 Score6 Cov.7 #pep.8 EmPAI9 Comp.10 Loc.11 Immun.12 Metabolism Energy production and conversion formate acetyltransferase 81, 83, 82 011_0041 046_0511 SAOV_0163 5,31 84808 1301,60 34,31 22 1,57 CW C ldh L-lactate dehydrogenase 82, 99, 81, 83, 95 011_0136 046_0198 SAOV_2646c 4,80 34389 1749,37 63,64 26 21,75 CW C [82] L-LACTATE DEHYDROGENASE (O11) 64 011_0021 046_0531 SAOV_0178 5,00 34548 745,83 50,79 14 2,59 S C [82] bifunctional acetaldehyde-CoA/ alcohol dehydrogenase 79 011_0796 046_0709 SAOV_0095 5,68 94945 2090,64 45,45 33 2,26 CW C D-3-phosphoglycerate dehydrogenase 100 011_0585 046_1131 SAOV_2344 5,32 34652 717,85 43,53 11 1,72 CW C atpD F0F1 ATP synthase subunit beta 62, 90 011_2064 046_0898 SAOV_2144c 4,68 51368 631,03 30,21 10 0,98 S,CW C [83] Nucleotide transport and metabolism adk adenylate kinase 9 011_0510 046_1206 SAOV_2266c 4,80 23375 531,72 42,38 9 2,80 S C [84] deoD purine nucleoside phosphorylase 9 011_1051 046_0577 SAOV_2178 4,85 25892 470,94 53,39 8 1,97 S C [4] guaB inositol-monophosphate dehydrogenase 102, 129 011_0077 046_0960 SAOV_0412 5,61 52818 1237,95 54,30 20 3,52 CW,T C [4] Carbohydrate transport and metabolism gpmA 2,3-bisphosphoglycerate- dependent phosphoglycerate mutase 131 011_1952 046_0264 SAOV_2463c 5,23 26663 271.65 30.26 5 0.80 S,T C eno enolase 2-phosphoglycerate dehydratase 62, 88, 90, 96, 137 011_2336 046_2241 SAOV_0818 4,55 47088 1868,52 63,59 25 7,09 S,CW,T C [4,52,85-87] fda fructose-1,6-bisphosphate aldolase 107, 94, 72, 91, 95 011_0131 046_0202 SAOV_2650 5,06 32878 852,09 46,62 13 2,48 CW,S C [85-88] gap glyceraldehyde-3-phosphate dehydrogenase 99, 57, 81, 82, 83 011_2340 046_2237 SAOV_0814 4,89 36258 940,06 46,13 13 3,04 CW,S C [82,86,87,89,90] putative translaldolase 9 011_1872 046_2451 SAOV_1765 4,72 25689 554,11 46,84 10 3,32 S C tpiA triosephosphate isomerase 9 011_2338 046_2239 SAOV_0816 4,81 27271 1396,16 76,28 21 16,78 S C [4,91,92] atl autolysin 23, 31 011_1991 046_0320 SAOV_0999c 9,59 136983 2567,68 42,14 42 1,87 S S [52,76,93] pyk pyruvate kinase 105 011_1282 046_0580 SAOV_1685 5,23 63067 271,45 11,62 5 0,29 CW C [94] Lipid metabolism fabZ 3R-hydroxymyristoyl ACP dehydratase 126 011_2356 046_1839 SAOV_2140c 5,71 16071 86,65 13,70 2 0,47 T C Proteins identified in this study on1 Spots2 O11 CDS3 O46 CDS3 ED133 CDS3 pI4 Mass (Da)5 Score6 Cov.7 #pep.8 EmPAI9 Comp.10 Loc.11 Immun.12 Le Maréchal et al. Detection and identification of immunogenic staphylococcal proteins by SERPA Veterinary Research 2011, 42:35 http://www.veterinaryresearch.org/content/42/1/35 Table 2 Proteins identified in this study (Continued) Table 2 Proteins identified in this study (Continued) acetoin reductase 106 011_1332 046_1393 SAOV_0074 5,04 27199 566,84 40,31 7 1,82 CW C Amino acid transport and metabolism dipeptidase PepV 62 011_1904 046_1329 SAOV_1737 4,56 52762 235.33 10.66 4 0,27 S C CYSTEINE SYNTHASE (O11) 64 011_1382 046_2402 SAOV_0535 5,38 32955 266.80 28.71 5 0,61 S C Information storage and processing Translation, ribosomal structure and biogenesis fus elongation factor G 95 011_0401 046_1769 SAOV_0582 4,80 76564 2173,77 68,11 30 4,11 CW C [3,90] prs 50S ribosomal protein L25/ general stress protein Ctc 94 011_1370 046_2414 SAOV_0523 4,39 23773 384,17 34,56 6 2,26 CW,S C [95] rplC 50S ribosomal protein L3 131 011_0531 046_1185 SAOV_2287c 9,72 22648 482,78 42,11 9 2,95 T C [95] rpsA 30S ribosomal protein S1 137, 62, 121, 171 011_2142 046_1743 SAOV_1482 4,51 43252 1555,46 71,36 23 6,25 S,CW,T C [39,95] rpsD 30S ribosomal protein S4 139 011_1260 046_1365 SAOV_1706 10,02 22999 428,22 40,50 8 1,96 T C [95] rplB 50S ribosomal protein L2 91 011_0528 046_1188 SAOV_2284c 10,77 30136 239,93 25,27 5 0,69 CW C [95] tsf elongation factor Ts 100, 64 011_0909 046_0755 SAOV_1259 5,05 32474 436,02 31,06 7 0,97 CW,S C [4,86,93,96] tuf elongation factor Tu 90, 88, 96, 10, 122, 137, 149, 81, 82, 83, 95 011_0402 046_1768 SAOV_0583 4,74 43077 2665,48 84,26 38 52,14 S,CW,T C [3,52,82,85,86,90,93,96] yfiA ribosomal subunit interface protein 106 011_2483 046_1255 SAOV_0789 5,25 21511 376,75 35,33 6 1,76 CW C aspS aspartyl-tRNA synthetase 63 011_2454 046_2303 SAOV_1627 4,99 66584 233,90 7,65 4 0,21 S C alaS alanyl-tRNA synthetase 82 011_2466 046_2291 SAOV_1616 5,00 98604 442,31 11,74 8 0,30 O C [97] Transcription nusA transcription elongation factor NusA 62 011_0919 046_0765 SAOV_1268 4,60 43701 247,74 11,00 4 0,34 S C DNA replication, recombination and repair dnaN DNA polymerase III subunit beta 90 011_1166 046_1471 SAOV_0002 4,66 41888 706,28 45,62 11 1,30 CW C nuc staphylococcal thermonuclease precursor 151, 108, 5, 153, 206, 207, 217 011_2070 046_2528 SAOV_0832 9,20 25089 967,00 50,00 20 14,46 S,CW PSE [98] ruvA Holliday junction DNA helicase 66 011_2442 046_2315 SAOV_1639 5,77 22249 137,72 17,00 3 0,52 S C Posttranslational modification, protein turnover, chaperones ahpC alkyl hydroperoxide reductase subunit C 67, 82, 83, 99, 95 011_0085 046_0968 SAOV_0404c 4,88 20963 667,36 56,08 11 4,95 S,CW C [93] AhpF ALKYL HYDROPEROXIDE REDUCTASE SUBUNIT F (O11) 97, 86 011_0086 046_0969 SAOV_0403c 4,68 54655 2242,14 67,06 34 9,91 CW C Table 2 Proteins identified in this study (Continued) dnaK chaperone protein 96, 173, 137 011_2230 046_2216 SAOV_1580 4,63 46021 2907,83 80,29 43 24,66 S,CW,T C [90,96,99] peptidyl-prolyl cis-isomerase 212, 1, 68, 91 011_2089 046_2477 SAOV_1837 9,01 35602 574,32 31,56 11 1,66 S,CW PSE tig trigger factor 105 011_1304 046_0602 SAOV_1664 4,34 48565 1061,87 61,43 17 2,25 CW C [94] TrxB THIOREDOXIN REDUCTASE (O11) 64 011_2580 046_2228 SAOV_0801 5,21 33595 681,95 33,76 11 1,81 S C [93] Cellular processes Cell envelope biogenesis, outer membrane IsaA IMMUNODOMINANT ANTIGEN A (O46) 185, 8, 161, 186, 9, 164, 189 011_0168 046_0166 SAOV_2614c 5,91 24219 1516,56 59,23 22 40,99 S S [3,75] isdA iron-regulated cell wall- anchored protein 31, 27, 73, 74, 75, 83, 118, 79, 81, 82, 95 011_1476 046_1296 SAOV_1125c 8,69 70445 2288,12 57,87 38 6,04 S,CW PSE [100-102] isdB cell surface transferrin-binding protein 212, 211, 210, 208, 193, 156, 138, 110, 70, 86, 131, 156, 193 011_1477 046_1295 SAOV_1126c 9,54 39197 886,30 45,48 15 4,45 S,CW,T PSE [54,100,103] IRON-REGULATED SURFACE DETERMINANT PROTEIN H (O11) 55, 31 011_1248 046_1353 SAOV_1717 5,05 100650 2209,72 42,11 37 2,58 S PSE [75] IsdD iron-regulated protein 15, 16 011_1480 046_1292 SAOV_1128 8,51 41357 278,05 15,36 5 0,47 S PSE Cell motility and secretion N-acetylmuramoyl-L-alanine amidase 52, 51, 187 011_1090 046_1546 SAOV_2693 5,87 69226 3097,93 71,57 43 16,52 S S [57,75,76] Inorganic ion transport and metabolism nasE assimilatory nitrite reductase 10 011_1932 046_0245 SAOV_2445c 4,95 11430 126,05 23,08 2 0,70 S C mntC Manganese/iron transport system substrate-binding protein 94 011_2274 046_0062 SAOV_0666c 8,68 34719 1183,08 51,46 20 10,68 S,CW PSE sirA iron-regulated lipoprotein 135, 64 011_1345 046_1405 SAOV_0062 9,20 36735 609,79 35,45 11 1,58 S,T PSE [104] fhuD2 ferrichrome-binding protein 91 011_0566 046_1150 SAOV_2323c 9,16 33990 406,75 30,13 8 1,10 CW PSE [105] ferrichrome ABC transporter lipoprotein 91 011_1857 046_1674 SAOV_2224c 9,44 36751 600,45 38,41 11 1,57 CW PSE Signal transduction mechanisms SA1540 GAF domain-containing protein 10 011_1261 046_1366 SAOV_1705 5,09 17042 139,34 22,73 3 0,72 S C Universal stress response protein 7, 123 011_1269 046_1374 SAOV_1697 5,60 18463 973,68 78,31 12 19,34 S,CW C [106] Toxins and haemolysins beta-hemolysin 15, 19, 205 011_1750 046_2394 SAOV_2040 8,75 37386 1308,79 61,03 20 4,92 S S [57,107] hla alpha-hemolysin precursor 13, 1, 15, 19, 21, 68, 72, 107, 146, 153 011_1514 046_1259 SAOV_1161c 8,87 36329 2238,72 76,71 34 44,90 S,CW,T S [93,107] hlgC gamma-hemolysin component C 68, 1 011_1956 046_0268 SAOV_2469 9,29 35562 765,07 31,43 12 1,90 S,CW S [57] Le Maréchal et al. Detection and identification of immunogenic staphylococcal proteins by SERPA Veterinary Research 2011, 42:35 http://www.veterinaryresearch.org/content/42/1/35 Table 2 Proteins identified in this study (Continued) Le Maréchal et al. ording to annotation of available S. aureus sequence genomes. (O11) indicates O11 specific proteins, (O46) indicates O46 specific proteins. ucts of the accessory seroproteome are indicated in bold capital letters, (O11) indicates O11-specific proteins, (O46) indicates O46-specific proteins. 5 O11, S. aureus O46, and ED133, respectively. Detection and identification of immunogenic staphylococcal proteins by SERPA Veterinary Research 2011, 42:35 http://www.veterinaryresearch.org/content/42/1/35 Table 2 Proteins identified in this study (Continued) Table 2 Proteins identified in this study (Continued) Virulence/defence mechanisms Sbi IGG-BINDING PROTEIN SBI (O11) 216, 217, 212 011_1954 046_0266 SAOV_2466 9,38 49998 984,16 41,74 18 2,80 S S [76] SspB CYSTEINE PROTEASE PRECURSOR (O11) 58, 57, 64, 182, 215 011_2154 046_0327 SAOV_0993c 5,45 42714 2027,86 65,52 30 11,52 S S [4] SplF serine proteinase 4 011_0672 046_2496 SAOV_1795 9,36 25638 255,88 23,01 5 0,84 S S [108] lukD leukotoxin D subunit 1 011_0685 046_2484 SAOV_1812 9,14 36936 365,37 22,02 7 0,98 S S [109] lukE leukotoxin E subunit 1, 68 011_0686 046_2483 SAOV_1813c 9,38 34126 451,11 17,65 7 1,77 S,CW S [109] leukocidin chain lukM precursor 68, 1, 120, 145, 177, 194, 201, 202, 94 011_1215 046_2777 SAOV_1909 9,41 35054 2504,69 71,43 35 29,80 S,CW,T S [58,110] leukocidin F subunit 16, 70 011_1752 046_1972 SAOV_2041 8,29 38639 1154,65 45,27 19 11,64 S,CW S [58,110] leukocidin S subunit 199, 5, 70, 108, 109, 197, 200, 211 011_1753 046_1973 SAOV_2042 9,38 40379 1399,91 55,56 23 6,70 S,CW S [58,110] Panton-Valentine leukocidin LukF- PV chain precursor 1, 68, 70, 15, 31, 119, 195, 203, 204, 10, 91 011_1216 046_2776 SAOV_1908 9,16 36496 965,38 50,31 15 3,37 S,CW S [58,110] plc 1-phosphatidylinositol phosphodiesterase 11, 12, 19 011_1424 046_1419 SAOV_0049 7,12 37030 2759,20 71,95 44 84,00 S S [4] Aur ZINC METALLOPROTEINASE AUREOLYSIN (O11) 220, 63 011_1083 046_1553 SAOV_2686c 4,98 54947 921,93 47,39 17 1,68 S C [76,111] Opp1A OLIGOPEPTIDE TRANSPORTER SUBSTRATE BINDING PROTEIN (O11) 86 011_2424 046_2102 SAOV_2517c 8,33 59224 1398,01 47,53 24 3,28 CW PSE SspA GLUTAMYL ENDOPEPTIDASE SERINE PROTEASE (O11) 56, 184, 214 011_2155 046_0325 SAOV_0994c 4,68 32250 1575,55 69,26 24 24,18 S C [57] SECRETED VON WILLEBRAND FACTOR-BINDING PROTEIN (O11) 22 011_2679 046_0987 SAOV_2051c 8,39 57935 1122,89 36,67 17 1,70 S S epidermal cell differentiation inhibitor B 208, 209, 193, 156, 4 011_0489 046_2741 9,51 27969 1385,18 69,32 22 22,31 S S Miscellaneous adhA alcohol dehydrogenase 100 011_1554 046_0086 SAOV_0640 5,34 36025 1167,82 66,37 16 4,30 CW C [87,93] exported secretory antigen precursor 67 011_0584 046_1132 SAOV_2343 5,77 17388 397,17 33,13 5 1,43 S S lip triacylglycerol lipase precursor 31, 27 011_1119 046_1518 SAOV_2721c 8,13 76637 1723,17 50,95 29 2,36 S S [93] mqo malate:quinone oxidoreductase 128 011_0130 046_0203 SAOV_2651c 6,12 55964 1047,99 46,79 17 2,50 T C putative staphylococcal enterotoxin 151 011_0097 046_0981 SAOV_0394 9,54 23311 173,20 18,72 4 0,71 S S Unknown HYPOTHETICAL PROTEIN (O11) 220 011_0736 046_1969 SAOV_0454 4,89 56229 823,11 39,40 14 1,21 S S hypothetical protein (Similar to truncated map-w protein (91%)) 88, 10, 62 011_1749 046_2393 9,91 53686 899,64 34,52 18 2,09 S,CW S [112] Le Maréchal et al. ns belong to the core seroproteome. fied in Gene Ontology functional classes. Names are given according to annotation of available S. aureus sequence genomes. fi ) Detection and identification of immunogenic staphylococcal proteins by SERPA Gels run in parallel were immunoblotted using the pools of sera obtained from group 1 (infected with O11) animals (middle panels) or from group 2 (infected with O46) animals (right panels). Samples were run in parallel on 13 cm gels (pI 3-10; 14% SDS-PAGE). Spots identified by MS/MS are labeled. Figure 2 Representative 2-DE gels and SERPA on supernatant fractions of S. aureus O11 (A) and S. aureus O46 (B). Supernatant samples were prepared from stationary phase cultures of S. aureus strains grown anaerobically on iron-depleted RPMI. Preparative 2-DE gels were Coomassie blue stained (left panel). Gels run in parallel were immunoblotted using the pools of sera obtained from group 1 (infected with O11) animals (middle panels) or from group 2 (infected with O46) animals (right panels). Samples were run in parallel on 13 cm gels (pI 3-10; 14% SDS-PAGE). Spots identified by MS/MS are labeled. proteolytic cascade, was detected in the culture superna- tant of strains isolated from clinical mastitis cases whereas it was not found in the strains isolated from subclinical mastitis (Figure 5A). The other protein tested was O46_2740 gene product (with similarity to exfolia- tive toxin family), which was specifically recognized in O46 infections and did not yield any significant signal in O11 infections (Figure 4B). When considering its pre- sence in the culture supernatant of other S. aureus strains isolated from clinical or subclinical mastitis in ewes, we observed that it was only detected in the subclinical strains (100%) whereas it was undetectable in any of the strains isolated from clinical mastitis (Figure 5B). These results show that at least these tested proteins are differentially produced by S. aureus strains isolated from clinical or subclinical mastitis cases. Whether these differences are involved or not in the onset and the acuteness of the subclinical vs. clinical mastitis remains to be demonstrated. Selected S. aureus immunoreactive proteins are widely distributed among a panel of strains isolated from clinical vs. subclinical ewe mastitis In order to test the hypothesis that the seroproteomic variations identified by SERPA on the two S. aureus iso- lates were more widely distributed among isolates of a specific host- and clinical-association, we screened an additional 10 strains isolated from subclinical (n = 5) and severe (i.e. clinical or gangrenous mastitis; n = 5) cases of ewe mastitis for the presence of 2 selected pro- teins by proteome analysis of supernatant samples (i.e. Detection and identification of immunogenic staphylococcal proteins by SERPA Veterinary Research 2011, 42:35 http://www.veterinaryresearch.org/content/42/1/35 Table 2 Proteins identified in this study (Continued) Le Maréchal et al. Veterinary Research 2011, 42:35 http://www.veterinaryresearch.org/content/42/1/35 y hypothetical protein (Similar to beta-lactamase (84%)) 10 011_0679 046_2490 6,84 20800 726,52 47,15 10 7,09 S S HYPOTHETICAL PROTEIN (O46) 157, 160, 175, 189, 193 011_0490 046_2740 8,88 30854 2343,27 74,29 41 65,07 S S hypothetical protein (Similar to probable glutamyl-endopeptidase (76%)) 66 011_0488 046_2742 5,44 20552 741,93 47,62 12 5,11 S C SA0570 HYPOTHETICAL PROTEIN (O46) 219,154, 127 011_2290 046_0078 SAOV_0649 9,17 18554 815,59 69,05 13 15,93 S,CW S SA0663 hypothetical protein 10, 164, 94 011_2561 046_2636 SAOV_0742c 9,15 16047 457,11 35,62 8 3,63 S PSE SA0914 hypothetical protein 6 011_2000 046_0311 SAOV_1008c 6,55 11338 522,48 57,14 8 13,10 S S pathogenicity island protein 55 011_2741 046_0985 SAOV_0429 9,55 12071 204,61 19,64 3 1,12 S S SA1607 hypothetical protein 91 011_1867 046_2456 SAOV_1770 6,04 34973 232,41 16,56 4 0,43 CW S SA1402 hypothetical protein 91 011_2223 046_2209 SAOV_1573 5,60 35160 448,93 28,27 6 0,72 CW S Gene products of the accessory seroproteome are indicated in bold capital letters (O11) indicates O11 specific proteins (O46) indicates O46 specific proteins 7: % of the protein sequence covered by the peptides identified. 10: Subproteome compartment where the spot was identified. CW, cell wall; S, supernatant; T, total fraction. Page 11 of 20 Le Maréchal et al. Veterinary Research 2011, 42:35 http://www.veterinaryresearch.org/content/42/1/35 Page 12 of 20 Figure 2 Representative 2-DE gels and SERPA on supernatant fractions of S. aureus O11 (A) and S. aureus O46 (B). Supernatant samples were prepared from stationary phase cultures of S. aureus strains grown anaerobically on iron-depleted RPMI. Preparative 2-DE gels were Coomassie blue stained (left panel). Gels run in parallel were immunoblotted using the pools of sera obtained from group 1 (infected with O11) animals (middle panels) or from group 2 (infected with O46) animals (right panels). Samples were run in parallel on 13 cm gels (pI 3-10; 14% SDS-PAGE). Spots identified by MS/MS are labeled. Figure 2 Representative 2-DE gels and SERPA on supernatant fractions of S. aureus O11 (A) and S. aureus O46 (B). Supernatant samples were prepared from stationary phase cultures of S. aureus strains grown anaerobically on iron-depleted RPMI. Preparative 2-DE gels were Coomassie blue stained (left panel). Detection and identification of immunogenic staphylococcal proteins by SERPA 2-DE and Coomassie blue staining). The O11 (severe mastitis) protein selected was SspB, which belongs to a proteolytic cascade where a metalloprotease aureolysin (Aur) activates a serine protease zymogen proSspA, which in turn activates the SspB cysteine protease [25]. SspB and the two other proteins, SspA and Aur of the cascade, were recognized in O11 and yielded a very faint signal in O46 (Figure 4A). SspB, one element of this Le Maréchal et al. Veterinary Research 2011, 42:35 http://www.veterinaryresearch.org/content/42/1/35 Page 13 of 20 0 2 4 6 8 10 12 14 Energy production and conversion Nucleotide transport and metabolism Carbohydrate transport and metabolism Lipid metabolism Amino acid transport and metabolism Translation, ribosomal structure and biogenesis Transcription DNA replication, recombination and repair Posttranslational modification, protein turnover, chaperones Cell envelope biogenesis, outer membrane Cell motility and secretion Inorganic ion transport and metabolism Signal transduction mechanisms Toxins and haemolysins Virulence/defence mechanisms Miscellaneous Unknown Observed localisation (on gels) Predicted localisation (SurfG+) A 74 12 3 O11 O46 Supernatant (52.4%) Total (11.3%) Cell wall (36.3%) Surface exposed (13.5%) Cytoplasmic (52.8%) Secreted (33.7%) B C Figure 3 (A) Distribution of the immunoreactive proteins based on their experimentally observed (upper panel) or predicted (lower panel) subcellular localization. (B) Distribution of the immunoreactive proteins based on their GO functional classes. (C) Venn diagram of immunoreactive proteins defining the core and the accessory seroproteomes. CB O11 SERPA Group 1 (O11) SspB SspA Aur CB O46 O46-2740 A B SERPA Group 2 (O46) SERPA Group 1 (O11) SERPA Group 2 (O46) Figure 4 SERPA of O11-specific (A) and O46-specific (B) immunoreactive proteins. Protein specific signals were zoomed in on Coomassie blue stained gels (CB) and membranes revealed using sera obtained from animals infected with O11 (Group1) or O46 (Group 2). Aur, aureolysin; SspA, glutamyl endopeptidase serine protease; SspB, staphopain B; O46-2740, gene product similar to exfoliative toxin family. Detection and identification of immunogenic staphylococcal proteins by SERPA 0 2 4 6 8 10 12 14 Energy production and conversion Nucleotide transport and metabolism Carbohydrate transport and metabolism Lipid metabolism Amino acid transport and metabolism Translation, ribosomal structure and biogenesis Transcription DNA replication, recombination and repair Posttranslational modification, protein turnover, chaperones Cell envelope biogenesis, outer membrane Cell motility and secretion Inorganic ion transport and metabolism Signal transduction mechanisms Toxins and haemolysins Virulence/defence mechanisms Miscellaneous Unknown Observed localisation (on gels) Predicted localisation (SurfG+) A 74 12 3 O11 O46 Supernatant (52.4%) Total (11.3%) Cell wall (36.3%) Surface exposed (13.5%) Cytoplasmic (52.8%) Secreted (33.7%) B C Figure 3 (A) Distribution of the immunoreactive proteins based on their experimentally observed (upper panel) or predicted (lower panel) subcellular localization. (B) Distribution of the immunoreactive proteins based on their GO functional classes. (C) Venn diagram of immunoreactive proteins defining the core and the accessory seroproteomes. B A Cell wall (36.3%) Secreted (33.7%) Predicted localisation (SurfG+) Predicted localisation (SurfG+) Figure 3 (A) Distribution of the immunoreactive proteins based on their experimentally observed (upper panel) or predicted (lower panel) subcellular localization. (B) Distribution of the immunoreactive proteins based on their GO functional classes. (C) Venn diagram of immunoreactive proteins defining the core and the accessory seroproteomes. CB O11 SERPA Group 1 (O11) SspB SspA Aur CB O46 O46-2740 A B SERPA Group 2 (O46) SERPA Group 1 (O11) SERPA Group 2 (O46) Figure 4 SERPA of O11-specific (A) and O46-specific (B) immunoreactive proteins. Protein specific signals were zoomed in on Coomassie blue stained gels (CB) and membranes revealed using sera obtained from animals infected with O11 (Group1) or O46 (Group 2). Aur, aureolysin; SspA, glutamyl endopeptidase serine protease; SspB, staphopain B; O46-2740, gene product similar to exfoliative toxin family. A SERPA Group 2 (O46) SERPA Group 1 (O11) B Figure 4 SERPA of O11-specific (A) and O46-specific (B) immunoreactive proteins. Protein specific signals were zoomed in on Coomassie blue stained gels (CB) and membranes revealed using sera obtained from animals infected with O11 (Group1) or O46 (Group 2). Aur, aureolysin; SspA, glutamyl endopeptidase serine protease; SspB, staphopain B; O46-2740, gene product similar to exfoliative toxin family. Le Maréchal et al. Veterinary Research 2011, 42:35 http://www.veterinaryresearch.org/content/42/1/35 Page 14 of 20 clinical clinical subclinical subclinical Figure 5 Distribution of SspB (A) and O46-2740 product (B) in a panel of S. aureus strains isolated from clinical (a) and subclinical (b) mastitis. Discussion Two closely related S. aureus ovine strains reproducibly induce distinct mastitis types in ewe and mouse models. Contrarily to Escherichia coli mastitis, which severity is mainly determined by host factors and not by the strains features [26], it seems that in S. aureus, inter-strain var- iations exist in terms of virulence potential. Such varia- tions have been experimentally observed but the involved staphylococcal factors have not been clearly identified yet [27-29]. S. aureus content in virulence fac- tors reportedly varies from strain to strain and this may account for the large panel of symptoms encountered in ruminant mastitis [30]. It was also shown that some ele- ments of the core genome could account for host adap- tation in S. aureus ruminant isolates [8]. Here, we show that two closely related S. aureus strains are able to induce dramatically different mastitis outcomes in ani- mal model. The experimental mastitis induced in ewes confirmed that strain O11, originally isolated from a gangrenous mastitis, induced more severe infections than strain O46 in ewes. To corroborate the finding of the diversity of the clinical signs caused by O11 and O46, the mouse model of mastitis was employed for further investigation [31]. Despite variable susceptibility to viral, fungal or bacterial infections among the differ- ent mouse lines [32], the mouse model was validated as an experimental approach to the study of bovine masti- tis [33]. In this study, CD-1 mice infected with O11 or O46 revealed diverse clinical signs and showed different cytokine profiles: O46 induced higher IL-1b and TNF-a Detection and identification of immunogenic staphylococcal proteins by SERPA Supernatant samples were prepared on stationary phase cultures of strains grown on iron-depleted RPMI. 2-DE gels were Coomassie blue stained. clinical subclinical clinical subclinical Figure 5 Distribution of SspB (A) and O46-2740 product (B) in a panel of S. aureus strains isolated from clinical (a) and subclinical (b) mastitis. Supernatant samples were prepared on stationary phase cultures of strains grown on iron-depleted RPMI. 2-DE gels were Coomassie blue stained. levels in the mammary gland lysates and higher serum levels of IL-1b and MCP-1. It has been shown that the cytokines play a pivotal role in the development of the mastitis [34-36]. Recent investigation revealed that the proinflammatory cytokines, TNF-a and IL-1b, increased the capacity of bovine endothelial cells to eliminate intracellular S. aureus [37]. Endothelial cells may so represent a functional and active defence barrier against bacteria invasion of infected quarters. Higher synthesis of proinflammatory cytokines in O46-infected mice might thus reflect a higher anti-staphylococcal response and may allow more effective elimination of strain O46. Further investigation has to be conducted to confirm this hypothesis. Altogether, our findings demonstrated that these two well-characterized strains reproducibly induce drastically different mastitis in terms of severity, and will prove useful tools to gain further insights into host-pathogen interactions. The core seroproteome b l h Proteins belonging to the core seroproteome are immu- nogenic regardless the severity of the induced mastitis. These proteins are therefore good targets for the devel- opment of new strategies against S. aureus mastitis. Some of them have been tested as vaccine target to pre- vent staphylococcal infections e.g., Enolase (Eno) [52], IsdA (an iron-regulated cell wall-anchored protein) and IsdB (a cell surface transferrin-binding protein) [53,54], GapC/B protein (glyceraldehyde-3-phosphate dehydro- genase) [55], Hla (alpha-hemolysin) [56]. These vaccines seem at least to limit infection damage (by notably decreasing mortality) but not to provide total effective protection. Well-known virulence factors, such as Hla, Hlb (beta- hemolysin), SspA (V8 serine protease), ScpA (Staphopain A), and Plc (phosphatidylinositol phosphodiesterase), were identified here in a mastitis context and were pre- viously identified as immunogenic in human infections [4,57]. Hla and leukotoxins were reportedly produced in vivo during mastitis [58] but to our knowledge this is the first time that the other proteins listed in Table 2 are shown to be produced in vivo during mastitis. These pro- teins deserve more attention to test their role in the mas- titis onset and to check their potential use as target for vaccine development. Of note, we found 5 iron-related Inventory of mastitis-associated core and accessory seroproteomes in S. aureus reveals new mastitis-related antigens This study reports the application of SERPA to deter- mine the core and accessory seroproteomes resulting from experimental ewe mastitis induced by two different S. aureus strains, isolated from a subclinical mastitis (O46) and a gangrenous mastitis (O11). To determine which proteins are recognized by the ewe immune sys- tem during mastitis with different severity, experimental infections were carried out on primiparous ewes using Le Maréchal et al. Veterinary Research 2011, 42:35 http://www.veterinaryresearch.org/content/42/1/35 Le Maréchal et al. Veterinary Research 2011, 42:35 http://www.veterinaryresearch.org/content/42/1/35 Page 15 of 20 these two S. aureus strains. By comparison of the SERPA profiles, this study allowed, for the first time, the determination of core and accessory seroproteomes for S. aureus in a mastitis context. Eighty nine proteins were immunogenic in ewe mastitis implying they were produced during the infection. Seventy four were found in both seroproteome profiles and composed a core ser- oproteome. Fifteen of them were found strain-specific and composed the accessory seroproteome. Among the 74 proteins belonging to the core seroproteome, only 44 (59.5%) had already been reported to be immunogenic in infections caused by S. aureus or other pathogens (e.g. Bacillus anthracis, Staphylococcus epidermidis, Clos- tridium perfringens, Schistosoma japonicum) (Table 2). Among these previously described antigens, only 30S ribosomal S1 and LukM/LukF’-PV were previously reported to be immunogenic in S. aureus bovine mastitis [38,39]. These 44 proteins include most of the immunor- eactive proteins categorized in Toxins-Haemolysins and Virulence/Defence mechanisms. The remaining 30 pro- teins (40.5%) are described for the first time as potential staphylococcal antigens in a mastitis context and, to our knowledge, were not described as immunogenic else- where. In the accessory seroproteome, 8 out the 15 pro- teins identified were already described as immunogenic elsewhere whereas 7 of them (AhpF, Opp1A, VVbp, Mqo, cysteine synthase and two hypothetical proteins) are described as such for the first time (Table 2). A majority of the proteins identified here are thus reported as immunogenic for the first time in a mastitis context. Furthermore, 37 out of 89 proteins of the total seropro- teome (~42%) correspond to proteins described as sta- phylococcal antigens for the first time whatever the infection considered. Only four of these new antigens are categorized in Toxins-Haemolysins and Virulence/ defense mechanisms. The other ones are mostly found in Metabolism (n = 9), Information storage (n = 7), and Unknown function (n = 13). Inventory of mastitis-associated core and accessory seroproteomes in S. aureus reveals new mastitis-related antigens Only a few studies pre- viously analyzed S. aureus proteome and all of them were carried out on human isolates, which reportedly differ from ruminant isolates [8,9,40] and the strains were grown in laboratory culture conditions [4,41-46]. Here we used two ovine isolates grown in culture conditions mimicking the mastitis context [11]. These two criteria might account for the abundance of new staphylococcal antigens identified in this study. in protein samples prepared from supernatant and cell wall fractions revealing, somehow, discrepancies between experimental (gels of total, cell wall and super- natant protein fractions) and theoretical localization (as determined in silico). Indeed, among the 47 immunor- eactive proteins that were predicted cytoplasmic, only 10 were experimentally found in the total fraction and 37 were found in supernatant or cell wall fractions (Table 2). Such discrepancies were observed in other studies as well [4,47,48]. Some proteins are multifunc- tional and found both intra- and extracellularly. For example, glyceraldehyde-3-phosphate dehydrogenase [49] and enolase [50] were shown to be both cytoplas- mic and surface exposed. In addition to their metabolic role in the cytoplasm, they play a role as adhesins when exposed on the bacterial surface. Furthermore, a new mechanism of protein exportation in Gram positive bac- teria was recently described and not included yet in pre- diction tools for protein localization. It has actually been demonstrated that S. aureus, like some Gram negative bacteria, secretes membrane vesicles, which contained at least 90 different proteins [51]. Twelve of the proteins identified here were found in the vesicle-secreted pro- teins identified by Lee et al. [51]. Whether the other immunoreactive proteins identified here are secreted through a membrane vesicle mechanism remains undetermined. these two S. aureus strains. By comparison of the SERPA profiles, this study allowed, for the first time, the determination of core and accessory seroproteomes for S. aureus in a mastitis context. Eighty nine proteins were immunogenic in ewe mastitis implying they were produced during the infection. Seventy four were found in both seroproteome profiles and composed a core ser- oproteome. Fifteen of them were found strain-specific and composed the accessory seroproteome. Among the 74 proteins belonging to the core seroproteome, only 44 (59.5%) had already been reported to be immunogenic in infections caused by S. aureus or other pathogens (e.g. Bacillus anthracis, Staphylococcus epidermidis, Clos- tridium perfringens, Schistosoma japonicum) (Table 2). Inventory of mastitis-associated core and accessory seroproteomes in S. aureus reveals new mastitis-related antigens Among these previously described antigens, only 30S ribosomal S1 and LukM/LukF’-PV were previously reported to be immunogenic in S. aureus bovine mastitis [38,39]. These 44 proteins include most of the immunor- eactive proteins categorized in Toxins-Haemolysins and Virulence/Defence mechanisms. The remaining 30 pro- teins (40.5%) are described for the first time as potential staphylococcal antigens in a mastitis context and, to our knowledge, were not described as immunogenic else- where. In the accessory seroproteome, 8 out the 15 pro- teins identified were already described as immunogenic elsewhere whereas 7 of them (AhpF, Opp1A, VVbp, Mqo, cysteine synthase and two hypothetical proteins) are described as such for the first time (Table 2). A majority of the proteins identified here are thus reported as immunogenic for the first time in a mastitis context. Furthermore, 37 out of 89 proteins of the total seropro- teome (~42%) correspond to proteins described as sta- phylococcal antigens for the first time whatever the infection considered. Only four of these new antigens are categorized in Toxins-Haemolysins and Virulence/ defense mechanisms. The other ones are mostly found in Metabolism (n = 9), Information storage (n = 7), and Unknown function (n = 13). Only a few studies pre- viously analyzed S. aureus proteome and all of them were carried out on human isolates, which reportedly differ from ruminant isolates [8,9,40] and the strains were grown in laboratory culture conditions [4,41-46]. Here we used two ovine isolates grown in culture conditions mimicking the mastitis context [11]. These two criteria might account for the abundance of new staphylococcal antigens identified in this study. The accessory seroproteome Some proteins were shown to be specifically produced by strain O11 or by strain O46 in infected ewes. None of 12 proteins specifically produced by O11 in vivo had previously been reported as produced during mastitis. Their role in mastitis is thus unknown. Nevertheless most of them have been described as immunogenic in S. aureus infections in humans. Their function is mainly linked to resistance to host immune response like for IsdH [59], AhpF and TrxB, implied in oxidative stress responses [60] that may confer O11 resistance to neu- trophils and so be an advantage compared to O46, Sbi that forms complexes with immunoglobulins Fc regions [61], Aur and SspB. Aureolysin is essential for activation of SspA [25], which in turn activates the SspB zymogen [62]. Aur, SspA and SspB seemed to be more produced in O11 than in O46. They can degrade conjunctive tis- sue [63]. Aureolysin has been shown to be involved in resistance to macrophage phagocytosis [64] and to sig- nificantly contribute to the activation of the fibrinolytic system [65]. It might thus reinforce the degradation of extracellular matrix in the mammary gland and promote bacterial spread and invasion. SspB can activate the che- moattractant chemerin, which results in a local inflam- mation of the tissue [66]. Moreover it induces the depletion of functional phagocytes at the site of infec- tion by blocking phagocytosis by neutrophils and inhi- biting their chemotactic activity [67]. SspB may so take part in the observed swelling of the mammary gland observed during gangrenous mastitis. Moreover, it has been shown that SspA and SspB play an important role in virulence in a mouse abscess model [68]. Opp1A was found to be produced by strain O11 in vivo. Opp pro- teins seem to take part in virulence in several infection models [69]. Although the role of Opp has not been clearly demonstrated until now, Opp proteins have also been reported to be involved in virulence in other Gram positive pathogens such as group A streptococci [70], Streptococcus agalactiae [71] or Listeria monocytogenes [72]. A variant of von Willebrand factor-binding protein gene has recently been located on the pathogenicity island SaPIov2, characteristic of small ruminant isolates [9]. Interestingly, besides being a coagulase, it is also an activator of pro-thrombin [73] that is present in cow milk [74]. Conclusion To the best of our knowledge, this study provides the first comparative and comprehensive serological pro- teome analysis in a mastitis context. The proteins identi- fied are immunogenic in ewes implying that they are also produced in the udder during infection. Many of them are found immunogenic for the first time and a great proportion was found in the supernatant and cell wall fractions even though they were predicted as cyto- plasmic proteins. Whether or how these proteins are really involved in the mastitis infection and or the sever- ity of the mastitis remains to be elucidated. This study provides a handful of interesting candidates for further investigations on their potential use as new targets for prophylactic or curative strategies such as vaccine or drug target as some appear to be involved in important virulence-associated functions (toxins, immune evasion, iron uptake). The accessory seroproteome Pro-thrombin activation in thrombin may have a pro-inflammatory effect during mastitis and thus take part in the symptoms observed in animals infected by O11. Whether and how these proteins are involved Importance of secreted and exported proteins in seroproteomic patterns It is commonly assumed that surface exposed proteins play a role in host-pathogen interactions and that, because of their cellular localization, they are preferen- tially recognized by the host immune system. In this study, immunoreactive proteins were mainly identified Le Maréchal et al. Veterinary Research 2011, 42:35 http://www.veterinaryresearch.org/content/42/1/35 Page 16 of 20 in the acuteness of the disease induced by O11 remains unknown. O46 specifically produced 3 immunoreactive proteins when compared to O11. IsaA has been reported to be immunogenic in many S. aureus infec- tions [3,52,57,75-77]. It presents autolytic activity and is necessary for complete virulence [78] but its role in mastitis is not known. Interestingly, a protein (encoded by O46-2740) which shows similarity to exfoliative toxin D (ETD) was found produced by O46 and not by O11. Three amino-acids were shown to constitute the active site common to all the exfoliative toxins [79]. These amino-acids are present in O46_2740 gene (O46 strain) product but one is missing in O11-0490 gene product. The corresponding gene is not found in the recently released sequence of ovine strain ED133 [9] although we found its product in the exoproteome of 5 additional S. aureus strains isolated from subclinical mastitis. ETD is associated with cutaneous abscesses and furuncles [80]. Interestingly this toxin is also produced by Coagu- lase Negative Staphylococci species like Staphylococcus hyicus, Staphylococcus pseudintermedius and Staphylo- coccus chromogenes. CNS are highly prevalent in ovine subclinical mastitis [81]. Whether this particular toxin is specifically produced and plays a role during subclinical mastitis remains to be tested. Altogether, proteins differ- entially produced by O11 and O46 may be considered as potential marker of gangrenous or subclinical mastitis but this has still to be further demonstrated. proteins (IsdA, IsdB, IsdH, MntC and SirA, 4 of which belonged to the core seroproteome) consistent with the culture conditions (iron depletion) and with a role in the physiologically important and difficult iron uptake in the mastitis conditions. Additional file 1: Table S1: Criteria used to define the acuteness of mastitis symptoms. Additional file 2: Figure S1: Dynamics of gangrenous mastitis onset in ewes infected by S. aureus O11 (argyles) and O46 (squares). References H l T Gels run in parallel were immunoblotted using the pools of sera obtained from group 1 (infected with O11) animals (middle panels) or from group 2 (infected with O46) animals (right panels). Samples were run in parallel on 13 cm gels (pI 4-7; 12% SDS-PAGE). Spots identified by MS/MS are labeled. Additional file 6: Figure S5: Representative 2-DE gels and SERPA on cell wall fraction (upper panels) and total proteins (lower panels) of S. aureus O11. Supernatant samples were prepared from late exponential phase cultures of S. aureus strains grown anaerobically on iron-depleted RPMI. Preparative 2-DE gels were Coomassie blue stained (left panel). Gels run in parallel were immunoblotted using the pools of sera obtained from group 1 (infected with O11) animals (middle panels) or from group 2 (infected with O46) animals (right panels). Samples were run in parallel on 13 cm gels (pI 4-7; 12% SDS-PAGE). Spots identified by MS/MS are labeled. 6. Vautor E, Abadie G, Guibert JM, Huard C, Pepin M: Genotyping of Staphylococcus aureus isolated from various sites on farms with dairy sheep using pulsed-field gel electrophoresis. Vet Microbiol 2003, 96:69-79 7. 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Supernatant samples were prepared from late exponential phase cultures of S. aureus strains grown aerobically on iron-depleted RPMI. Preparative 2-DE gels were Coomassie blue stained (left panel). Gels run in parallel were immunoblotted using the pools of sera obtained from group 1 (infected with O11) animals (middle panels) or from group 2 (infected with O46) animals (right panels). Additional material Additional file 2: Figure S1: Dynamics of gangrenous mastitis onset ewes infected by S. aureus O11 (argyles) and O46 (squares). Page 17 of 20 Page 17 of 20 Le Maréchal et al. Veterinary Research 2011, 42:35 http://www.veterinaryresearch.org/content/42/1/35 Le Maréchal et al. Veterinary Research 2011, 42:35 http://www.veterinaryresearch.org/content/42/1/35 Le Maréchal et al. Veterinary Research 2011, 42:35 http://www.veterinaryresearch.org/content/42/1/35 Received: 18 October 2010 Accepted: 15 February 2011 Published: 15 February 2011 Received: 18 October 2010 Accepted: 15 February 2011 Published: 15 February 2011 Additional file 3: Figure S2: (A) Intramammary growth of S. aureus strain O11 and O46 in CD-1 mice. Populations are the mean values of S. f Additional file 3: Figure S2: (A) Intramammary growth of S. aureus strain O11 and O46 in CD-1 mice. Populations are the mean values of S. aureus counts in homogenates of 12 mice mammary gland. (B) Temperature of infected mice 24h post-infusion. The mean value of the groups of mice infected with S. aureus O11 or O46 is given. The dashed line indicates the temperature of the animals at T0, before infection. Asterisks indicate statistically significant values. Authors’ contributions CLM participated in the experimental mastitis in ewes, carried out the 2-DE analyses, participated in the identification of the immunoreactive proteins and drafted the manuscript. JJ and GJ participated in the identification of the immunoreactive proteins by mass spectrometry, SE, NB and RT participated in the design of the study and in the results analyses, CP, JMG and EV carried out the experimental mastitis in ewes, DD and EM carried out the experimental mastitis in mice, DH, PF and JS carried out the genome sequencing and genome sequence analyses, EV and YLL conceived the study, and participated in its design and coordination. All authors read and approved the final manuscript. 17. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227:680-685. 17. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227:680-685. 18. 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Acknowledgements C li L M é h l g Caroline Le Maréchal is the recipient of a PhD grant from the French National Institute for Agricultural Research (INRA) and the Agence Nationale de Sécurité Sanitaire (ANSES), IMISa Project. CLM received a 3-month grant from Université Européenne de Bretagne (UEB). 12. Trivier D, Davril M, Houdret N, Courcol RJ: Influence of iron depletion on growth kinetics, siderophore production, and protein expression of Staphylococcus aureus. FEMS Microbiol Lett 1995, 127:195-199. 13. Hernandez D, Francois P, Farinelli L, Osteras M, Schrenzel J: De novo bacterial genome sequencing: millions of very short reads assembled on a desktop computer. Genome Res 2008, 18:802-809. References H l T 1. Halasa T, Nielen M, Huirne RBM, Hogeveen H: Stochastic bio-economic model of bovine intramammary infection. Livest Sci 2009, 124:295-305. 1. Halasa T, Nielen M, Huirne RBM, Hogeveen H: Stochastic bio-economic model of bovine intramammary infection. 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Acta Pathol Microbiol Immunol Scand B 1983, 91:351-356. 108. doi:10.1186/1297-9716-42-35 Cite this article as: Le Maréchal et al.: Staphylococcus aureus seroproteomes discriminate ruminant isolates causing mild or severe mastitis. Veterinary Research 2011 42:35. Competing interests Holtfreter S, Kolata J, Broker BM: Towards the immune proteome of Staphylococcus aureus - The anti-S. aureus antibody response. Int J Med Microbiol 2010, 300:176-192. 109. Verkaik NJ, Dauwalder O, Antri K, Boubekri I, de Vogel CP, Badiou C, Bes M, Vandenesch F, Tazir M, Hooijkaas H, Verbrugh HA, van Belkum A, Etienne J, Lina G, Ramdani-Bouguessa N, van Wamel WJ: Immunogenicity of toxins during Staphylococcus aureus infection. Clin Infect Dis 2010, 50:61-68. 110. Rainard P, Corrales JC, Barrio MB, Cochard T, Poutrel B: Leucotoxic activities of Staphylococcus aureus strains isolated from cows, ewes, and goats with mastitis: importance of LukM/LukF’-PV leukotoxin. Clin Diagn Lab Immunol 2003, 10:272-277. 111. 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https://openalex.org/W4292324214	https://www.nature.com/articles/s41598-022-17846-0.pdf	English	null	Association between prognostic factors and the clinical deterioration of preterm neonates with necrotizing enterocolitis	Scientific reports	2,022	cc-by	4,485	Ibnu Sina Ibrohim1, Henggar Allest Pratama1, Aditya Rifqi Fauzi1, Kristy Iskandar2, Nunik Agustriani1 & Gunadi1* Ibnu Sina Ibrohim1, Henggar Allest Pratama1, Aditya Rifqi Fauzi1, Kristy Iskandar2, Nunik Agustriani1 & Gunadi1* Necrotizing enterocolitis (NEC) is responsible for most morbidity and mortality in neonates. Early recognition of the clinical deterioration in newborns with NEC is essential to enhance the referral and management and potentially improve the outcomes. Here, we aimed to identify the prognostic factors and associate them with the clinical deterioration of preterm neonates with NEC. We analyzed the medical records of neonates with NEC admitted to our hospital from 2016 to 2021. We ascertained 214 neonates with NEC. The area under the receiver operating characteristic (ROC) curve and cut-off level of age at onset, C-reactive protein (CRP), leukocyte count, and platelet count for the clinical deterioration of preterm neonates with NEC was 0.644 and 10.5 days old, 0.694 and 4.5 mg/L, 0.513 and 12,200/mm3, and 0.418 and 79,500/mm3, respectively. Late-onset, history of blood transfusion, thrombocytopenia, and elevated CRP were significantly associated with the clinical deterioration of neonates with NEC (p = < 0.001, 0.017, 0.001, and < 0.001, respectively), while leukocytosis, gestational age, and birth weight were not (p = 0.073, 0.274, and 0.637, respectively). Multivariate analysis revealed that late-onset and elevated CRP were strongly associated with the clinical deterioration of neonates with NEC, with an odds ratio of 3.25 (95% CI = 1.49–7.09; p = 0.003) and 3.53 (95% CI = 1.57–7.95; p = 0.002), respectively. We reveal that late-onset and elevated CRP are the independent prognostic factor for the clinical deterioration of preterm neonates with NEC. Our findings suggest that we should closely monitor preterm neonates with NEC, particularly those with late-onset of the disease and those with an elevated CRP, to prevent further clinical deterioration and intervene earlier if necessary. Abbreviations PRC Packed red cell CRP C-reactive protein CI Confidence interval OR Odds ratio NEC Necrotizing enterocolitis Necrotizing enterocolitis (NEC) is a severe gastrointestinal emergency that affects preterm neonates1,2. NEC is responsible for most perioperative fatalities in pediatric surgery, with a mortality rate of up to 19%3. However, studies from developing countries on the clinical deterioration in preterm neonates are minimal. p g p In addition, validated early indicators of clinical deterioration in preterm neonates with NEC are essential. Early predictors for surgery in premature neonates with NEC would help enhance referral and treatment path- ways and potentially improve outcomes4. www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| (2022) 12:13911 Association between prognostic factors and the clinical deterioration of preterm neonates with necrotizing enterocolitis OPEN Ibnu Sina Ibrohim1, Henggar Allest Pratama1, Aditya Rifqi Fauzi1, Kristy Iskandar2, Nunik Agustriani1 & Gunadi1* Methods Patients Patients. A retrospective study was conducted using medical records of neonates with NEC at our institu- tion from January 2016 to June 2021. We included 237 diagnosed premature neonates with NEC, with the Inter- national Classification of Diagnosis (ICD) X code of P.77. Subsequently, we excluded 10 and 13 neonates due to term neonates and incomplete medical records, respectively. We investigated 214 neonates for final analysis5. Staging of NEC. According to modified Bell’s staging, the diagnosis and staging of NEC were established, consisting of the severity of systemic, intestinal, radiographic, and laboratory findings6. Prognostic factors and ROC curve. We evaluated the following prognostic factors for the clinical dete- rioration of preterm neonates with NEC: age at onset, C-reactive protein (CRP), leukocyte count, platelet count, history of packed red cell (PRC) transfusion, gestational age, and birth weight. The CRP level was determined using an immunochemical assay (NycoCard™ CRP, Abbott, US) with the normal value of < 5 mg/L. We defined clinical outcomes of NEC into two categories: worsened and improved. The clinical deterioration was defined as worsening of the modified Bell’s staging. According to gestational age, the preterm birth was classified into the following: extremely preterm (< 28 weeks), very preterm (28 to < 32 weeks), and moderate to late preterm (32 to < 37 weeks)7. The birth weight was defined as normal birth weight (NBW) (≥ 2500 g), low birth weight (LBW) (< 2500 g), very low birth weight (VLBW) (< 1500 g), and extremely low birth weight (ELBW) (< 1000 g)8.hf The cut-off value of age at onset, CRP, leukocyte count, and platelet count was analyzed by receiver operating characteristics (ROC) curves. According to a previous study, we assumed the cut-off level for late-onset NEC in preterm infants was ≥ 14 days9. Statistical analysis. The Chi-square test was used to determine the association between the prognostic factors and the clinical deterioration of premature neonates with NEC. The multivariate regression test was used to look for the strong prognostic factors for clinical deterioration of premature neonates with NEC. The p-value of < 0.05 was determined as a significant level. The IBM SPSS Statistics version 16 (SPSS Chicago, USA) was used for all statistical analyses. Ethics approval and consent to participate. This study was approved by the Institutional Review Board of the Faculty of Medicine, Universitas Gadjah Mada/Dr. Sardjito Hospital, Yogyakarta, Indonesia (KE/ FK/0464/EC/2021). www.nature.com/scientificreports/ www.nature.com/scientificreports/ Table 1. Baseline characteristics of neonates with NEC in our institution. NEC necrotizing enterocolitis, PRC packed red cell. s. Characteristics N (%) Sex Male 98 (45.8) Female 116 (54.2) History of PRC transfusion Yes 112 (52.3) No 102 (47.7) Clinical outcomes Worsened 49 (23) Improved 165 (77) Characteristics N (%) Sex Male 98 (45.8) Female 116 (54.2) History of PRC transfusion Yes 112 (52.3) No 102 (47.7) Clinical outcomes Worsened 49 (23) Improved 165 (77) Table 1. Baseline characteristics of neonates with NEC in our institution. NEC necrotizing enterocolitis, PR packed red cell. s. Table 1. Baseline characteristics of neonates with NEC in our institution. NEC necrotizing enterocolitis, PRC packed red cell. s. Methods Patients Written informed consent was obtained from all parents for participating in this study. The research has been performed following the Declaration of Helsinki. Ibnu Sina Ibrohim1, Henggar Allest Pratama1, Aditya Rifqi Fauzi1, Kristy Iskandar2, Nunik Agustriani1 & Gunadi1* Here, we aimed to identify the prognostic factors and associate them with the clinical deterioration of preterm neonates with NEC. 1Pediatric Surgery Division, Department of Surgery, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada/Dr, Sardjito Hospital, Yogyakarta 55281, Indonesia. 2Department of Child Health, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, UGM Academic Hospital, Yogyakarta 55291, Indonesia. email: drgunadi@ugm.ac.id Scientific Reports \| (2022) 12:13911 \| https://doi.org/10.1038/s41598-022-17846-0 Results Results Baseline characteristics. A total of 214 preterm neonates with NEC were included in the study, with 77% improved clinical outcomes (Table 1). Baseline characteristics. A total of 214 preterm neonates with NEC were included in the study, with 77% mproved clinical outcomes (Table 1). Association between prognostic factors and clinical deterioration of preterm neonates with NEC. The area under the receiver operating characteristic (ROC) curve and cut-off level of age at onset, C-reactive protein (CRP), leukocyte count, and platelet count for the clinical deterioration of preterm neonates with NEC was 0.644 and 10.5 days old, 0.694 and 4.5 mg/L, 0.513 and 12,200/mm3, and 0.418 and 79,500/mm3, respectively (Fig. 1).i y g Late-onset, history of blood transfusion, thrombocytopenia, and elevated CRP were significantly associated with the clinical deterioration of neonates with NEC (p = < 0.001, 0.017, 0.001, and < 0.001, respectively), while leukocytosis, gestational age, and birth weight were not (p = 0.073, 0.274, and 0.637, respectively). (Table 2). Multivariate analysis of prognostic factors for clinical deterioration of preterm neonates with NEC. Multivariate analysis revealed that late-onset and elevated CRP were strongly associated with the clini- Scientific Reports \| (2022) 12:13911 \| https://doi.org/10.1038/s41598-022-17846-0 www.nature.com/scientificreports/ Figure 1. ROC curve of leucocyte count (A), platelet count (B), C-reactive protein (C), and the onset of NEC (D) for distinguishing between worsened and improved clinical deterioration of NEC, with the area under the ROC curve of 0.513, 0.418, 0.694, and 0.644, respectively. Figure 1. ROC curve of leucocyte count (A), platelet count (B), C-reactive protein (C), and the onset of NEC (D) for distinguishing between worsened and improved clinical deterioration of NEC, with the area under the ROC curve of 0.513, 0.418, 0.694, and 0.644, respectively. cal deterioration of neonates with NEC, with an odds ratio of 3.25 (95% CI = 1.49–7.09; p = 0.003) and 3.53 (95% CI = 1.57–7.95; p = 0.002), respectively (Table 3). cal deterioration of neonates with NEC, with an odds ratio of 3.25 (95% CI = 1.49–7.09; p = 0.003) and 3.53 (95% CI = 1.57–7.95; p = 0.002), respectively (Table 3). cal deterioration of neonates with NEC, with an odds ratio of 3.25 (95% CI = 1.49–7.09; p = 0.003) and 3.53 (95% CI = 1.57–7.95; p = 0.002), respectively (Table 3). Discussion Since the survival of preterm newborns is continuously enhanced, it is advised that clinicians search for the modi- fiable prognostic factors for NEC to intervene earlier and prevent further clinical deterioration10,11. The frequency of clinical deterioration of our patients was 23%. It is similar to a previous study12. In addition, our study shows that late-onset and elevated CRP were the strong prognostic factors for the clinical deterioration of NEC with the OR of ~ 4 and fivefold, respectively. It is similar to a previous study13. CRP is an acute-phase reactant that rises in the bloodstream in response to infection or tissue damage. The liver generates it in response to infection or tissue damage that causes inflammation. CRP was shown to be higher in 83% of neonates with confirmed NEC at the time of diagnosis compared to those without NEC in a previous study14. Pourcyrous et al.15 recently used serial CRP measures to identify preterm infants with NEC in a large cohort. CRP levels were abnormal in both stages II and III of NEC15. They concluded that persistently high CRP levels in neonates with NEC might indicate persisting illness and/or complications and suggested serial CRP tests for NEC follow-up15. Notably, https://doi.org/10.1038/s41598-022-17846-0 Scientific Reports \| (2022) 12:13911 \| www.nature.com/scientificreports/ Table 2. Association between prognostic factors and clinical deterioration with NE p<0 05; CI confidence interval OR odds ratio NEC necrotizing enterocolitis PRC Variables Clinical outcomes OR (95% CI) p-value Worsened (N, %) Improved (N, %) Age at onset (days) < 0.001 ≥ 10.5 29 (38.2) 47 (61.8) 2.63 (1.6–4.32) < 10.5 20 (14.5) 118 (85.5) History of PRC transfusion 0.017* Yes 33 (29.5) 79 (70.5) 2.25 (1.15–4.39) No 16 (15.8) 86 (84.2) Leukocyte count (/mm3) 0.073 ≥ 12,200 27 (28.7) 67 (71.3) 1.57 (0.96–2.57) < 12,200 22 (18.3) 98 (81.7) Platelet count (/mm3) 0.001* ≤ 79,500 27 (35.1) 50 (64.9) 2.18 (1.34–3.56) > 79,500 22 (16.1) 115 (83.9) CRP (mg/L) < 0.001* ≥ 4.5 40 (36.4) 70 (63.6) 4.2 (2.15–8.23) < 4.5 9 (8.7) 95 (91.3) Gestational age 0.274 Extremely preterm 1 (7.1) 13 (92.9) 0.22 (0.03–1.78) Very preterm 16 (21.3) 59 (78.3) 0.79 (0.39–1.56) Moderate to late preterm 32 (25.6) 93 (74.4) Birth weight 0.637 ELBW 12 (30) 28 (70) 2.14 (0.41–11.29) VLBW 15 (20.3) 59 (79.7) 1.27 (0.25–6.43) LBW 20 (22.7) 68 (77.3) 1.47 (0.29–7.27_ NBW 2 (16.7) 10 (83.3) Table 2. Discussion Association between prognostic factors and clinical deterioration with NEC in our institution. , p < 0.05; CI, confidence interval, OR odds ratio, NEC necrotizing enterocolitis, PRC packed red cell, CRP C-reactive protein, NBW normal birth weight, LBW low birth weight, VLBW very low birth weight, ELBW extremely low birth weight. Variables Clinical outcomes OR (95% CI) p-value Worsened (N, %) Improved (N, %) Age at onset (days) < 0.001 ≥ 10.5 29 (38.2) 47 (61.8) 2.63 (1.6–4.32) < 10.5 20 (14.5) 118 (85.5) History of PRC transfusion 0.017* Yes 33 (29.5) 79 (70.5) 2.25 (1.15–4.39) No 16 (15.8) 86 (84.2) Leukocyte count (/mm3) 0.073 ≥ 12,200 27 (28.7) 67 (71.3) 1.57 (0.96–2.57) < 12,200 22 (18.3) 98 (81.7) Platelet count (/mm3) 0.001* ≤ 79,500 27 (35.1) 50 (64.9) 2.18 (1.34–3.56) > 79,500 22 (16.1) 115 (83.9) CRP (mg/L) < 0.001* ≥ 4.5 40 (36.4) 70 (63.6) 4.2 (2.15–8.23) < 4.5 9 (8.7) 95 (91.3) Gestational age 0.274 Extremely preterm 1 (7.1) 13 (92.9) 0.22 (0.03–1.78) Very preterm 16 (21.3) 59 (78.3) 0.79 (0.39–1.56) Moderate to late preterm 32 (25.6) 93 (74.4) Birth weight 0.637 ELBW 12 (30) 28 (70) 2.14 (0.41–11.29) VLBW 15 (20.3) 59 (79.7) 1.27 (0.25–6.43) LBW 20 (22.7) 68 (77.3) 1.47 (0.29–7.27_ NBW 2 (16.7) 10 (83.3) Table 2. Association between prognostic factors and clinical deterioration with NEC in our institution. , p < 0.05; CI, confidence interval, OR odds ratio, NEC necrotizing enterocolitis, PRC packed red cell, CRP C-reactive protein, NBW normal birth weight, LBW low birth weight, VLBW very low birth weight, ELBW extremely low birth weight. Table 2. Association between prognostic factors and clinical deterioration with NEC in our institution. , p < 0.05; CI, confidence interval, OR odds ratio, NEC necrotizing enterocolitis, PRC packed red cell, CRP C-reactive protein, NBW normal birth weight, LBW low birth weight, VLBW very low birth weight, ELBW extremely low birth weight. Table 3. Multivariate analysis of the clinical deterioration of premature neonates with NEC in our institution. , p < 0.05; CI confidence interval, OR odds ratio, NEC necrotizing enterocolitis, PRC packed red cell, CRP C-reactive protein. Data availability y All data generated or analyzed during this study are included in the submission. The raw data are available from the corresponding author on reasonable request. Received: 22 November 2021; Accepted: 2 August 2022 Received: 22 November 2021; Accepted: 2 August 2022 Conclusions We reveal that late-onset and elevated CRP are the independent prognostic factor for the clinical deterioration of preterm neonates with NEC. Our findings suggest that we should closely monitor preterm neonates with NEC, particularly those with late-onset of the disease and those with an elevated CRP, to prevent further clinical deterioration and intervene earlier if necessary. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Thrombocytopenia is frequently found in NEC neonates and is a poor prognostic variable when platelet counts drop quickly26. Lower platelet counts were linked to the development of surgical NEC26. On the other hand, low platelet count was not found to be a reliable predictor of surgical NEC17. Intestinal ischemia can result in platelet thrombi and platelet consumption at the local level27. Furthermore, these platelet thrombi may hasten the bowel’s necrosis process. Our study did not support thrombocytopenia as a significant prognostic factor for clinical deterioration of NEC. PRC transfusion has been linked to the development of NEC in premature infants28–30. However, our study did not reveal a significant association between the history of PRC transfusion and clinical deterioration of preterm neonates with NEC. Christensen et al.29 found that neonates who had surgery for NEC after PRC transfusion had a greater mortality rate than infants who had surgery for NEC without PRC transfusion. However, these differences were not statistically significant. In addition, PRC transfusion is linked to an increased risk of surgi- cal intervention31. This might be caused by the donated PRCs that are exposed to significant stress during the collection, preservation, and storage32. Red blood cells go through structural and metabolic changes that might impair intestinal oxygen transport33,34. The production of cytokines and other pro-inflammatory substrates after infusion of these products might cause an inflammatory response33. gl y In our cohort, leukocytosis did not correlate with the deterioration of premature NEC neonates. In most cases, leukocytosis is thought to be a response to inflammatory processes. Various etiologies, including infection, inflammation, medications, and stress, may lead to a change of leucocyte counts in newborns35. l y g y Our study has limitations, such as a retrospective design and a single-center report, indicating that more multicenter research is needed to validate our findings. These facts should be considered during the interpreta- tion of our results. Discussion Variables OR (95% CI) p-value Late-onset 3.25 (1.49–7.09) 0.003 History of PRC transfusion 2.04 (0.95–4.39) 0.07 Thrombocytopenia 1.27 (0.59–2.75) 0.539 Elevated CRP 3.53 (1.57–7.95) 0.002* Variables OR (95% CI) p-value Late-onset 3.25 (1.49–7.09) 0.003* History of PRC transfusion 2.04 (0.95–4.39) 0.07 Thrombocytopenia 1.27 (0.59–2.75) 0.539 Elevated CRP 3.53 (1.57–7.95) 0.002* Table 3. Multivariate analysis of the clinical deterioration of premature neonates with NEC in our institution. *, p < 0.05; CI confidence interval, OR odds ratio, NEC necrotizing enterocolitis, PRC packed red cell, CRP C-reactive protein. we do not have any data on blood culture. Therefore, our findings should be interpreted cautiously; mainly, the CRP might reflect the intestinal necrosis that occurs in NEC rather than infection.fi we do not have any data on blood culture. Therefore, our findings should be interpreted cautiously; mainly, the CRP might reflect the intestinal necrosis that occurs in NEC rather than infection.fi l In this study, the cut-off level of age at onset was 10.5 days. Our findings were compatible with another study16. We showed that premature neonates with late-onset NEC are fivefold more likely to deteriorate clinically than those with early-onset NEC. Our findings are compatible with a previous study17. However, previous studies revealed that the early onset of NEC is an independent prognostic factor for NEC to be managed surgically16,18,19. The timing of NEC development in term and preterm infants has been studied extensively in a prior study20. Early-onset NEC individuals were more likely to require surgery20. A previous study suggested that a more widespread immaturity of the intestinal innate immune response might contribute to the observed increased inflammation in the juvenile gut and hence play a role in the onset of NEC21.h l j g p y There was a strong inverse association between gestational age and birth weight with NEC mortality22,23. However, our study did not show any association between gestational age and birth weight with the clinical deterioration of NEC. These differences might be due to several factors, including broad discrepancies in patient populations, disease degree, coexisting illnesses, and severity classification among institutions24. Moreover, the following important variables might explain the NEC occurring in neonates with LBW, such as the immaturity of the gastrointestinal tract and its function, barrier function, regulation of circulation, and immune defense25. Scientific Reports \| (2022) 12:13911 \| https://doi.org/10.1038/s41598-022-17846-0 References 1. 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(Accessed on June 9, 2022). y GT. Preterm birth: Definitions of prematurity, epidemiology, and p 9. Berrington, J. E. & Embleton, N. D. Time of onset of necrotizing enterocolitis and focal perforation in preterm infants: impact on clinical, surgical, and histological features. Front. Pediatr. 9, 724280 (2021).h 9. Berrington, J. E. & Embleton, N. D. Time of onset of necrotizing enterocolitis and focal perforation in preterm infants: impact on clinical, surgical, and histological features. Front. Pediatr. 9, 724280 (2021).h g g 0. Alsaied, A., Islam, N. & Thalib, L. Global incidence of necrotizing enterocolitis: A systematic review and meta-analysis. BMC Pediatr. 20, 344 (2020). 1. Siahaan, E. S. E. D. et al. www.nature.com/scientificreports/ Am. J. Perinatol. 23, 451–458 (2006). 5. Morag, I., Dunn, M., Nayot, D. & Shah, P. S. Leukocytosis in very low birth weight neonates: Associated clinical factors and neonata outcomes. J. 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Prevention of necrotizing enterocolitis in preterm very low birth weight infants: f bl ? J F M d A ( ) ( ) 5. Lin, H. Y., Chang, J. H., Chung, M. Y. & Lin, H. C. Prevention of necrotizing enterocolitis in preterm very low birth weight infants is it feasible?. J. Formos. Med. Assoc. 113(8), 490–497 (2014). 26. Dominguez, K. M. & Moss, R. L. Necrotizing enterocolitis. Clin. Perinatol. 39, 387–401 (2012). 27. Hutter, J. J. Jr., Hathaway, W. E. & Wayne, E. R. Hematologic abnormalities in severe neonatal necrotizing enterocolitis. J. Pe 88, 1026–1031 (1976).t 8. Blau, J. et al. Transfusion-related acute gut injury: necrotizing enterocolitis in very low birth weight neonates after packed red blood cell transfusion. J. Pediatr. 158, 403–409 (2011). 9. Christensen, R. D. et al. Is “transfusion-associated necrotizing enterocolitis” an authentic pathogenic entity?. Transfusion 50 1106–1112 (2010). 30. Josephson, C. D. et al. Do red cell transfusions increase the risk of necrotizing enterocolitis in premature infants?. J. Pediatr. 157, 972–978 (2010). ( ) 31. Cunningham, K. E., Okolo, F. C., Baker, R., Mollen, K. P. & Good, M. Red blood cell transfusion in premature infants leads to worse necrotizing enterocolitis outcomes. J. Surg. Res. 213, 158–165 (2017). g g 2. La Gamma, E. F., Feldman, A., Mintzer, J., Lakshminrusimha, S. & Alpan, G. Red blood cell storage in transfusion-related acute gut injury. Neo Rev. 16, e420–e430 (2015). 33. Sharma, R. & Hudak, M. L. A clinical perspective of necrotizing enterocolitis: Past, present, and future. Clin. Perinatol. 40, 27–51 (2013). ( ) 34. Mally, P. et al. Association of necrotizing enterocolitis with elective packed red blood cell transfusions in stable, growing, premature neonates. Acknowledgements g We extend our thanks to physicians and nurses who provided excellent management for the patients. Some results for the manuscript are from Ibnu Sina Ibrohim’s thesis. g We extend our thanks to physicians and nurses who provided excellent management for the patients. Some results for the manuscript are from Ibnu Sina Ibrohim’s thesis. Author contributions Author contributions I.S.I., N.A., and G. conceived the study. I.S.I., A.R.F., K.I., and G. drafted the manuscript. I.S.I. and G. analyzed the data. H.A.P., I.S.I., N.A., and G. facilitated all project-related tasks. All authors read and approved the final manuscript. I.S.I., N.A., and G. conceived the study. I.S.I., A.R.F., K.I., and G. drafted the manuscript. I.S.I. and G. analyzed the data. H.A.P., I.S.I., N.A., and G. facilitated all project-related tasks. All authors read and approved the final manuscript. I.S.I., N.A., and G. conceived the study. I.S.I., A.R.F., K.I., and G. drafted the manuscript. I.S.I. and G. analyzed the data. H.A.P., I.S.I., N.A., and G. facilitated all project-related tasks. All authors read and approved the final manuscript. References Outcomes and prognostic factors for survival of neonates with necrotizing enterocolitis. Front. Pediatr 9, 744504 (2021). ( ) 12. Wang, Z. L. et al. Probiotics may not prevent the deterioration of necrotizing enterocolitis from stage I to II/III. BMC Pediatr. 19, 1–7 (2019). 13. Luo, L. et al. Correlative factors of the deterioration of necrotizing enterocolitis in small for gestational age newborns. Sci Rep. 8, 1–6 (2018).fi 14. Çetinkaya, M., Özkan, H., Köksal, N., Akacı, O. & Özgür, T. Comparison of the efficacy of serum amyloid A, C-reactive protein, and procalcitonin in the diagnosis and follow-up of necrotizing enterocolitis in premature infants. J. Pediatr. Surg. 46, 1482–1489 (2011). ( ) 15. Pourcyrous, M., Korones, S. B., Yang, W., Boulden, T. F. & Bada, H. S. C-reactive protein in the diagnosis, management prognosis of neonatal necrotizing enterocolitis. Pediatrics 116, 1064–1069 (2005). y , , , , g, , , , p g , g , rognosis of neonatal necrotizing enterocolitis. Pediatrics 116, 1064–1069 (2005). p g g 16. Berman, L. & Moss, R. L. Necrotizing enterocolitis: an update. Semin. Fetal Neonatal. Med. 16, 145–150 (2011). 7. Short, S. S. et al. Late onset of necrotizing enterocolitis in the full-term infant is associated with increased mortality: Results from a two-center analysis. J. Ped. Surg. 49, 950–953 (2014). 18. El Hassani, S. E. et al. Predictive factors for surgical treatment in preterm neonates with necrotizing enterocolitis: A multicenter case-control study. Eur. J. Ped. 180, 617–625 (2021). y ( ) 19. Duci, M. et al. Neonatal independent predictors of severe NEC. Ped. Surg. Int. 34, 663–669 (2018). p p g 20. Yee, W. H. et al. Canadian neonatal network. Incidence and timing of presentation of necrotizing enterocolitis in preterm infants. Pediatrics 129(2), e298-304 (2012). https://doi.org/10.1038/s41598-022-17846-0 Scientific Reports \| (2022) 12:13911 \| Competing interests h g The authors declare no competing interests. Additional information Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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. 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https://openalex.org/W4376654406	https://zenodo.org/records/7943641/files/111.pdf	English	null	LEXICAL-SEMANTIC ANALYSIS OF OCCUPATION NAMES USED IN SOME TURKISH DICTIONARIES OF THE XI-XIV CENTURY	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	1,266	06 International scientific journal «MODERN SCIENCE АND RESEARCH» VOLUME 2 / ISSUE 5 / UIF:8.2 / MODERNSCIENCE.UZ XI-XIV ASRDAGI BA'ZI TURKIY LUGʻATLARDA QOʻLLANGAN KASB NOMLARINING LEKSIK-SEMANTIK TAHLILI Amiriddinova Mohinur Xonmurotova Munisa Termiz davlat universiteti talabalari Xidirova Iroda Ilmiy rahbar, Termiz davlat universiteti https://doi.org/10.5281/zenodo.7943641 Annotatsiya. Ushbu maqolada ayrim lug'atlarda qo'llanilgan kasb nomlari haqida ma'lumotlar keltirilgan. Kalit soʻzlar: Lug'atshunoslik, "Devoni lug'atit turk", “At-tuhfatuz zakiyati fil lug‘atit turkiya”, "Muhokamat ul-lug'atayn". LEXICAL-SEMANTIC ANALYSIS OF OCCUPATION NAMES USED IN SOME TURKISH DICTIONARIES OF THE XI-XIV CENTURY Abstract. This article provides information about occupational names used in some dictionaries. Key words: Lexicography, "Devoni lugatit turk", "At-tuhfatuz zakiyati fil lugatit turkiya", "Muhokamat ul-lugatayn". ЛЕКСИКО-СЕМАНТИЧЕСКИЙ АНАЛИЗ НАЗВАНИЙ ЗАНЯТИЙ, ИСПОЛЬЗУЕМЫХ В НЕКОТОРЫХ ТУРЕЦКИХ СЛОВАРЯХ XI-XIV ВЕКА Аннотация. В данной статье представлена информация о названиях профессий, используемых в некоторых словарях Annotatsiya. Ushbu maqolada ayrim lug atlarda qo llanilgan kasb nomlari haqida ma'lumotlar keltirilgan. Kalit soʻzlar: Lug'atshunoslik, "Devoni lug'atit turk", “At-tuhfatuz zakiyati fil lug‘atit turkiya”, "Muhokamat ul-lug'atayn". Abstract. This article provides information about occupational names used in some dictionaries. Key words: Lexicography, "Devoni lugatit turk", "At-tuhfatuz zakiyati fil lugatit turkiya", "Muhokamat ul-lugatayn". ЛЕКСИКО-СЕМАНТИЧЕСКИЙ АНАЛИЗ НАЗВАНИЙ ЗАНЯТИЙ, ЛЕКСИКО-СЕМАНТИЧЕСКИЙ АНАЛИЗ НАЗВАНИЙ ЗАНЯТИЙ, ЛЕКСИКО-СЕМАНТИЧЕСКИЙ АНАЛИЗ НАЗВАНИЙ ЗАНЯТИЙ, СПОЛЬЗУЕМЫХ В НЕКОТОРЫХ ТУРЕЦКИХ СЛОВАРЯХ XI-XIV ВЕКА ИСПОЛЬЗУЕМЫХ В НЕКОТОРЫХ ТУРЕЦКИХ СЛОВАРЯХ XI-XIV ВЕКА Аннотация. В данной статье представлена информация о названиях профессий, используемых в некоторых словарях. Аннотация. В данной статье представлена информация о названиях профессий, используемых в некоторых словарях. Ключевые слова: Лексикография, "Девони лугатит турк", "Ат-тухфатуз закияти фил лугатит туркия", "Мухокамат ул-лугатайн". Lug‘atshunoslik lugʻat tuzish ishi sifatida turli xalqlarda yozuv taraqqiyotining ilk bosqichlarida tushunarsiz (eskirgan, dialektal, maxsus yoki chet tilga mansub) soʻzning qanday maʼno anglatishini bilish ehtiyoji natijasida paydo boʻldi. Dastlabki tuzilgan lugʻatlar umumlashgan, universal xususiyatga ega edi. Lugʻatlarning turli shakllari, koʻrinishlari esa keyingi davrlarda yuzaga kelgan hodisadir. XIX asr oxiri va XX asrning birinchi choragida turli xil sabab va ehtiyojlar, davr taqozosi bilan Turkistonda ikki tilli lug‘atshunoslik rivoj topdi, oʻnlab ruscha-oʻzbekcha, oʻzbekcha-ruscha lugʻatlar,soʻzlashgichlar tuzilib nashr etildi. Oʻzbek leksikografiyasi tarixi Mahmud Koshgʻariyning “Devonu lugʻotit turk” asaridan boshlangan deb aytish mumkin. Ushbu lugʻat faqat soʻzlar va ularning maʼnolarini tavsiflab qolmay, turkiy xalqlar tarixi, urf-odatlari, geografik joylashuvi kabi keng maʼlumotlar manbayi hamdir. Shu bilan birga “Devonu lugʻotit turk” dastlabki ikki tilli (turkiycha-arabcha) lugʻatlardandir. Mahmud Zamaxshariy ham oʻzining “Asos ul-balogʻa”, “Muqaddimat ul-adab” asarlari bilan amaliy ham nazariy leksikografiyaning rivojiga ulkan hissa qoʻshdi. Shuningdek, Alisher Navoiy ijodiga boʻlgan katta qiziqish XV asrdan keyingi davrda bir qancha lugʻatlarning yaratilishiga sabab boʻldi: bunga XVI asrda Turkiyada yaratilgan “Abushqa” izohli lugʻati, Tole Imon Hiraviyning “Badoye ul-lugʻat”, Muhammad Rizo Xoksorning “Muntaxab ul-lugʻot”, Mirzo Mehdixonning “Sangloh”, Muhammad Yoqub Chingiyning “Kelurnoma”, Is’hoqxon Ibratning International scientific journal «MODERN SCIENCE АND RESEARCH» VOLUME 2 / ISSUE 5 / UIF:8.2 / MODERNSCIENCE.UZ International scientific journal «MODERN SCIENCE АND RESEARCH» VOLUME 2 / ISSUE 5 / UIF:8.2 / MODERNSCIENCE.UZ Xulosa qilib aytadigan bo'lsak, turkiy lug'atlarni bir-biri bilan taqqoslaganimizda bir so'zning har bir lug'atlarda oldingisini takrorlamagan holda turfa xil bo'lib kelishini tadqiq qilish biz uchun juda manfaatli va qiziqarli kechdi. REFERENCES 1. Khidirova, I. (2023). PEYORATIVE, DEGORATIVE VOCABULARY IN FAMILY SPEECH (EXAMPLE OF THE KHIDIROV AND SHAYMATOV FAMILIES). Modern Science and Research, 2(3). 1. Khidirova, I. (2023). PEYORATIVE, DEGORATIVE VOCABULARY IN FAMILY SPEECH (EXAMPLE OF THE KHIDIROV AND SHAYMATOV FAMILIES). Modern Science and Research, 2(3). 2. Xidirova, I., & Xidirova, N. (2021). Gender Characteristics of Family Speech Speech (On the Example of the Uzbek Family). EUROPEAN JOURNAL OF INNOVATION IN NONFORMAL EDUCATION, 1(2), 196-199. 2. Xidirova, I., & Xidirova, N. (2021). Gender Characteristics of Family Speech Speech (On the Example of the Uzbek Family). EUROPEAN JOURNAL OF INNOVATION IN NONFORMAL EDUCATION, 1(2), 196-199. 3. Raxmonjonova, G., & Xidirova, I. (2022). CHORTOQ SO ‘ZINING KELIB CHIQISHIGA OID ILMIY QARASHLAR. Eurasian Journal of Academic Research, 2(11), 188-189. 3. Raxmonjonova, G., & Xidirova, I. (2022). CHORTOQ SO ‘ZINING KELIB CHIQISHIGA OID ILMIY QARASHLAR. Eurasian Journal of Academic Research, 2(11), 188-189. 4. Jo’rayeva, M., & Xidirova, I. (2023). QOYALAR HAM YIG’LAYDI” ASARIDA QO’LLANILGAN DIALEKTIZMLARNING LEKSIK-SEMANTIK TAHLILI. Development of pedagogical technologies in modern sciences, 2(2), 42-48. 4. Jo’rayeva, M., & Xidirova, I. (2023). QOYALAR HAM YIG’LAYDI” ASARIDA QO’LLANILGAN DIALEKTIZMLARNING LEKSIK-SEMANTIK TAHLILI. Development of pedagogical technologies in modern sciences, 2(2), 42-48. 5. qizi Jo‘rayeva, M. S., & Xidirova, I. X. (2023, January). “TEMIR XOTIN” DRAMASINING FONETIK-FONOLOGIK TAHLILI. In INTERNATIONAL CONFERENCES (Vol. 1, No. 1, pp. 492-496). 5. qizi Jo‘rayeva, M. S., & Xidirova, I. X. (2023, January). “TEMIR XOTIN” DRAMASINING FONETIK-FONOLOGIK TAHLILI. In INTERNATIONAL CONFERENCES (Vol. 1, No. 1, pp. 492-496). 6. qizi Jo‘rayeva, M. S., & Xidirova, I. X. (2023, January). SHOYIM BO ‘TAYEVNING “SHO ‘RODAN QOLGAN ODAMLAR” ASARIDA NOADABIY QATLAM SO ‘ZLARINING QO ‘LLANILISHI. In INTERNATIONAL CONFERENCES (Vol. 1, No. 1, pp. 487-491). 7. Xidirova, I. ., & Jo‘rayeva, M. (2023). LEXICAL-SEMANTIC ANALYSIS OF DIALECTISMS USED IN THE WORK OF ART (IN THE EXAMPLE OF THE WORK "ROCKS ALSO CRY"). Modern Science and Research, 2(3), 142–144. 8. Xidirova, I. ., & Dobilova, M. (2023). IMLO MUAMMOLARI VA YECHIM. Modern Science and Research, 2(3), 138–141. 9. Alisher Navoiy. Muhokamat ul-lug'atayn. – Toshkent: 9. Alisher Navoiy. Muhokamat ul-lug'atayn. – Toshkent: 10. At-tuhfatuz zakiyatu fil lug’otit turkiya. – Toshkent: Fan, 1968. – B. 123. tuhfatuz zakiyatu fil lug’otit turkiya. – Toshkent: Fan, 1968. – B. 123. 11. Devonu lug’oti-t-turk. G'afur G'ulom nomidagi matbaa -nashriyot uyi,Toshkent, 2 12. Norboyevna Q. S., Shohista M. O‘ZBEK TILIDA KOSMONIMLARNING HOSIL BO‘LISH BOSQICHLARI //E Conference Zone. – 2022. – С. 48-50. 12. International scientific journal «MODERN SCIENCE АND RESEARCH» VOLUME 2 / ISSUE 5 / UIF:8.2 / MODERNSCIENCE.UZ “Lugʻati sitta as-sina” (“Olti tilli lugʻat”) kabi lugʻatlarini koʻrsatish mumkin. Mirzo Mehdixonning “Maboni al-lug‘at” asarini fors tilidan o‘zbek tiliga tarjima qilgan Zikrillo Umarov asar xususida shunday yozadi: “XVIII asrda fors turkologi Mirzo Mehdixonning “Maboni al- lug‘at” grammatik ocherki Navoiy asarlari asosida yozilgan birinchi eski o‘zbek tili grammatikasidir. Uning tadqiqi o‘zbek tili tarixiy grammatikasini tuzishda, eski o‘zbek tilining fonetik, grammatik xususiyatlarini va turkiy tillar tarixini o‘rganishda katta ahamiyatga ega [Mirzo Mehdixon 2008]. Barchamizga ma’lumki, tilshunoslikning leksikografiya bo’limi alohida e’tiborga molik. Sababi leksikografiya ya’ni lug’atchilik yaratgan asarlar shu jumladan lug’atlar barchamizga, yoshimiz, kasb-hunarimiz, qiziqishimizdan qat’i nazar hamisha kerakli kitoblar deb, bugungi kunda rivojlanib, kengayib borayotganligi fikrimning dalili bo’la oladi. Ushbu o'rganayotgan ishimizning asosiy oʻrganish obyekti sifatida XI–XIV asrlarda yaratilgan qadimgi lug‘atlar (“Devonu lug‘otit turk” (keying o‘rinlarda MK), “At-tuhfatuz zakiyati fil lug‘atit turkiya” (keying o‘rinlarda “At-tuhfat”) asarlarida soʻzlarning fonetikasi, fonologiyasi farqli hamda muqobil jihatlari, anglatgan maʼnolari ochib boriladi. "Devoni lug'atit turk" – tabib- atasag'un (114) jїрагу-cholg'uchi(43) D. 3-Jild yalnguq- choʻri (454) "Muhokamat ul-lug'atayn" – Atibbo- tabib(32) jorubkash (Жорўбкаш) — xizmatkor maʼnosida (34) darzchilik (дарзилик) — tikuvchilik (33) Saroyinda(Сароянда) — kuylovchi (45) "At-tuhfatuz zakiyatu fil- lugʻatit turk"– ichägi- tabib (214) tabu- xizmatchi (251) toquchï- toʻquvchi (261), bezirkan- savdogar (182), tejik- savdogar (256). Yuqorida keltirilgan lug'atlardan yana ayrim kasb nomlarini misol qilib keltiradigan bo'lsak; 1.avlavchï- ovchi (163) 3.bezirkan- savdogar (182) 4. dilmäch- tarjimon (194) 5. do'ro'tchi- yaratuvchi (194) 6.javurchi- etikdo'z (208) 7. ichägi- tabib (214) ISSN: 2181-3906 2023 International scientific journal «MODERN SCIENCE АND RESEARCH» VOLUME 2 / ISSUE 5 / UIF:8.2 / MODERNSCIENCE.UZ ISSN: 2181-3906 2023 REFERENCES Norboyevna Q. S., Shohista M. O‘ZBEK TILIDA KOSMONIMLARNING HOSIL BO‘LISH BOSQICHLARI //E Conference Zone. – 2022. – С. 48-50. 13. XIX asr oxiri XX asr boshlarida leksikografiya rivoji va unda “Lug‘oti salos”ning o‘rni. Odina Mahkamova. 13. XIX asr oxiri XX asr boshlarida leksikografiya rivoji va unda “Lug‘oti salos”ning o‘rni. Odina Mahkamova. 14. Normamatov S. Leksikografiya asoslari. – T., 2020. 14. Normamatov S. Leksikografiya asoslari. – T., 2020. 15. Xidirova, I. ., & Dobilova, M. (2023). IMLO MUAMMOLARI VA YECHIM. Modern Science and Research, 2(3), 138–141. 16. www.ziyonet.uz 15. Xidirova, I. ., & Dobilova, M. (2023). IMLO MUAMMOLARI VA YECHIM. Modern Science and Research, 2(3), 138–141. 16. www.ziyonet.uz 477
https://openalex.org/W4221091343	https://zenodo.org/records/6316510/files/IJISRT22JAN441%20(1).pdf	English	null	Research Gaps in Management Sciences: An X-Ray of Literature	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	5,209	Volume 7, Issue 1, January – 2022 Volume 7, Issue 1, January – 2022 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Research Gaps in Management Sciences: An X-Ray of Literature Azeez, F.T. Assistant Lecturer, Department of Insurance, Faculty of Management Science, Lagos State University, Ojo Elegunde, A.F. Senior Lecturer, Department of Business Administration, Faculty of Management Sciences, Lagos State University, Ojo gaps requires rigorous reading and analyzing of materials from various literatures (Farooq, 2018). Abstract:- Researchers and academia often have difficulties identifying the research gap in literature in various fields of study. Hence, exploring research gap is one of the most arduous tasks for researchers especially those at the preliminary stage. The explicit identification of research gap is an inevitable step in developing a research agenda including decision about funding and the design of informative studies. Thus, to identify the research gap, the researcher needs to prune down his area of interest as identifying research gaps requires a lot of reading and analyzing of materials from various literatures. Hence, this study explores literatures regarding the method of identifying research gaps in management sciences. This was done by extensively examining various literatures on the method of identifying research gaps from previous researchers. However, the study made use of content analysis to identify research gaps in some articles. This study revealed that researchers are focused on a single type of research gap, leaving other research gaps unexplored. Also, there are some methods of research identification that has remained understandable by researchers as there are little or no knowledge about them. Hence, the study recommended among others that the various research identification methods be explored by researchers who intend to engage in studies in this field of management sciences. Research gap according to Robinson, Saldanha and Mckoy (2011) arises when the ability of the systematic reviewer to draw conclusions is limited. Research gap can be referred to as a starting point for research (Mueller-Bloch &Kranz, 2015). Robinson et al., (2011) opined that research gap represents an output of literature reviews while Mueller- Bloch and Kranz (2015) perceives research gap as an input as it can be motivation for further research. However, regardless of how salient research gap seem to be to research development, there exist no specific research gap process defined in literature. Hence, this study aims at examining the literature development of the process of identifying research gap in research study. I. INTRODUCTION Currently, the body of research is growing and new concepts and constructs keep evolving. However, the meaning of the term research gap differs depending on the research context as there is no standardized meaning (Nyanchoka, Tudur-Smith, Thu, Iversen, Tricco & Porcher, 2019). A research question which has not been properly addressed known as a research gap (Farooq, 2018). Researchers and academician often find it difficult to identify the research gap in literature in their various fields (Farooq, 2018). Hence, exploring research gap is one of the most difficult tasks for researchers especially those at the preliminary stage (Farooq, 2018). Robinson, Saldanha and Mckoy (2011) cited in Farooq (2018) opined that the identification of research gaps in a clear and explicit manner is a salient step in developing a research agenda including decision regarding funding and designing informative studies. Thus, to identify the research gap, the researcher has to prune down his area of interest as identifying research Hence, there is a paucity of research about research gap analysis. There are rare studies which have conceptualized research gap based on certain dimension and propositions. Thus, the need for this study. II. STATEMENT OF THE PROBLEM The idea of establishing gaps in research has been a major concern for many researchers for some time as there were no formal or defined frameworks for identifying or characterizing research gaps (Miles, 2017). What constitutes a research gap to a researcher might not be what constitutes a research gap to another; hence, research gaps seem to be a case of “in the eye of the beholder” (Miles, 2017). However, the majority of the conflicts regarding research gaps are mostly based on perception (Miles, 2017). Identifying research gap from literature is a common practice but the criteria used seems to be ambiguous and vague (Farooq, 2018). Research gap analysis is ambiguous and equivocal for novice, young researchers as researchers finds it difficult and challenging to explore research gaps because of lack of criteria or predetermined procedures.  Objective of the Study  Objective of the Study This study examined how to identify research gaps for proper clarity in statement of the problem. III. CONCEPTUAL REVIEW gap does not lead to the expected outcome then the research gap identified is referred to as being vague and indefinite and as such has to be revised. A. Research Gaps A. Research Gaps Research gaps are defined as gaps in sets of information (Mueller-Bloch & Kranz, 2015). Also the National Collaborating Center for Methods and Tools in Canada described research gap as a research question for which paucity of information limits the ability to reach a conclusion. Scott, Carmen, Christa and Jacques (2008) also posited that research gaps are evidence missing from a body of research on a particular topic that could otherwise potentially answer the question of decision makers. Also research gap is when there are little or no information available and/or there is a high level of uncertainty about the accuracy of the existing estimate (Rudan et al., 2015), where additional research is needed from policy-makers perspective to address the gap in the available primary research (Scott et al., 2008). C. Identification of Research Gaps According to Farooq (2018) there are various conceptualizations regarding the identification of research gaps or methods of identifying research gaps. These can be through; a) Citation Analysis: Citation analysis is one of the most effective ways to identify and analyse research gap (Farooq, 2018), as studies which are frequently cited provide the basic understanding about problem identification. However, Hoffmann & Doucette (2012) opined that “Citation analysis is a branch of bibliometric which studies the citations found in publications such as journal articles and books to ascertain the patterns of use.  Literature Review  Literature Review This section gives an overview of the studies of various researchers. The section consists of the conceptual review, theoretical review and empirical review. 955 www.ijisrt.com IJISRT22JAN441 Volume 7, Issue 1, January – 2022 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 B. Research Gap Process Citation analysis is the most used method of identifying research gap with the aid of Google scholar, Scopus, Web-of-Science, CiteSeer, other scholarly database including Ebsco, ProQuest, Emerald etc. The keywords and nature of study are used in analyzing the citations. According to Farooq (2018) the current research gap process is based on five elements, which are; identifying the research gap, Methods of identifying the research gap, Feasibility of research gap, Selection of research gap and Expected Outcome.  Identifying the research gap: The method of identifying research gap in literature remains debatable as there is no generally accepted opinion among researchers and academicians. In order to identify research gaps the research will have to engage in a lot of reading and analyze the materials from literature and these have been made simplified with the existence of some online and electronic database. b) Content Analysis: Content analysis is a research technique used in qualitative research to make decisions and conclusions by interpreting the texts, images and documents. Content analysis reports can be very supportive for identifying the research gap in a qualitative research (Farooq, 2018). b) Content Analysis: Content analysis is a research technique used in qualitative research to make decisions and conclusions by interpreting the texts, images and documents. Content analysis reports can be very supportive for identifying the research gap in a qualitative research (Farooq, 2018).  Methods of identifying the research gaps: According to Tom (2012) the first step is to identify and select relevant information sources (such as books, catalogs, database, internet etc.). However, there are a number of methods used in identifying the research gap, these are via; Citation analysis report, meta-analysis reports, content analysis report and systematic review. “Content analysis is a class of methods at the intersection of the qualitative and quantitative traditions, is promising for the rigorous exploration of many important but difficult-to-study issues of interest to management researchers” (Duriau; Reger & Pfarrer, 2007).  Feasibility of research gap: Feasibility of research gap depends upon the availability of both primary and secondary data, sufficient literature available and statistical tools available. However, after exploring different methods of identifying research gaps, the research gap identified has to check if it is feasible, if not the method of identification will have to be modified and repeated. c) Meta-Analysis: Meta-analysis is the process of integrating the findings from previous studies by statistically analyzing the literature (Farooq, 2017). B. Research Gap Process Meta- Analysis report provides an overview regarding a particular construct; that is the measurement of the construct and the different findings of that particular construct. c) Meta-Analysis: Meta-analysis is the process of integrating the findings from previous studies by statistically analyzing the literature (Farooq, 2017). Meta- Analysis report provides an overview regarding a particular construct; that is the measurement of the construct and the different findings of that particular construct. d) Systematic Reviews: This is a scientific tool used in appraising, summarizing and communicating the result and implications of otherwise unmanageable quantities of research (Green, 2005). A systematic review collates, analyse literatures regarding a research problem from different studies. Systematic reviews are quantitative in nature whereby the researcher explores the literature that could support or contradict a finding depending on the form of study (Farooq, 2018).  Selection of research gap: The selection of research gaps depends on the available literature, the researcher’s own interest and the contribution to the study. Selection of research gap is similar to the decision making process whereby decision is taken based on the various alternatives.  Expected Outcomes: A researcher must have a prior knowledge of the expected outcome of the research being carried out, which should lead to contribution to knowledge or study. Thus, if the identified research 956 IJISRT22JAN441 www.ijisrt.com Volume 7, Issue 1, January – 2022 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 7, Issue 1, January 2022 International Journal of Innovative Science and Research Technolog ISSN No:-2456-21 Fig.1: Framework for identifying research gaps in literature review  Identifying Research Gap Fig. B. Research Gap Process 2: Framework for Identifying Research Gap Adopted from (Mueller-Bloch &Kranz, 2015) Localization Characterization Localization strategy depends on the type of research gap After locating a research gap, characterizing its type can help to specify what kind of research is required to address the research gap Presentation Verification Substantiating the existence of the research gap Adequately presenting the research gap in a literature review Fig.1: Conceptual Model of Research Gap Adapted from Farooq (2018) Citation Analysis Meta- Analysis Content Analysis Report Future Research and Limitations Systematic Reviews Research Gap Problem Identification Fi 1 F k f id tif i h i lit t i Fig.1: Conceptual Model of Research Gap Adapted from Farooq (2018) Citation Analysis Meta- Analysis Content Analysis Report Future Research and Limitations Systematic Reviews Research Gap Problem Identification Fi 1 F k f id if i h i li i Fig.1: Conceptual Model of Research Gap Adapted from Farooq (2018) Citation Analysis Meta- Analysis Content Analysis Report Future Research and Limitations Systematic Reviews Research Gap Problem Identification Citation Analysis Content Analysis Problem Identification Research Gap Meta- Analysis Identification Systematic Reviews Future Research and Limitations Fig.1: Conceptual Model of Research Gap Adapted from Farooq (2018) Fig.1: Conceptual Model of Research Gap Adapted from Farooq (2018) Fig.1: Framework for identifying research gaps in literature review  Identifying Research Gap  Identifying Research Gap Identifying Research Gap Localization Characterization Localization strategy depends on the type of research gap After locating a research gap, characterizing its type can help to specify what kind of research is required to address the research gap Fi 2 F k f Id if i R h G Localization Characterization Localization strategy depends on the type of research gap After locating a research gap, characterizing its type can help to specify what kind of research is required to address the research gap Presentation Verification Substantiating the existence of the research gap Adequately presenting the research gap in a literature review Localization Characterization Localization strategy depends on the type of research gap After locating a research gap, characterizing its type can help to specify what kind of research is required to address the research gap Presentation Verification Substantiating the existence of the research gap Adequately presenting the research gap in a literature review Localization strategy depends on the type of research gap Localization Characterization Char After locating a research gap, characterizing its type can help to specify what kind of research is required to address the research gap Substantiating the existence of the research gap Presentation Fig. 2: Framework for Identifying Research Gap Adopted from (Mueller-Bloch &Kranz, 2015) Adopted from (Mueller-Bloch &Kranz, 2015) 957 IJISRT22JAN441 F. Verification of Research Gap After research gaps have been localized, verification is needed. However, verification herein means to ensure the research gap does really exist (Mueller-Bloch &Kranz, 2015). Conducting an extensive search based on the articles from which the various research gaps emerged or closely linked to the research gap is necessary for verification of research gap. The reason for this approach is that other researchers who may have closed the research gap would have quoted the articles from which the research gap emerged from to justify their studies. However, in case where the research gap did not directly emanate from a specific study, it might help to search relevant databases or scan prevailing textbooks for search terms that refer to the research gap. In addition, the researcher might have to undertake further efforts beyond this proposed approach if there is an indication that the prospective research gap may have been filled already. Miles (2017) building on the foundation of the two aforementioned theorist, a theoretical framework that synergizes both aforementioned theories was developed. This model comprises of seven core research gaps; a) Evidence Gap b) Knowledge Gap c) Practical-Knowledge Conflict Gap d) Methodological Gap e) Empirical Gap H. Theoretical Review The theoretical review of this research work is premised on the theory formulated by Miles (2017). However, Miles (2017) built his theory based on the frameworks suggested by Mueller-Bloch and Kranz (2014) and Robinson et al., (2011).The work of Robinson et al., (2011) was the initial article that developed the framework for defining research gap. This framework identified and described five types of research gaps, known as the (PICOS; (a) Population (b) Intervention (c) Comparison (d) Outcomes and (d) Setting). Afterwards, Mueller-Bloch and Kranz (2014) formulated a research gap model that was developed from the Robinson et al., (2011) framework. This framework was based on Jacob (2011) theory on research problems. Mueller-Bloch and Kranz (2014) framework of research gaps comprises of six gaps: (a) Contradictory Evidence Gap (b) Knowledge Void Gap (c) Action-knowledge conflict gap (d) Methodological gap (e) Evaluation Void Gap and (f) Theory Application Void Gap. D. Localization of Research Gap Mueller-Blochand Kranz (2015) posited that localization of research gaps begins when literature is being synthesized. As researchers frequently examine concepts that become visible from a literature, they start to uncover potential gaps in the literature. However, the process of localizing research gaps is being informed strongly by the characterization of research gaps. IJISRT22JAN441 IJISRT22JAN441 Volume 7, Issue 1, January – 2022 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Parallel Presentation (Mueller-Bloch &Kranz, 2015). Sequential Presentation describes the research gaps after the synthesis, in that the research gaps are presented differently from the synthesis. Hence, making it possible for the reader to swiftly locate research gaps in the review. Sequential presentation of research gap according to Mueller-Bloch and Kranz (2015) is more structured compared to Parallel presentation of research gaps. Parallel Presentation of research gap is a more natural approach to presenting research gaps. Parallel Presentation allows the researcher to fully describe how the research gaps unfold as a result from the synthesis. Parallel presentation facilitates the detailed divulgence of the sets of information that the research gap stem from, allowing researchers concede the origins of the respective research gap and to garner better perception for what reasons the gap exists. The above framework by Mueller-Bloch &Kranz (2015) established a distinction between the identification of research gap in the broader sense and the localization of research gaps in the narrower sense. The framework was constructed based on the findings of the analysis of literature reviews. However, from the framework it is evidenced that the begining stage is the localization of research gap, which is intiated by the characterization of research gaps. After the initial stage has been completed, the verification of research gaps may be necessary. E. Characterization of Research Gap Characterization of research gaps means to classify research gaps owing to the reasons of their existence (Robinson et al., 2011). Also, Muller-Bloch and Kranz (2015) assume that this is an integral aspect of identifying research gaps. Source: Miles (2017) Source: Miles (2017) Source: Miles (2017) e) Empirical Gap: An empirical gap is the type of research gap that has to do with gaps in the previous researches. This type of research gap deals with the research findings or propositions which requires evaluation or empirical verification (Mueller-Bloch &Kranz, 2014 cited in Miles, 2017). e) Empirical Gap: An empirical gap is the type of research gap that has to do with gaps in the previous researches. This type of research gap deals with the research findings or propositions which requires evaluation or empirical verification (Mueller-Bloch &Kranz, 2014 cited in Miles, 2017). a) Evidence Gap: An evidence gap occurs with a provocative exception if a new study finding contradicts widely accepted conclusion. The evidence gap involves divergence in findings of the prior research. This type of gap occurs when conclusions from the result of a study are accepted in its own right but is contradictory when viewed from a more abstract point of view, that is it is contradicting when compared to the result of similar researches. The identification of contradictory evidence starts with analyzing each research stream (Mueller-Bloch & Kranz, 2014 cited in Miles, 2017). f) Theoretical Gap: The theoretical gap deals with the gap in theory in previous research. Theoretical gaps are a common occurrence in examining prior research on a phenomenon (Mueller-Bloch &Kranz, 2014 cited in Miles, 2017). f) Theoretical Gap: The theoretical gap deals with the gap in theory in previous research. Theoretical gaps are a common occurrence in examining prior research on a phenomenon (Mueller-Bloch &Kranz, 2014 cited in Miles, 2017). g) Population Gap: A population gap is a commonly identified gap among researchers. There always exist under-served populations that have been under- researched. This gap exists when there are populations that were not properly represented or under-researched in the evidence based on previous researches (Robinson, et al, 2011cited in Miles, 2017). g) Population Gap: A population gap is a commonly identified gap among researchers. There always exist under-served populations that have been under- researched. This gap exists when there are populations that were not properly represented or under-researched in the evidence based on previous researches (Robinson, et al, 2011cited in Miles, 2017). b) Knowledge Gap: Knowledge gap is a common form of research gap. Source: Miles (2017) There are two situations in which a knowledge gap or knowledge void may occur; first, there may not be existing knowledge in the particular field of study regarding theories and literatures from related research domain; second, a possibility that the result of the study is different from the expected outcome (Mueller-Bloch &Kranz, 2014 cited in Miles, 2017). G. Presentation of Research Gap f) Theoretical Gap There are two approaches to present research gaps in literature reviews. These are Sequential Presentation and g) Population Gap IJISRT22JAN441 958 IJISRT22JAN441 www.ijisrt.com International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 7, Issue 1, January – 2022 Evidence Gap Knowledge Gap Practical- Knowledge Gap Empirical Gap Methodology Gap Theoretical Gap Population Gap The Seven Research Gaps Evidence Gap Knowledge Gap Population Gap The Seven Research Gaps Practical- Knowledge Gap Theoretical Gap Methodology Gap Empirical Gap IV. METHODS Also, Farooq (2017) opined that researchers and academicians often lack preliminary knowledge regarding the identification and exploration of research gap using content analysis. Meanwhile, Duriau, et al, (2007) is of the opinion that content analysis if well implemented should be of particular interest for management researchers because of several factors such as access to deep structures of managers, non-intrusiveness, analytical flexibility and the ability to implement longitudinal designs. Having reviewed several literatures and discovering the different types of research gaps that exists in literature, this study choose a simplified way of identifying research gaps in some selected articles. Content analysis was carried out on ten article papers from two Journal publications (LASU Journal of Employment Relations and Human Resources Management and LASU Journal of Business Review), to examine the types of research gaps that were used by the researchers in their studies. The selection and inclusion is based on convenient sampling. S/N ARTICLE TITLE AUTHOR(S) TYPES OF GAPS Remarks Structural challenges and local government administration in Nigeria. Afegbua, S.I.; Osakede, K.O. & Nkomah, B.B. Knowledge Gap The gap identified in the study substantially reflected knowledge gap Frauds and forgeries on the performance of the Nigerian banking industry. Araga, A.S. & Sufian, J.B. Empirical Gap From the study it can also be inferred that there is an evidence gap in the study which was not filled Entrepreneurship skills development, risk taking propensity and students’ self- employment intention. Ojapinwa, A.F.; Fapohunda, T.M. & Jayeoba, F.I Practical- Knowledge Gap In the study practical- knowledge gap was identified but was not fully filled. An empirical gap was established Workforce retention strategies and corporate development: An empirical Study. Abioro,M.A; Oladejo, D.A. & Ashogbon, F.O. Empirical Gap From the study it was discovered that the empirical gap identified was filled. Impact of staff welfare on job commitment in Tuyil Pharmaceutical company, Ilorin. Kwara State. Jimoh, A.L. & Kadiri, I.B. Empirical Gap The gap identified in the study substantially reflected empirical gap and was filled The impact of advertising and sales promotions on Brand equity: A study of the Nigerian Bottling Company (NBC). Olumoko, T.A. Empirical Gap The gap identified in the study substantially reflected empirical gap and was slightly filled Assessment of the effect of multiple tax on the survival of selected small and medium enterprise in Abuja. Ibrahim, M.G. I. Empirical Review C Common consent is lacking regarding what constitutes the most suitable methodological approach to identifying research gaps, determining research priorities and displaying research gaps or priorities (Robinson, et al, (2011); Gadsby, et al., (2012); Rudan, et al, (2015); and Nasser (2018)). Research gaps significantly differs across research contexts and no common methodological guidance on which approach should be used to identify research gaps or determine research priorities (Nyanchoka, et al, 2019). However, Nyanchoka et al, (2019) posited that half of the methods used to display research gaps are traditional ways of presenting findings. Hence, the study of Nyanchoka et al,(2019) provided a synopsis of different methods that can be used to report the identification of research gaps. The findings of the study can be adopted to inform the c) Practical-Knowledge Gap: Practical-knowledge gap refers to discrepancies that can bring about new research. A practical-knowledge (action-knowledge) conflict arises when the actual behaviour of professionals differs from their advocated behaviour (Mueller-Bloch &Kranz, 2014 cited in Miles, 2017). d) Methodological Gap: A methodological gap is the gap refers to the conflict that arises in research as a result of the influence of methodology on a result. This gap seek to address the conflicts which arise as a result of the research methods in initial studies and offers a new line of research that is different from the previous research methods(Mueller-Bloch &Kranz, 2014 cited in Miles, 2017). www.ijisrt.com 959 IJISRT22JAN441 Volume 7, Issue 1, January – 2022 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 ISSN No:-2456-2165 ISSN No:-2456-2165 development of standardized methods of identifying, prioritizing and displaying of research gaps. Also, the findings informed the need for further studies and evidence- based-decision making by providing description of different methods that can be adopted to identify research gaps which will guide the development of qualitative study needed in identifying, communicating and displaying gaps in research. Robinson, et al,(2011) formulated a framework to facilitate the identification and characterization of research gaps from systematic review. The researchers were of the opinion that mere exploration of literatures from database is not a lead to research gap, because literature explored requires extensive reading and understating particular problem or research question. I. Empirical Review C Study by Farooq (2017) identified some method of identifying research gaps but also was of the opinion that meta-analysis (which is one of the methods designed by the researcher) is the least preferred by researchers because there is lack of knowledge or expertise regarding the method while systematic review is the most widely accepted method of identifying research gap as it just requires the researcher to review and analyze literature over a period of time. Hence, Mueller-Bloch and Kranz (2015) identified the need to formulate a framework that will help scholars to identify research gaps in literature review whose objective is to summarize extant theory to identify gaps in theory or research. As lack of methodological literature on the area of research suggests that the procedures are often creative, implicit and informal. V. CONCLUSIONS [6.] Mueller-Bloch, C. &Kranz, J. (2015). A framework for rigorously identifying research gaps in qualitative literature reviews. Thirty sixth International conference on Information Systems, Fort Worth, Texas-USA, 1-9. In the literature being reviewed for this study, it was discovered that some research platforms such as Google Scholar, Scopus and some other scholarly databases can be used to identify research gaps through citation analysis, these method should be empirically tested and confirmed for usage as it will help researchers especially those at the preliminary stage to better understand and identify research gaps. This will help improve the quality of research. [7.] Naseer, M. (2018).Setting priorities for conducting and updating systematic reviews. UK: Peninsula schools of Medicine and dentistry, University of Plymouth. [8.] Nyanchoka, L. Tudur-Smith, C.; Thu, V.N.; Iversen, V; Tricco, A.C.; Porcher, R. (2019). A scoping review describes methods used to identify, prioritize and display gaps in health insurance. Journal of Clinical Epidemiology, 109, 99-110. IV. METHODS Empirical Gap The gap identified in the study substantially reflected empirical gap and was appropriately filled Effect of supply chain management on organizational productivity. A study of Nigeria Solomon, J.; Marcus, G.O. & Akhaine, M.E. Knowledge Gap The gap identified in the study substantially reflected empirical gap and was www.ijisrt.com IJISRT22JAN441 960 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 7, Issue 1, January – 2022 ISSN No:-2456-2165 Table 1: Some Examples of Gaps in Literature in some Selected Articles Published Bottling Company. Abuja. adequately filled Effect of forensic accounting on fraud management in Nigeria public service (A study of Ogun State Board of Internal Revenue). Idowu, K.A.; Olagunju, A. & Atere, A. Empirical Gap The gap identified in the study substantially reflected empirical gap and was slightly filled Effect of branding on the corporate image of selected insurance companies in Lagos State, Nigeria. Hassan, A.R.; Balogun, T.M. & Yusuf, O.A. Empirical Gap The gap identified in the study substantially reflected empirical gap and was substantially filled Table 1: Some Examples of Gaps in Literature in some Selected Articles Published Table 1: Some Examples of Gaps in Literature in some Selected Articles Published  Contribution to Study [3.] Gadsby, R.; Snow, R.; Daly, A.C.; Crowe, S.; Matyka, K. &Hall, B. (2012). Setting research priorities for Type 1 diabetes. Diabet Med, 29, 1321-1326. From this review, it was observed that recent literature probably seem not to be in existence regarding research gaps and previous ones had many limitations and suggestions for studies that were hoped to be filled by other researcher. Hence, this review had been able to achieve the research objective of this study by exploring the various research gaps in literature. [4.] Green, S. (2005). Systematic review and meta-analysis. Singapore Medical Journal, 46(6), 270-274. [5.] Miles, D.A. (2017). A taxonomy of research gaps: identifying and defining the seven research gaps. Doctoral student workshop: Finding Research Gaps- Research Methods and Strategies, Dallas, Texas, 1-10. REFERENCES [1.] Duriau, V.J.; Reger, R.K. & Pfarrer M.D. (2007). A content analysis of the content analysis literature in organization studies: Research themes, data sources and methodological refinements.Organizational Research Methods, 10(1), 5-34. [2.] Farooq, R. (2018). A framework for identifying research gap in social sciences: Evidence from the past. The IUP Journal of Management Research, XVI( 4), 67-76 Source: Author (2021) be well designed and its understanding should be emphasized. From analysis carried out above, it was discovered that researcher majorly built their research on what previous researchers have done, they must empirically verified what previous researcher have done. Hence, seven out of ten articles that were reviewed showed that the researchers identified adopted the empirical gap in their study and only two researchers identified a knowledge gap (i.e. tried to investigate the inexistence of knowledge in the study carried out) while one researcher identified practical-knowledge gap (i.e. had a conflicting view regarding what is previously obtained in the study carried out and investigated another perspective to the study). the value of clinical research. Health Policy, 3(3), 109- 127. [12.] Tom (2012). Identifying gaps in the literature is a critical part of a review. Available at http://www.literaturereviewof.com/identifying-gaps, html. Accessed on September 14, 2015. RECOMMENDATIONS From the study it was discovered from the content analysis carried out that very few types of research are being identified and emphasized by authors or researchers. All of types of research gaps in existence identified in literature that were not captured in the any of the articles reviewed during the content analysis will be left lagging. Hence, this research recommends are follows; [9.] Robinson, K.A, Saldanha, I.J. &Mckoy, N.A. (2011). Development of a framework to identify research gaps from systematic reviews. Journal of Clinical Epidemiology, 64(1), 1325-1330. [10.] Rudan, I., Campbell, H., Marusic, A., Sridhar, D., Nair, H., &Adeloye, D., (2015). Assembling GHERG: could academic crowd-sourcing address gap in global health estimate? Journal of Global Health, 5(1).  All existing research gaps should be well investigated by researchers who want to further investigate into research gaps. [11.] Scott, N.A.; Carmen, M.; Christa, H. & Jacques, M. (2008). Using health technology assessment to identify research gaps: An unexploited resource for increasing  Research gaps such as theoretical gap, population gaps, methodological gaps; methods of identifying them should www.ijisrt.com 961 IJISRT22JAN441 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 7, Issue 1, January – 2022 the value of clinical research. Health Policy, 3(3), 109- 127. [12.] Tom (2012). Identifying gaps in the literature is a critical part of a review. Available at http://www.literaturereviewof.com/identifying-gaps, html. Accessed on September 14, 2015. IJISRT22JAN441 IJISRT22JAN441 962 www.ijisrt.com
https://openalex.org/W4312617991	https://ejournal.mandalanursa.org/index.php/JIME/article/download/256/247	Indonesian	null	PEMBERDAYAAN REMAJA PUTUS SEKOLAH MELALUI KETERAMPILAN SCREEN PRINTING	Jurnal Ilmiah Mandala Education	2,017	cc-by-sa	3,196	Hadi Gunawan Sakti; Muh Husein Baysha, & Endah Resnandari Puji Astuti Dosen FIP IKIP Mataram Baysha234@gmail.com Hadi Gunawan Sakti; Muh Husein Baysha, & Endah Resnandari Puji Astuti Dosen FIP IKIP Mataram Baysha234@gmail.com Abstrak: Perekonomian masyarakat yang tergolong masih rendah, tingkat pendidikan orang tua yang masih rendah, dan tingginya angka pernikahan dini di Desa Lepak Timur menyebabkan banyaknya remaja putus sekolah. Kebanyakan dari remaja yang putus sekolah di Desa Lepak Timur tidak memiliki bekal keterampilan yang memadai sehingga tidak dapat terserap di dunia kerja. Upaya yang harus dilakukan dalam mengatasi permasalahan remaja putus sekolah yang tidak memiliki keterampilan yaitu dengan memberikan bekal keterampilan kepada mereka berupa pendidikan kecakapan hidup (life skill) melalui keterampilan screen printing. Remaja putus sekolah di Desa Lepak Timur akan diarahkan untuk menghasilkan produk berupa souvenir hasil screen printing. Tujuan pelaksanaan program KKN-PPM ini adalah memberikan keterampilan kepada remaja putus sekolah. Target khusus dari pelaksanaan program KKN-PPM ini yakni memberikan keterampilan screen printing pada remaja putus sekolah. Metode yang digunakan dalam melaksanakan program KKN-PPM ini yaitu pemberdayaan remaja putus sekolah ini dilakukan secara partisipatif. Adapun teknik pelaksanaan program yaitu dengan membuat kelompok mahasiswa (1 kelompok berjumlah 8 mahasiswa) dan akan dilaksanakan selama 6 hari dalam satu minggu dan akan dilakukan selama 3 bulan. Kelompok mahasiswa KKN-PPM ditempatkan secara tersentral di Desa Lepak Timur. Kegiatan pendampingan ini terus dibimbing oleh Dosen Pembimbing Lapangan (DPL) yang secara periodik akan melakukan mekanisme monitoring dan evaluasi untuk memperoleh deskripsi penyelenggaraan program yang lebih komprehensif dan sebagai bahan memperoleh masukan terhadap kekurangan program dilapangan. Luaran dari pelaksanaan program KKN-PPM ini antara lain; (1) Publikasi Ilmiah di Jurnal/ Prosiding; (2) Publikasi pada Media Masa (Cetak); (3) Peningkatan Kesehatan/ Pendidikan/ Ketentraman Masyarakat; (4) Peningkatan Pendapatan dan Partisipasi Masyarakat; (5) Peningkatan Swadana dan Swadaya Masyarakat; (6) Dihasilkannya produk souvenir hasil screen printing; dan (7) Buku panduan keterampilan Screen Printing. Kata kunci : Pemberdayaan, Putus Sekolah, Screen Printing Jurnal Ilmiah Mandala Education JIME, Vol. 3. No. 2 ISSN 2442-9511 Oktober 2017 PEMBERDAYAAN REMAJA PUTUS SEKOLAH MELALUI KETERAMPILAN SCREEN PRINTING JIME, Vol. 3. No. 2 ISSN 2442-9511 Oktober 2017 PENDAHULUAN banyaknya anak putus sekolah (drop out). Tingkat pendidikan orang tua yang masih rendah di Desa Lepak Timur ini juga turut menjadi pemicu banyaknya anak usia sekolah yang tidak melanjutkan sekolah. Selain itu, tingginya angka pernikahan dini (merariq kodeq) di Desa Lepak Timur semakin menambah tingginya tingkat anak putus sekolah. Desa Lepak Timur merupakan salah satu desa yang berada di Kecamatan Sakra Timur, Lombok Timur. Masyarakat desa Lepak Timur banyak bermatapencaharian sebagai petani dan buruh tani. Lahan pertanian yang dimiliki sebagian besar merupakan lahan tadah hujan yang masa tanamnya sangat bergantung pada pergantian musim. Musim kering yang berkepanjangan menyebabkan daerah pertanian di Desa Lepak, Sakra Timur mengalami gagal panen. Hal ini sangat berpengaruh terhadap perekonomian masyarakat di Desa Lepak Timur. Berdasarkan data di Desa Lepak Timur sebanyak 24% anak usia SMP dan 35% anak usia SMA yang mengalami putus sekolah. Banyaknya remaja usia sekolah yang putus sekolah dengan berbagai alasan dapat menyebabkan munculnya berbagai permasalahan sosial seperti pengangguran, minuman keras, pergaulan bebas, perilaku Perekonomian masyarakat yang tergolong masih rendah, menyebabkan 216 Jurnal Ilmiah Mandala Education ISSN 2442-9511 JIME, Vol. 3. No. 2 Oktober 2017 anak menyimpang, dan lain sebagainya. Kebanyakan dari remaja yang putus sekolah di Desa Lepak Timur tidak memiliki bekal keterampilan yang memadai sehingga tidak dapat terserap di dunia kerja. Pada akhirnya remaja yang putus sekolah tersebut akan mengikuti orang tuanya menjadi petani atau buruh tani. Keterampilan screen printing dapat memberikan kesempatan yang luas bagi terciptanya wirausaha baru. Hal ini sesuai dengan pendapat Horvath et al (2012: 134) the tecnology of screen printing has large aplication field as veried as decal fabrication, balloon, and cloths patterning, textile fabrication, producing signs and displays, decorative automobile trim and truck signs and last but not least printed electronic, including circuit board printing and thick film technology. Upaya yang harus dilakukan dalam mengatasi permasalahan anak putus sekolah yang tidak memiliki keterampilan yaitu dengan memberikan bekal keterampilan kepada mereka. Bekal keterampilan yang diberikan berupa pendidikan kecakapan hidup (life skill). PENDAHULUAN Syamsi (2012: 443) berpendapat bahwa, pendidikan kecakapan hidup diberikan kepada remaja putus sekolah agar (1) memiliki kesadaran tinggi tentang pentingnya pendidikan keterampilan untuk dirinya sendiri maupun anggota keluarganya; (2) meningkatkan pengetahuan, keterampilan dan sikap remaja putus sekolah di bidang kecakapan hidup; (3) memiliki pengetahuan, keterampilan, dan sikap yang dibutuhkan dalam memasuki dunia kerja baik bekerja mandiri (wirausaha) dana tau bekerja pada suatu perusahaan produksi/jasa dengan penghasilan yang semakin layak untuk memenuhi kebutuhan hidupnya; (4) memiliki motivasi dan etos kerja yang tinggi serta dapat menghasilkan karya-karya yang unggul dan mampu bersaing di pasar global; (5) menghasilkan model pendidikan kecakapan hidup (life skill) yang tepat diterapkan bagi remaja putus sekolah. Melalui keterampilan screen printing, remaja putus sekolah di desa Lepak Timur akan diarahkan untuk menghasilkan produk berupa souvenir hasil screen printing. Souvenir yang dapat dihasilkan antara lain berupa kaos sablon, gantungan kunci sablon, dan kipas sablon. Kegiatan ini akan dilaksanakan melalui kegiatan pemberdayaan, pelatihan dan pendampingan kepada anak putus sekolah. Dalam pelaksanaan program KKN- PPM ini akan bermitra dengan Karangtaruna Juita. Karangtaruna Juita merupakan organisasi sosial wadah pengembangan generasi muda yang tumbuh dan berkembang atas dasar kesadaran dan tanggung jawab sosial dari, oleh dan untuk masyarakat terutama generasi muda di wilayah Desa Lepak Timur, Sakra Timur, Lombok Timur. Karangtaruna Juita berdiri pada tahun 2014 sesuai dengan Surat Keputusan Kepala Desa Lepak Timur No. 02/KRT/DSLT/XII/2014. Visi Karangtaruna Juita yaitu tauladan, kreatif, dan mandiri. Sedangkan misinya antara lain; 1) menjadi tauladan bagi masyarakat, 2) mampu menciptakan lapangan kerja, dan 3) tidak tergantung pada orang lain. Salah satu cara pemberian bekal pendidikan kecakapan hidup kepada remaja putus sekolah di Desa Lepak Timur yaitu dengan pemberdayaan remaja putus sekolah melalui keterampilan screen printing. Horvath et al (2014: 30) Screen printing is the most widespread and common additive layer deposition and patterning method because of its ability to print on many kinds of substrates with the widest range of inks and because, considering any modern print process, it can deposit the greatest thickness of ink film. Pemilihan keterampilan screen printing dimaksud agar remaja-remaja tersebut memiliki keterampilan screen printing sehingga dapat membentuk suatu kelompok wirausaha di Desa Lepak Timur. Sebagaimana mewujudkan misi tersebut, maka kelompok KKN-PPM IKIP Mataram akan melaksanakan kemitraan dengan karangtaruna Juita. Bentuk kemitraan dalam pelaksanaan program KKN-PPM dengan Karangtaruna Juita ini yaitu berupa kegiatan pelatihan, pendampingan, penyuluhan, dan pemberdayaan untuk memberikan keterampilan screen printing kepada remaja putus sekolah di Desa Lepak Timur. Jurnal Ilmiah Mandala Education PENDAHULUAN Dengan diadakannya kegiatan ini diharapkan remaja putus sekolah memiliki bekal keterampilan sehingga dapat 217 Jurnal Ilmiah Mandala Education ISSN 2442-9511 JIME, Vol. 3. No. 2 Oktober 2017 Oktober 2017 berwirausaha, terserap di dunia kerja, dan tidak tergantung lagi pada orang lain. baru untuk mengurangi jumlah pengangguran di Desa Lepak Timur; (2)Terciptanya alternative solusi dalam menangani remaja putus sekolah di Desa Lepak Timur; (3)Tumbuhnya motivasi dan etos kerja yang tinggi bagi remaja putus sekolah; (4)Meningkatnya jumlah wirausahawan baru di Desa Lepak Timur; (5)Peningkatan pendidikan kecakapan hidup (life skill) bagi remaja putus sekolah: (6)Meningkatnya partisipasi masyarakat dalam mengikuti kegiatan ini; (7)Dihasilkannya produk souvenir hasil screen printing dari Desa Lepak Timur; (8)Buku panduan keterampilan Screen Printing. Program KKN-PPM ini akan dilaksanakan selama 6 hari dalam satu minggu dan akan dilakukan selama 3 bulan. Kelompok mahasiswa KKN-PPM yang terdiri dari 32 orang akan dibagi menjadi 4 kelompok di 4 dusun. Setiap kelompok mahasiswa yang berjumlah 8 orang akan mendampingi 1 (satu) kelompok pemuda yang terdiri dari 15 orang di setiap dusun. Jumlah seluruh pemuda yang akan mengikuti pendampingan ini adalah 60 orang. Selanjutnya, kegiatan pendampingan ini akan terus dibimbing oleh Dosen Pembimbing Lapangan (DPL) yang secara periodik akan melakukan mekanisme monitoring dan evaluasi untuk memperoleh deskripsi penyelenggaraan program yang lebih komprehensif dan sebagai bahan memperoleh masukan terhadap kekurangan program dilapangan. Selain itu, dalam proses tersebut juga akan dilengkapi dengan instrument monitoring dan evaluasi program sebagai alat untuk mengukur pencapaian tujuan program. Jurnal Ilmiah Mandala Education PRINTING Metode pelaksanaan program KKN- PPM ini dilakukan secara terpadu dan partisipatif dengan tahapan sebagai berikut. Persiapan dan Pembekalan Persiapan dan Pembekalan p Mekanisme Pelaksanaan Program KKN-PPM Mekanisme dalam pelaksanaan program KKN-PPM ini dilakukan dengan langkah- langkah sebagai berikut: (a) Melakukan pendataan dan verifikasi akademik mahasiswa IKIP Mataram sebagai calon peserta KKN- PPM dengan melibatkan 10 program studi yang meliputi program studi Bimbingan dan Konseling, Teknologi Pendidikan, Administrasi Pendidikan, Pendidikan Luar Sekolah, Pendidikan Bahasa Inggris, Pendidikan Olah Raga, Pendidikan Matematika, Pendidikan Biologi, Pendidikan Kimia, dan Pendidikan Fisika dengan syarat telah menempuh minimal 110 SKS dengan IPK minimal 2,75 dan telah memprogramkan (KRS) mata kuliah KKN. Adapun mahasiswa yang akan dilibatkan dalam pelaksanaan program KKN-PPM ini adalah sebanyak 32 orang dari sepuluh program studi yang ada di IKIP Mataram; (b) Adapun mahasiswa yang dilibatkan dalam pelaksanaan program KKN- PPM ini adalah sebanyak 32 orang; (c)Memberikan pembekalan materi KKN- PPM kepada mahasiswa sebagai peserta dengan pendampingan oleh Dosen Pembimbing Lapangan (DPL); (d) Melakukan survey kesiapan lokasi KKN-PPM dan koordinasi oleh Tim KKN-PPM dan LPPM IKIP Mataram dengan Kepala Desa Lepak Timur, Ketua Karangtaruna beserta g p p j p g Program KKN-PPM “Pemberdayaan Remaja Putus Sekolah melalui Keterampilan Screen Printing” ini memiliki target sebagai berikut: (1) Program ini dilaksanakan untuk menumbuhkan kesadaran remaja putus sekolah mengenai pentingnya pendidikan keterampilan hidup (life skill) baik bagi dirinya sendiri maupun bagi anggota keluarganya; (2) Meningkatkan pengetahuan, keterampilan dan sikap remaja putus sekolah di bidang kecakapan hidup; (3) Memiliki pengetahuan, keterampilan, dan sikap yang dibutuhkan dalam memasuki dunia kerja baik bekerja mandiri (wirausaha) dana tau bekerja pada suatu perusahaan produksi/jasa dengan penghasilan yang semakin layak untuk memenuhi kebutuhan hidupnya; (4) Memiliki motivasi dan etos kerja yang tinggi serta dapat menghasilkan karya-karya yang unggul dan mampu bersaing di pasar global; (5) Remaja Putus sekolah di Desa Lepak Timur memiliki keterampilan screen printing; dan (6) tersusunnya Buku panduan keterampilan screen printing Sementara luaran dari pelaksanaan program pengabdian kepada masyarakat ini adalah: (1) terciptanya lapangan pekerjaan 218 Jurnal Ilmiah Mandala Education ISSN 2442-9511 JIME, Vol. 3. No. 2 Oktober 2017 Pelaksanaan Program KKN-PPM Adapun langkah-langkah pelaksanaan program KKN-PPM sebagai berikut: (a)Analisis aktivitas rutinitas (kegiatan) remaja di Desa Lepak Timur; (b)Analisis kebutuhan program berdasarkan pada permasalahan yang ada di Desa Lepak Timur berkaitan dengan remaja putus sekolah; (c)Pemetaan anak putus sekolah di Desa Lepak Timur. Persiapan dan Pembekalan Pada proses ini juga sebagai dasar pembagian kelompok mahasiswa KKN untuk mendampingi sasaran dalam pelaksanaan program; (d)Satu kelompok KKN-PPM yang terdiri dari 8 orang mahasiswa akan mendampingi 1 kelompok remaja yang terdiri dari 15 orang; (e)Kelompok remaja akan didampingi oleh mahasiswa KKN-PPM dalam melaksanakan kegiatan-kegiatan penyuluhan, pemberdayaan, pelatihan, dan pendampingan dalam penguasaan keterampilan screen printing hingga menghasilkan produk souvenir hasil screen printing; (f)Kegiatan pendampingan oleh mahasiswa KKN-PPM akan dilakukan 6 hari dalam seminggu dengan waktu 3 jam per setiap pertemuan. Artinya, akan dilakukan sebanyak 24 kali pertemuan atau 72 jam selama 1 bulan dan 216 jam dalam 3 bulan; (g) Metode yang digunakan dalam pemberdayaan kelompok sasaran antara lain; partisipatif yakni metode yang ditujukan untuk melibatkan sasaran secara bersama-sama mulai dari pra-proses- pasca program. Metode ini juga bertujuan untuk membuat khalayak sasaran merasa memiliki dan bertanggungjawab atas keberlangsungan program; penyuluhan yakni metode yang digunakan untuk menyampaikan materi-materi terkait dengan pemberian materi screen printing; pendampingan yakni metode yang digunakan untuk mendampingi sasaran dalam penguasaan keterampilan screen printing; dan pelatihan yakni metode yang digunakan untuk kegiatan yang bersifat aplikasi dan praktek dalam penguasaan keterampilan screen printing hingga menghasilkan produk souvenir screen printing; (h) Kegiatan monitoring akan dilakukan 1 kali dalam seminggu oleh Dosen Pembimbing Lapangan (DPL) untuk mengamati proses kinerja mahasiswa KKN jajarannya; (e) Melakukan acara penerimaan mahasiswa KKN-PPM IKIP Mataram di Desa Lepak Timur; (f) Melakukan sosialisasi kepada warga masyarakat dan tokoh-tokoh masyarakat Desa Lepak Timur terkait dengan pelaksanaan program KKN-PPM; (g) Merumuskan pola pelaksanaan program KKN-PPM bersama kepala desa, tokoh masyarakat dan agama, Ketua Karangtaruna Juita, perwakilan anggota karangtaruna Juita, perwakilan remaja putus sekolah, mahasiswa, dan DPL; (h) Melaksanakan program KKN- PPM yang meliputi kegiatan penyuluan, pelatihan, pendampingan, pemberdayaan, dan pembinaan lapangan; (i) Melakukan monitoring dan evaluasi program KKN-PPM; (j) Melakukan acara penarikan mahasiswa KKN-PPM IKIP Mataram di Desa Lepak Timur. Pelaksanaan Program KKN-PPM Adapun langkah-langkah pelaksanaan program KKN-PPM sebagai berikut: (a)Analisis aktivitas rutinitas (kegiatan) remaja di Desa Lepak Timur; (b)Analisis kebutuhan program berdasarkan pada permasalahan yang ada di Desa Lepak Timur berkaitan dengan remaja putus sekolah; (c)Pemetaan anak putus sekolah di Desa Lepak Timur. Persiapan dan Pembekalan Pada proses ini juga sebagai dasar pembagian kelompok mahasiswa KKN untuk mendampingi sasaran dalam pelaksanaan program; (d)Satu kelompok KKN-PPM yang terdiri dari 8 orang mahasiswa akan mendampingi 1 kelompok remaja yang terdiri dari 15 orang; (e)Kelompok remaja akan didampingi oleh mahasiswa KKN-PPM dalam melaksanakan kegiatan-kegiatan penyuluhan, g g p y , pemberdayaan, pelatihan, dan pendampingan dalam penguasaan keterampilan screen printing hingga menghasilkan produk souvenir hasil screen printing; (f)Kegiatan pendampingan oleh mahasiswa KKN-PPM akan dilakukan 6 hari dalam seminggu dengan waktu 3 jam per setiap pertemuan. Artinya, akan dilakukan sebanyak 24 kali pertemuan atau 72 jam selama 1 bulan dan 216 jam dalam 3 bulan; (g) Metode yang digunakan dalam pemberdayaan kelompok sasaran antara lain; partisipatif yakni metode yang ditujukan untuk melibatkan sasaran secara bersama-sama mulai dari pra-proses- pasca program. Metode ini juga bertujuan untuk membuat khalayak sasaran merasa memiliki dan bertanggungjawab atas keberlangsungan program; penyuluhan yakni metode yang digunakan untuk menyampaikan materi-materi terkait dengan pemberian materi screen printing; pendampingan yakni metode yang digunakan untuk mendampingi sasaran dalam penguasaan keterampilan screen printing; dan pelatihan yakni metode yang digunakan untuk kegiatan yang bersifat aplikasi dan praktek dalam penguasaan keterampilan screen printing hingga menghasilkan produk souvenir screen printing; (h) Kegiatan monitoring akan dilakukan 1 kali dalam seminggu oleh Dosen Pembimbing Lapangan (DPL) untuk mengamati proses kinerja mahasiswa KKN- PPM dalam pelaksanaan program serta Materi dan Pembekalan KKN-PPM Kegiatan pembekalan KKN-PPM dilaksanakan oleh LPPM IKIP Mataram dengan tetap melakukan koordinasi dengan Tim Pengusul KKN-PPM, serta pelaksanaan kegiatan pembekalannya sesuai dengan jadwal kalender akademik IKIP Mataram. Adapun materi dalam pembekalan KKN-PPM yang akan disampaikan kepada mahasiswa meliputi: (a) Materi Umum yakni Konsep KKN-PPM, Penyusunan Program KKN-PPM berbasis Partisipatif (PRA), Pelaporan, Penilaian, Peraturan dan Tata Tertib pelaksanaan KKN-PPM; (b) Materi Isi, pada pembekalan materi isi, mahasiswa akan diberikan pembekalan materi isi yang sama, namun ada beberapa materi yang akan disesuaikan dengan disiplin ilmu (program studi). Pembekalan meteri ini akan dilakukan oleh penanggungjawab program, DPL KKN- PPM, Pengelola Karangtaruna Juita, dan dosen sesuai dengan bidang ilmu yang dibutuhkan. Pembekalan materi isi yang diberikan berupa: Analisis Kebutuhan dan permasalahan yang ada di Desa Lepak Timur, pendidikan keterampilan hidup (life skill), pengenalan peralatan dan bahan screen printing kepada mahasiswa calon peserta KKN-PPM, praktek pembuatan film pada screen, cara membersihkan screen, praktek melakukan screen printing hingga memperoleh hasil souvenir berupa produk screen printing. 219 Jurnal Ilmiah Mandala Education JIME, Vol. 3. No. 2 ISSN 2442-9511 Oktober 2017 Oktober 2017 Berikut adalah rangkaian kegiatan sosialisasi, penyuluhan, pelatihan dan pendampingan program keterampilan screen printing Desa lepak Timur: j Tabal 2 Rangkaian Kegiatan sosialisasi, penyuluhan, pelatihan dan pendampingan program keterampilan screen printing Desa lepak Timur Oktober 2017 Sebelum mahasiswa diterjunkan ke lokasi KKN-PPM, mahasiswa terlebih dahulu diberikan pembekalan. Berikut jadwal pembekalan mahasiswa: melakukan pendampingan terhadap kendala atau hambatan selama pelaksanaan program KKN-PPM dilapangan; (i) Kegiatan evaluasi program dilakukan pada waktu program berakhir, hal ini untuk memberikan penilaian terhadap kinerja dan pelaporan mahasiswa KKN-PPM dalam melaksanakan program; (j) Publikasi Laporan KKN-PPM pada Jurnal Ilmiah dan Media Cetak (Koran) serta publikasi produk kegiatan; (k) Volume total pekerjaan yang dilakukan mahasiswa dalam pelaksanaan program KKN-PPM yaitu 144 jam/bulan/ 1 orang mahasiswa 4608 jam/ 3 bulan; (l) Publikasi Produk p Tabel 1 Pelaksanaan Pembekalan Mahasiswa KKN PPM KKN PPM Secara rinci langkah-langkah pelaksanaan program dapat dilihat pada gambar berikut. p g p p g Hasil yang Dicapai Program KKN-PPM pemberdayaan remaja putus sekolah melalui keterampilan screen printing dilaksanakan di Desa Lepak Timur, Kecamatan Sakra Timur, Kabupaten Lombok Timur. Kegiatan ini dimulai sejak tanggal 23 Juli 2017 dan berakhir tanggal 20 Oktober 2017. Selama kurang lebih 3 bulan 30 mahasiswa IKIP Mataram memberikan kegiatan penyuluhan, pelatihan, serta pendampingan mengenai keterampilan screen printing kepada remaja putus sekolah di Desa Lepak Timur, Sakra Timur, Lombok Timur. Berdasarkan table 1 tersebut diketahui bahwa mahasiswa yang mengikuti program KKN PPM di Desa Lepak Timur memperoleh materi pembekalan mulai bulan Juni hingga bulan Juli. Setelah pembekalan mahasiswa, pada tanggal 22 Juli 2017 mahasiswa yang mengikuti kegiatan PPL KKN termasuk mahasiswa dalam program KKN PPM dilepas secara resmi oleh rector IKIP Mataram untuk mengkuti kegiatan PPL KKN selama 3 bulan. Hasil yang Dicapai Program KKN-PPM pemberdayaan remaja putus sekolah melalui keterampilan screen printing dilaksanakan di Desa Lepak Timur, Kecamatan Sakra Timur, Kabupaten Lombok Timur. Kegiatan ini dimulai sejak tanggal 23 Juli 2017 dan berakhir tanggal 20 Oktober 2017. Selama kurang lebih 3 bulan 30 mahasiswa IKIP Mataram memberikan kegiatan penyuluhan, pelatihan, serta pendampingan mengenai keterampilan screen printing kepada remaja putus sekolah di Desa Lepak Timur, Sakra Timur, Lombok Timur. Mahasiswa KKN PPM di Desa Lepak Timur diterima secara resmi oleh Desa epak Timur pada tanggal 23 Juli 2017. Kegiatan penerimaan mahasiswa dilakukan secara sederhana dan dihadiri Dosen pembimbing lapangan (DPL), Ketua Karang Taruna Juita, Kepala Desa dan jajaran staf desa Lepak Timur. Setelah secara resmi diterima di Desa epak Timur, Tim KKN PPM 220 Jurnal Ilmiah Mandala Education JIME, Vol. 3. No. 2 ISSN 2442-9511 Oktober 2017 Oktober 2017 Tabel 3 Capaian Luaran dalam Laporan Kemajuan mulai mensosialisasikan program KKN PPM di Desa Lepak Timur. PENUTUP Kegiatan KKN PPM di Desa Lepak Timur masih dalam proses pelaksanaan kegiatan. Kegiatan ini dilaksanakan sejak tanggal 23 Juli hingga 20 ktober 2017. Kegiatan yang dilakukan yaitu memberikan penyuluhan, pelatihan dan pendampingan kepada remaja putus sekolah di Desa Lepak Timur sehingga memiliki keterampilan screen printing. Saran yang dapat disampaikan yaitu: (a) Karang taruna Juwita di desa Lepak Timur hendaknya terus melakukan kegiatan-kegiatan yang bermanfaat dan dapat meningkatkan pengetahuan serta keterampilan remaja di Desa Lepak Timur sehingga dpat memotivasi remaja untuk terus berkarya dan berwirausaha; (b) Keterampilan screen priting merupakan keterampilan yang baik dikembangkan untuk meningkatkan perekonomian masyarakat sehingga remaja di Desa Lepak Timur hendaknya terus memperdalam keterampilannya setelah kegiatan ini berakhir. Luaran yang telah tercapai pada pelaporan hasil kemajuan program KKN PPM ini adalah: (a) Publikasi pada media masa cetak: sudah terbit dalam Koran Lombok Pos tanggal 18 Agustus 2017 Halaman 22; (b) Peningkatan pendidikan bagi masyarakat khususnya remaja putus sekolah melalui keterampilan screen printing; (c) Peningkatan pendapatan dan partisipasi masyarakat khususnya remaja putus sekolah melalui keterampilan screen printing. Lebih jelasnya dapat dilihat dalam table berikut. 221 Jurnal Ilmiah Mandala Education Oktober 2017 Oktober 2017 JIME, Vol. 3. No. 2 ISSN 2442-9511 DAFTAR RUJUKAN Arifien, Koko K. 2011. Sangkil Merintis Usaha Percetaka Sablon. Bandung. Yrama Pustaka. Horvath, Eszter, A. Torok, P. Ficzere, dan I. Zador. 2014. Optimisation of Computer- aided Screen Printing Design. Acta Polytechnica Hungarica. 11 (8): 29-44 Horvath, Eszter, G. Henap, dan G. Harsanyi. 2012. Materials and Technological Developement of Screen Printing in Transportation. International Journal for Traffic and Transport Engineering. 2 (2): 133-141 Mahendra, Gunawan. 2013. Panduan Bisnis Cetak Sablon Manual & Digital. Smart Pustaka. Rahardjo, Beny Setiawan. 2009. Home Industry Screen Printing. Jakarta. PT Elex Media Komputindo. Syamsi, Ibnu. 2012. Model Pendidikan Kecakapan Hidup Remaja Miskin Putus Sekolah dengan Pelatihan Berwirausaha. Jurnal Cakrawala Pendidikan (Jurnal Ilmiah Pendidikan). XXXI (3): 441-452 222 Jurnal Ilmiah Mandala Education Jurnal Ilmiah Mandala Education Jurnal Ilmiah Mandala Education
https://openalex.org/W4388338143	https://www.biorxiv.org/content/biorxiv/early/2023/11/04/2023.11.01.565146.full.pdf	English	null	Melanophilin mediates the association of myosin-5a with melanosome via three distinct interactions	bioRxiv (Cold Spring Harbor Laboratory)	2,023	cc-by	22,287	Melanophilin mediates the association of myosin-5a with melanosome via three distinct interactions 1 Jiabin Pan‡§, Rui Zhou‡§, Lin-Lin Yao‡, Jie Zhang‡, Ning Zhang‡, Qin-Juan Cao‡, Shaopeng Sun‡§, and 2 Xiang-dong Li‡§1 3 ‡Group of Cell Motility and Muscle Contraction, State Key Laboratory of Integrated Ma Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 10010 §University of Chinese Academy of Sciences, Beijing 100049, China 1To whom correspondence should be addressed: Xiang-dong Li, Institute of Zoology, C of Sciences, Beijing 100101, China; Tel: +86-10-6480-6015; Email: lixd@ioz.ac.cn Running title: Myosin-5a and melanophilin interaction Key words: actin, melanophilin, melanosome, myosin-5a, Rab27a . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint Melanophilin mediates the association of myosin-5a with melanosome via three distinct interactions 1 Jiabin Pan‡§, Rui Zhou‡§, Lin-Lin Yao‡, Jie Zhang‡, Ning Zhang‡, Qin-Juan Cao‡, Shaopeng Sun‡§, and 2 Xiang-dong Li‡§1 3 4 ‡Group of Cell Motility and Muscle Contraction, State Key Laboratory of Integrated Management of Pest 5 Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China 6 §University of Chinese Academy of Sciences, Beijing 100049, China 7 1To whom correspondence should be addressed: Xiang-dong Li, Institute of Zoology, Chinese Academy 8 of Sciences, Beijing 100101, China; Tel: +86-10-6480-6015; Email: lixd@ioz.ac.cn 9 Running title: Myosin-5a and melanophilin interaction 10 Key words: actin, melanophilin, melanosome, myosin-5a, Rab27a 11 12 ABSTRACT 13 Transport and localization of melanosome at the periphery region of melanocyte are depended on 14 myosin-5a (Myo5a), which associates with melanosome by interacting with its adaptor protein melanophilin 15 (Mlph). Mlph contains four functional regions, including Rab27a-binding domain, Myo5a GTD-binding 16 motif (GTBM), Myo5a exon F-binding domain (EFBD), and actin-binding domain (ABD). The association 17 of Myo5a with Mlph is known to be mediated by two specific interactions: the interaction between the 18 exon-F-encoded region of Myo5a and Mlph-EFBD and that between Myo5a-GTD and Mlph-GTBM. Here, 19 we identify a third interaction between Myo5a and Mlph, i.e., the interaction between the exon-G-encoded 20 region of Myo5a and Mlph-ABD. The exon-G/ABD interaction is independent from the exon-F/EFBD 21 interaction and is required for the association of Myo5a with melanosome. Moreover, we demonstrate that 22 Mlph-ABD interacts with either the exon-G or actin filament, but cannot interact with both of them 23 simultaneously. Based on above findings, we propose a new model for the Mlph-mediated Myo5a 24 transportation of melanosomes. 25 ABSTRACT 26 27 Introduction Melanosome is a specialized organelle within which melanin pigments are synthesized and stored. The peripheral accumulation of melanosomes in melanocytes is essential for the intercellular transfer of the organelles from the dendrite tips of melanocytes to the adjacent keratinocytes (A. N. Hume & Seabra, 2011). Microtubule and the associated motor proteins are responsible for the long-range and bidirectional transport of melanosomes between the nucleus to cell periphery, and actin filament and the associated motor protein myosin-5a (Myo5a) are essential for the short-distance transport and the capture of melanosomes at cell periphery (X. Wu, Bowers, Rao, Wei, & Hammer, 1998; X. Wu & Hammer, 2014). The overall effect of the two transport systems is the peripheral accumulation of melanosomes in melanocytes. Lack of the latter causes the perinuclear accumulation of melanosomes in melanocytes, a phenotype called “dilute” (Provance, Wei, Ipe, & Mercer, 1996; Q. Wei, Wu, & Hammer, 1997). Myo5a is a processive motor that has been implicated in the transportation and the tethering of 39 organelles along actin filaments (Lindsay & McCaffrey, 2014; Mehta et al., 1999; Rudolf, Bittins, & Gerdes, 40 2011; Yoshimura et al., 2006; N. Zhang, Yao, & Li, 2018). Myo5a has two heavy chains, each containing 41 an N-terminal motor domain, six IQ motifs that act as the binding sites for calmodulin (CaM) or CaM-like 42 light chains, and a tail (Ikebe, 2008; N. Zhang et al., 2018). The tail of Myo5a can be further divided into 43 three segments. The proximal tail is a long coiled-coil with length about 200 residues. The distal tail is the 44 C-terminal globular tail domain (GTD). Between the proximal tail and the GTD is the middle tail, which is 45 comprised of several short coiled-coils and the connecting loops. At least three essential functions are 46 served by Myo5a tail. First, the coiled-coil regions enable Myo5a to form a dimer. Second, the GTD 47 inhibits the motor activity of myosin head. Third, the tail is the binding site for the cargo proteins. The 48 motor activity of Myo5a is tightly regulated (Krementsov, Krementsova, & Trybus, 2004; Li, Jung, 49 Mabuchi, Craig, & Ikebe, 2006; Li et al., 2008; Li, Mabuchi, Ikebe, & Ikebe, 2004; Liu, Taylor, 50 Krementsova, Trybus, & Taylor, 2006; Thirumurugan, Sakamoto, Hammer, Sellers, & Knight, 2006; Wang 51 et al., 2004). Introduction In the inhibited state, Myo5a forms a folded conformation, in which the GTDs fold back to 52 interact and inhibit the motor function. Upon being activated by high Ca2+, cargo or adaptor proteins, 53 Myo5a transforms from the folded conformation to the extended conformation, in which the GTD 54 dissociates from the motor domain and the inhibition is relieved. 55 Melanophilin (Mlph) is one of the best-studied cargo proteins of Myo5a. Together with Rab27a, Mlph 56 mediates the attachment of Myo5a to melanosomes. Rab27a is a small GTPase that, in the GTP-bound form, 57 is anchored into the melanosome membrane via its lipid moiety (Ishida, Arai, Ohbayashi, & Fukuda, 2014; 58 Wandinger-Ness & Zerial, 2014) and Mlph bridges the interaction between Rab27a and Myo5a (Fukuda, 59 Kuroda, & Mikoshiba, 2002; Nagashima et al., 2002; X. S. Wu et al., 2002). The tripartite complex of 60 Rab27a, Mlph, and Myo5a connects the melanosome to the actin filament. Lacking any one of these three 61 components in melanocytes causes dilute phenotype (perinuclear accumulation of melanosomes) (Fukuda et 62 al., 2002; A. N. Hume et al., 2001; Strom, Hume, Tarafder, Barkagianni, & Seabra, 2002; Q. Wei et al., 63 1997; X. Wu et al., 2001; X. S. Wu et al., 2002). 64 Mlph contains four functional regions, including RBD (Rab27a-binding domain), GTBM (Myo5a 65 GTD-binding motif), EFBD (Myo5a exon F-binding domain), and ABD (actin-binding domain)(Fukuda et 66 al., 2002; Geething & Spudich, 2007; Li, Ikebe, & Ikebe, 2005; Yao et al., 2015). The association of Myo5a 67 with Mlph involves two molecular interactions. One is the interaction between the exon-F-encoded region 68 (exon-F for simplification) of Myo5a and the EFBD of Mlph (Fukuda & Itoh, 2004; X. Wu, Wang, Rao, 69 Sellers, & Hammer, 2002; X. S. Wu et al., 2002). Exon-F is an alternatively spliced exon presents in the 70 middle tail of the melanocyte-spliced isoform of Myo5a (Huang et al., 1998). The exon-F/EFBD interaction 71 is absolutely essential for the localization of Myo5a to the melanosome (X. Wu et al., 2002). The other 72 interaction is between the GTD of Myo5a and the GTBM of Mlph (Geething & Spudich, 2007; Pylypenko 73 et al., 2013; Z. Wei, Liu, Yu, & Zhang, 2013). 1 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint Introduction 90 Biochemical analysis of Mlph show that the ABD is not essential for the interaction with Myo5a nor 91 the activation of Myo5a motor function, suggesting that a truncated Mlph protein containing the RBD and 92 the two Myo5a-binding domains (the GTBM and the EFBD) but lacking the ABD is able to form a tripartite 93 complex with Rab27a and Myo5a and thus sufficient for the recruitment of Myo5a to melanosome 94 membrane (T. S. Kuroda et al., 2003; X. Wu et al., 2002). However, using melan-ln cells (Mlph-null 95 melanocytes), Hume et al. have found that the ABD-deleted Mlph cannot to promote the recruitment of 96 Myo5a onto melanosomes, indicating that the ABD is necessary in situ for the association of Myo5a with 97 Mlph (A. N. Hume, A.K. Tarafder, J.S. Ramalho, E.V. Sviderskaya, and M.C. Seabra, 2006). This 98 discrepancy between in vitro and cell culture results promoted us to investigate the interaction between 99 Mlph-ABD and Myo5a. 100 In this study, we have identified a third interaction between Myo5a and Mlph, i.e., the interaction 101 between the exon-G-encoded region (exon-G for simplification) of Myo5a and Mlph-ABD. We find that the 102 exon-G/ABD interaction is independent from the exon-F/EFBD interaction, and that, similar to 103 exon-F/EFBD, exon-G/ABD is essential for the dilute-like phenotype induced by Myo5a-tail 104 overexpression. Moreover, we demonstrate that Mlph-ABD interacts with either the exon-G of Myo5a or 105 actin filament, but cannot interact with both of them simultaneously. Based on above findings, we propose a 106 new model for the Mlph-mediated Myo5a transportation of melanosomes. 107 108 109 activation of Myo5a by Mlph-GTBM is regulated by another Myo5a-binding protein RILPL2 77 (Rab-interacting lysosomal protein-like 2) and the small GTPase Rab36, a binding partner of RILPL2 (Cao, 78 Zhang, Zhou, Yao, & Li, 2019). 79 activation of Myo5a by Mlph-GTBM is regulated by another Myo5a-binding protein RILPL2 77 (Rab-interacting lysosomal protein-like 2) and the small GTPase Rab36, a binding partner of RILPL2 (Cao, 78 Zhang, Zhou, Yao, & Li, 2019). 79 Mlph-ABD is essential to proper melanosome transport (Fukuda & Kuroda, 2002; T. S. Kuroda, H. 80 Ariga, & M. Fukuda, 2003; Taruho S. Kuroda, Hiroyoshi Ariga, & Mitsunori Fukuda, 2003). Besides being 81 able to bind to actin filament, Mlph-ABD could interact with end-binding protein 1, which might facilitate 82 the transfer of melanosomes from microtubules to actin (X. S. Introduction Wu, Tsan, & Hammer, 2005). In vitro 83 motility assays at single-molecular level show that Mlph prolongs and slows the processive movements of 84 Myo5a, presumably via the interaction between ABD and actin filament (Sckolnick, Krementsova, 85 Warshaw, & Trybus, 2013). A cluster of conserved positively charged residues in ABD is essential for actin 86 binding and Melanophilin localization in melanocytes (T. S. Kuroda et al., 2003). An in vitro study suggests 87 that Mlph-ABD is a target of protein kinase A and that the phosphorylated Mlph preferred binding to actin 88 even in the presence of microtubules, whereas dephosphorylated Mlph bind to microtubules (Oberhofer et 89 al., 2017). 90 Biochemical analysis of Mlph show that the ABD is not essential for the interaction with Myo5a nor 91 the activation of Myo5a motor function, suggesting that a truncated Mlph protein containing the RBD and 92 the two Myo5a-binding domains (the GTBM and the EFBD) but lacking the ABD is able to form a tripartite 93 complex with Rab27a and Myo5a and thus sufficient for the recruitment of Myo5a to melanosome 94 membrane (T. S. Kuroda et al., 2003; X. Wu et al., 2002). However, using melan-ln cells (Mlph-null 95 melanocytes), Hume et al. have found that the ABD-deleted Mlph cannot to promote the recruitment of 96 Myo5a onto melanosomes, indicating that the ABD is necessary in situ for the association of Myo5a with 97 Mlph (A. N. Hume, A.K. Tarafder, J.S. Ramalho, E.V. Sviderskaya, and M.C. Seabra, 2006). This 98 discrepancy between in vitro and cell culture results promoted us to investigate the interaction between 99 Mlph-ABD and Myo5a. 100 In this study, we have identified a third interaction between Myo5a and Mlph, i.e., the interaction 101 between the exon-G-encoded region (exon-G for simplification) of Myo5a and Mlph-ABD. We find that the 102 exon-G/ABD interaction is independent from the exon-F/EFBD interaction, and that, similar to 103 exon-F/EFBD, exon-G/ABD is essential for the dilute-like phenotype induced by Myo5a-tail 104 overexpression. Moreover, we demonstrate that Mlph-ABD interacts with either the exon-G of Myo5a or 105 actin filament, but cannot interact with both of them simultaneously. Based on above findings, we propose a 106 new model for the Mlph-mediated Myo5a transportation of melanosomes. 107 109 Introduction We previously found that GTBM activates Myo5a motor 74 function by relieving the GTD inhibition on the motor domain and inducing a folded-to-extended 75 conformational transition of Myo5a (Li et al., 2005; Yao et al., 2015). Recently, we demonstrated that the 76 2 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint activation of Myo5a by Mlph-GTBM is regulated by another Myo5a-binding protein RILPL2 77 (Rab-interacting lysosomal protein-like 2) and the small GTPase Rab36, a binding partner of RILPL2 (Cao, 78 Zhang, Zhou, Yao, & Li, 2019). 79 Mlph-ABD is essential to proper melanosome transport (Fukuda & Kuroda, 2002; T. S. Kuroda, H. 80 Ariga, & M. Fukuda, 2003; Taruho S. Kuroda, Hiroyoshi Ariga, & Mitsunori Fukuda, 2003). Besides being 81 able to bind to actin filament, Mlph-ABD could interact with end-binding protein 1, which might facilitate 82 the transfer of melanosomes from microtubules to actin (X. S. Wu, Tsan, & Hammer, 2005). In vitro 83 motility assays at single-molecular level show that Mlph prolongs and slows the processive movements of 84 Myo5a, presumably via the interaction between ABD and actin filament (Sckolnick, Krementsova, 85 Warshaw, & Trybus, 2013). A cluster of conserved positively charged residues in ABD is essential for actin 86 binding and Melanophilin localization in melanocytes (T. S. Kuroda et al., 2003). An in vitro study suggests 87 that Mlph-ABD is a target of protein kinase A and that the phosphorylated Mlph preferred binding to actin 88 even in the presence of microtubules, whereas dephosphorylated Mlph bind to microtubules (Oberhofer et 89 al., 2017). The actin-binding domain of melanophilin (Mlph-ABD) contains a novel Myo5a-binding site. To detect interaction between the ABD of Mlph with Myo5a, we performed GST pulldown assay 112 using GST-Mlph-ABD (the GST-tagged ABD of Mlph, residues 400-590) and Flag-Myo5a-MTD (the 113 Flag-tagged middle tail domain of Myo5a, residues 1106-1469) (Figure 1). GST-Mlph-ABD was expressed 114 in E. coli and purified by GSH-Sepharose chromatography, and Flag-Myo5a-MTD was expressed in Sf9 115 cells and purified by anti-Flag affinity chromatography. GST pulldown assay shows that GST-Mlph-ABD 116 specifically interacted with Flag-Myo5a-MTD (Figure 1C, lane 2). Deleting the residues 400-437 of 117 Mlph-ABD had little effect on the interaction with Myo5a-MTD (Figure 1C, lane 3 and 4), indicating the 118 N-terminal 37 residues of Mlph-ABD are not essential for the interaction. Further deletion of 437-446 119 slightly decreased the interaction and deletion of 446-455 eliminated the interaction (Figure 1C, lane 5 and 120 6), indicating that residues 446-455 are essential for the interaction with Myo5a-MTD. Similarly, we 121 performed C-terminal truncation analysis of Mlph-ABD on the interaction with Myo5a-MTD and identified 122 residues 560-571 essential for the interaction (Figure 1D). Taking together, we identified the middle portion 123 of Mlph-ABD (residues 446-571) as the core of the novel binding site for Myo5a-MTD. 124 We then produced a number of truncated Myo5a-MTD constructs and performed GST pulldown 125 assays to narrow down the region in Myo5a-MTD binding to Mlph-ABD (Figure 2A). To simplify the 126 experiments, we first compared the bacterial-expressed Myo5a-MTD with the Sf9 cell-expressed 127 Myo5a-MTD in the interaction with Mlph-ABD. GST pulldown assay shows that both versions of 128 Myo5a-MTD specifically interacted with Mlph-ABD (Figure 2-figure supplement A). All the following 129 pulldown experiments were performed using the bacterial-expressed Myo5a-MTD, unless otherwise 130 indicated. C-terminal truncation of Flag-Myo5a-MTD shows that the C-terminal half of exon-G (residues 131 1453-1467) is essential for binding to Mlph-ABD (Figure 2-figure supplement B). Mlph-ABD interacted 132 strongly with Flag-Trx-Myo5a (1411-1467) (a short peptide containing exon-F and exon-G), but weakly 133 with Flag-Trx-Myo5a (1436-1467) (a short peptide containing exon-G) (Figure 2-figure supplement C, lane 134 3 and 4), suggesting that exon-F might be required for strong interaction of exon-G with Mlph-ABD. 135 However, deletion of exon-F from Myo5a-MTD did not affect the interaction with Mlph-ABD (Figure 2B, 136 lane 2), indicating that exon-F is not essential for the interaction. 3 C-terminal truncation of Flag-Myo5a-MTD shows that the C-terminal half of exon-G (residues 131 1453-1467) is essential for binding to Mlph-ABD (Figure 2-figure supplement B). Mlph-ABD interacted 132 strongly with Flag-Trx-Myo5a (1411-1467) (a short peptide containing exon-F and exon-G), but weakly 133 with Flag-Trx-Myo5a (1436-1467) (a short peptide containing exon-G) (Figure 2-figure supplement C, lane 134 3 and 4), suggesting that exon-F might be required for strong interaction of exon-G with Mlph-ABD. 135 However, deletion of exon-F from Myo5a-MTD did not affect the interaction with Mlph-ABD (Figure 2B, 136 lane 2), indicating that exon-F is not essential for the interaction. Deletion of the C-terminal portion 137 (residues 1454-1467) of exon-G abolished the binding of Myo5a-MTD with Mlph-ABD (figure 2B, lane 3), 138 but did not affect the interaction of Myo5a-MTD with Mlph-EFBD (Figure 2C). Figure 2A summaries the 139 truncation analysis of the interaction between Myo5a-MTD and Mlph-ABD. Based on above results, we 140 conclude that the exon-G of Myo5a binds to Mlph-ABD. 141 I i i t ti iti l f th G/ABD i t ti 142 The actin-binding domain of melanophilin (Mlph-ABD) contains a novel Myo5a-binding site. Deletion of the C-terminal portion 137 (residues 1454-1467) of exon-G abolished the binding of Myo5a-MTD with Mlph-ABD (figure 2B, lane 3), 138 but did not affect the interaction of Myo5a-MTD with Mlph-EFBD (Figure 2C). Figure 2A summaries the 139 truncation analysis of the interaction between Myo5a-MTD and Mlph-ABD. Based on above results, we 140 conclude that the exon-G of Myo5a binds to Mlph-ABD. 141 RESULTS n-binding domain of melanophilin (Mlph-ABD) contains a novel Myo5a-binding site. 3 3 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint RESULTS 110 The actin-binding domain of melanophilin (Mlph-ABD) contains a novel Myo5a-binding site. 111 To detect interaction between the ABD of Mlph with Myo5a, we performed GST pulldown assay 112 using GST-Mlph-ABD (the GST-tagged ABD of Mlph, residues 400-590) and Flag-Myo5a-MTD (the 113 Flag-tagged middle tail domain of Myo5a, residues 1106-1469) (Figure 1). GST-Mlph-ABD was expressed 114 in E. coli and purified by GSH-Sepharose chromatography, and Flag-Myo5a-MTD was expressed in Sf9 115 cells and purified by anti-Flag affinity chromatography. GST pulldown assay shows that GST-Mlph-ABD 116 specifically interacted with Flag-Myo5a-MTD (Figure 1C, lane 2). Deleting the residues 400-437 of 117 Mlph-ABD had little effect on the interaction with Myo5a-MTD (Figure 1C, lane 3 and 4), indicating the 118 N-terminal 37 residues of Mlph-ABD are not essential for the interaction. Further deletion of 437-446 119 slightly decreased the interaction and deletion of 446-455 eliminated the interaction (Figure 1C, lane 5 and 120 6), indicating that residues 446-455 are essential for the interaction with Myo5a-MTD. Similarly, we 121 performed C-terminal truncation analysis of Mlph-ABD on the interaction with Myo5a-MTD and identified 122 residues 560-571 essential for the interaction (Figure 1D). Taking together, we identified the middle portion 123 of Mlph-ABD (residues 446-571) as the core of the novel binding site for Myo5a-MTD. 124 We then produced a number of truncated Myo5a-MTD constructs and performed GST pulldown 125 assays to narrow down the region in Myo5a-MTD binding to Mlph-ABD (Figure 2A). To simplify the 126 experiments, we first compared the bacterial-expressed Myo5a-MTD with the Sf9 cell-expressed 127 Myo5a-MTD in the interaction with Mlph-ABD. GST pulldown assay shows that both versions of 128 Myo5a-MTD specifically interacted with Mlph-ABD (Figure 2-figure supplement A). All the following 129 pulldown experiments were performed using the bacterial-expressed Myo5a-MTD, unless otherwise 130 indicated. Mlph-ABD cannot interact with F-actin and exon-G simultaneously. In addition to interacting with the exon-G of Myo5a, Mlph-ABD is able to bind to actin. To investigate 184 whether Mlph-ABD interacts with actin and exon-G simultaneously, we performed actin co-sedimentation 185 of Mlph-ABD and Myo5a-MTD. As expected, substantial amount of Mlph-ABD, but essentially no 186 Myo5a-MTD, was co-sedimentated with F-actin (Figure 5A). Interestingly, Mlph-ABD did not increase the 187 amount of Myo5a-MTD co-sedimentated with F-actin (Figure 5A), suggesting that Mlph-ABD cannot 188 interact with F-actin and exon-G simultaneously. 189 The inability of Mlph-ABD to interact with F-actin and exon-G simultaneously suggests that F-actin 90 and exon-G compete in binding to Mlph-ABD. It is possible that Myo5a-MTD might antagonize the 91 interaction between Mlph-ABD and F-actin. To test this possibility, we performed F-actin 92 co-sedimentations of Mlph-ABD in the presence of different concentrations of Myo5a-MTD. As expected, 93 Myo5a-MTD substantially decreased the amount of Mlph-ABD co-sedimentated with F-actin (Figure 5B). 94 We therefore concluded that Mlph-ABD cannot bind to F-actin and exon-G simultaneously and those two 95 interactions are mutually exclusive. 96 FBD interaction and the exon-G/ABD interaction are independent from each other. Because exon-F and exon-G are in immediate vicinity, it is possible that the exon-F/EFBD interaction 163 and the exon-G/ABD interaction interfere with each other. To test this possibility, we performed GST 164 pulldown assays of GST-Mlph-ABD and Flag-Mlph-EFBD in the presence or absence of 165 Flag-Myo5a-MTD. If Myo5a-MTD is able to interact with both Mlph-ABD and Mlph-EFBD 166 simultaneously, a tripartite complex will be formed by Myo5a-MTD, Mlph-ABD, and Mlph-EFBD (Figure 167 4A, upper panel), and Flag-Mlph-EFBD will be pulled down with GST-Mlph-ABD in the presence of 168 Flag-Myo5a-MTD. As shown in Figure 4A, Flag-Mlph-EFBD was pulled down with GST-Mlph-ABD in 169 the presence of Flag-Myo5a-MTD, but not in the absence of Flag-Myo5a-MTD. This result is consistent 170 with a scenario that Myo5a-MTD binds to both Mlph-EFBD and Mlph-ABD simultaneously. We therefore 171 conclude that the exon-F/EFBD interaction and the exon-G/ABD interaction do not interfere with each 172 other. 173 As the exon-F/EFBD interaction and the exon-G/ABD interaction are independent to each other, we 174 expected that those two interactions act synergically for the binding of Myo5a-MTD to Mlph. As expected, 175 GST pulldown assays show that GST-Mlph-RBD, which contains both EFBD and ABD, strongly bound 176 to Flag-Myo5a-MTD, and deletion of either exon-F or exon-G from Myo5a-MTD substantially weakened 177 the interaction with GST-Mlph-RBD (Figure 4B). Conversely, Flag pulldown shows that substantial 178 amount of GST-Mlph-RBD could be pulled down with Flag-Myo5a-MTD, and deleting ABD from 179 GST-Mlph-RBD strongly decreased the amount of protein pulled down with Flag-Myo5a-MTD (Figure 180 4C). Above results indicate that the exon-F/EFBD interaction and the exon-G/ABD interaction act 181 synergically, resulting in a strong interaction between Myo5a-MTD and Mlph. 182 Ionic interactions are critical for the exon-G/ABD interaction. As expected, 175 GST pulldown assays show that GST-Mlph-RBD, which contains both EFBD and ABD, strongly bound 176 to Flag-Myo5a-MTD, and deletion of either exon-F or exon-G from Myo5a-MTD substantially weakened 177 the interaction with GST-Mlph-RBD (Figure 4B). Conversely, Flag pulldown shows that substantial 178 amount of GST-Mlph-RBD could be pulled down with Flag-Myo5a-MTD, and deleting ABD from 179 GST-Mlph-RBD strongly decreased the amount of protein pulled down with Flag-Myo5a-MTD (Figure 180 4C). Above results indicate that the exon-F/EFBD interaction and the exon-G/ABD interaction act 181 synergically, resulting in a strong interaction between Myo5a-MTD and Mlph. 182 Mlph-ABD cannot interact with F-actin and exon-G simultaneously. 183 In addition to interacting with the exon-G of Myo5a, Mlph-ABD is able to bind to actin. To investigate 184 whether Mlph-ABD interacts with actin and exon-G simultaneously, we performed actin co-sedimentation 185 of Mlph-ABD and Myo5a-MTD. As expected, substantial amount of Mlph-ABD, but essentially no 186 Myo5a-MTD, was co-sedimentated with F-actin (Figure 5A). Interestingly, Mlph-ABD did not increase the 187 amount of Myo5a-MTD co-sedimentated with F-actin (Figure 5A), suggesting that Mlph-ABD cannot 188 interact with F-actin and exon-G simultaneously. 189 The inability of Mlph-ABD to interact with F-actin and exon-G simultaneously suggests that F-actin 190 and exon-G compete in binding to Mlph-ABD. It is possible that Myo5a-MTD might antagonize the 191 interaction between Mlph-ABD and F-actin. To test this possibility, we performed F-actin 192 co-sedimentations of Mlph-ABD in the presence of different concentrations of Myo5a-MTD. As expected, 193 Myo5a-MTD substantially decreased the amount of Mlph-ABD co-sedimentated with F-actin (Figure 5B). 194 We therefore concluded that Mlph-ABD cannot bind to F-actin and exon-G simultaneously and those two 195 interactions are mutually exclusive. 196 The exon-G/ABD interaction is essential for Myo5a-tail to induce dilute-like phenotype in 197 melanocytes. 198 Myo5a associates with melanosome via its interaction with Mlph, which attaches to melanosome via 199 the interaction between its RBD and the melanosome-bound Rab27a. Overexpression of the GFP fusion 200 protein of Mlph-RBD in the melanocyte cell line, melan-a, caused dilute-like phenotype, i.e., the 201 perinuclear distribution of melanosomes in melanocyte (Figure 6A). Generation of dilute-like phenotype by 202 Ionic interactions are critical for the exon-G/ABD interaction. 189 The inability of Mlph-ABD to interact with F-actin and exon-G simultaneously suggests that F-actin 190 and exon-G compete in binding to Mlph-ABD. It is possible that Myo5a-MTD might antagonize the 191 interaction between Mlph-ABD and F-actin. To test this possibility, we performed F-actin 192 co-sedimentations of Mlph-ABD in the presence of different concentrations of Myo5a-MTD. As expected, 193 Myo5a-MTD substantially decreased the amount of Mlph-ABD co-sedimentated with F-actin (Figure 5B). 194 We therefore concluded that Mlph-ABD cannot bind to F-actin and exon-G simultaneously and those two 195 interactions are mutually exclusive. 196 The exon-G/ABD interaction is essential for Myo5a-tail to induce dilute-like phenotype in 197 We expected the conserved acidic residues E449 and E452 of Mlph to interact with the conserved 156 basic residues in the exon-G of Myo5a. Sequence alignment of the three Myo5 isoforms of mammals and 157 birds reveals two highly conserved basic residues (K1456 and K1460) in the C-terminal portion of exon-G 158 of Myo5a (Figure 3D). GST pulldown assay shows that both K1456A and K1460A mutations substantially 159 decreased the amount of Flag-Myo5a-MTD pulled down with GST-Mlph-ABD (Figure 3E), suggesting that 160 both K1456 and K1460 of Myo5a play a key role in the interaction with Mlph-ABD. 161 The exon-F/EFBD interaction and the exon-G/ABD interaction are independent from each other. 162 Because exon-F and exon-G are in immediate vicinity, it is possible that the exon-F/EFBD interaction 163 and the exon-G/ABD interaction interfere with each other. To test this possibility, we performed GST 164 pulldown assays of GST-Mlph-ABD and Flag-Mlph-EFBD in the presence or absence of 165 Flag-Myo5a-MTD. If Myo5a-MTD is able to interact with both Mlph-ABD and Mlph-EFBD 166 simultaneously, a tripartite complex will be formed by Myo5a-MTD, Mlph-ABD, and Mlph-EFBD (Figure 167 4A, upper panel), and Flag-Mlph-EFBD will be pulled down with GST-Mlph-ABD in the presence of 168 Flag-Myo5a-MTD. As shown in Figure 4A, Flag-Mlph-EFBD was pulled down with GST-Mlph-ABD in 169 the presence of Flag-Myo5a-MTD, but not in the absence of Flag-Myo5a-MTD. This result is consistent 170 with a scenario that Myo5a-MTD binds to both Mlph-EFBD and Mlph-ABD simultaneously. We therefore 171 conclude that the exon-F/EFBD interaction and the exon-G/ABD interaction do not interfere with each 172 other. 173 As the exon-F/EFBD interaction and the exon-G/ABD interaction are independent to each other, we 174 expected that those two interactions act synergically for the binding of Myo5a-MTD to Mlph. Ionic interactions are critical for the exon-G/ABD interaction. We characterized the exon-G/ABD interaction by investigating the effect of ionic strength on their 143 interaction. GST pulldown assays of GST-Mlph-ABD and Flag-Myo5a-MTD were conducted in the 144 presence different concentrations of NaCl. Compared with 100 mM NaCl, 300 mM and 500 mM NaCl 145 greatly decreased the amount of Flag-Myo5a-MTD pulled down with GST-Mlph-ABD (Figure 3A), 146 suggesting that ionic interactions are critical for the exon-G/ABD interaction. 147 Sequence alignment among the Mlph of mammals and birds reveals several highly conserved charged 148 residues in the two regions of Mlph-ABD essential for the interaction with Myo5a-MTD, including four 149 acidic residues (D447, E449, E452, and E454) in the residues 446-455 and two basic residues (R562 and 150 R568) in the residues 560-571 (Figure 3B). To determine the roles of those conserved charged residues of 151 Mlph on the exon-G/ABD interaction, we mutated them individually into alanine. GST pulldown assays 152 show that both E449A and E452A mutations abolished the interaction with Myo5a-MTD, whereas other 153 mutations (D447A, E454A, R562A, R568A) had little effect on the interaction (Figure 3C), indicating that 154 both E449 and E452 of Mlph are essential for the exon-G/ABD interaction. 155 4 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint We expected the conserved acidic residues E449 and E452 of Mlph to interact with the conserved 156 basic residues in the exon-G of Myo5a. Sequence alignment of the three Myo5 isoforms of mammals and 157 birds reveals two highly conserved basic residues (K1456 and K1460) in the C-terminal portion of exon-G 158 of Myo5a (Figure 3D). GST pulldown assay shows that both K1456A and K1460A mutations substantially 159 decreased the amount of Flag-Myo5a-MTD pulled down with GST-Mlph-ABD (Figure 3E), suggesting that 160 both K1456 and K1460 of Myo5a play a key role in the interaction with Mlph-ABD. Ionic interactions are critical for the exon-G/ABD interaction. 161 The exon-F/EFBD interaction and the exon-G/ABD interaction are independent from each other. 162 Because exon-F and exon-G are in immediate vicinity, it is possible that the exon-F/EFBD interaction 163 and the exon-G/ABD interaction interfere with each other. To test this possibility, we performed GST 164 pulldown assays of GST-Mlph-ABD and Flag-Mlph-EFBD in the presence or absence of 165 Flag-Myo5a-MTD. If Myo5a-MTD is able to interact with both Mlph-ABD and Mlph-EFBD 166 simultaneously, a tripartite complex will be formed by Myo5a-MTD, Mlph-ABD, and Mlph-EFBD (Figure 167 4A, upper panel), and Flag-Mlph-EFBD will be pulled down with GST-Mlph-ABD in the presence of 168 Flag-Myo5a-MTD. As shown in Figure 4A, Flag-Mlph-EFBD was pulled down with GST-Mlph-ABD in 169 the presence of Flag-Myo5a-MTD, but not in the absence of Flag-Myo5a-MTD. This result is consistent 170 with a scenario that Myo5a-MTD binds to both Mlph-EFBD and Mlph-ABD simultaneously. We therefore 171 conclude that the exon-F/EFBD interaction and the exon-G/ABD interaction do not interfere with each 172 other. 173 As the exon-F/EFBD interaction and the exon-G/ABD interaction are independent to each other, we 174 expected that those two interactions act synergically for the binding of Myo5a-MTD to Mlph. As expected, 175 GST pulldown assays show that GST-Mlph-RBD, which contains both EFBD and ABD, strongly bound 176 to Flag-Myo5a-MTD, and deletion of either exon-F or exon-G from Myo5a-MTD substantially weakened 177 the interaction with GST-Mlph-RBD (Figure 4B). Conversely, Flag pulldown shows that substantial 178 amount of GST-Mlph-RBD could be pulled down with Flag-Myo5a-MTD, and deleting ABD from 179 GST-Mlph-RBD strongly decreased the amount of protein pulled down with Flag-Myo5a-MTD (Figure 180 4C). Above results indicate that the exon-F/EFBD interaction and the exon-G/ABD interaction act 181 synergically, resulting in a strong interaction between Myo5a-MTD and Mlph. 182 Mlph-ABD cannot interact with F-actin and exon-G simultaneously. 183 In addition to interacting with the exon-G of Myo5a, Mlph-ABD is able to bind to actin. To investigate 184 whether Mlph-ABD interacts with actin and exon-G simultaneously, we performed actin co-sedimentation 185 of Mlph-ABD and Myo5a-MTD. As expected, substantial amount of Mlph-ABD, but essentially no 186 Myo5a-MTD, was co-sedimentated with F-actin (Figure 5A). Interestingly, Mlph-ABD did not increase the 187 amount of Myo5a-MTD co-sedimentated with F-actin (Figure 5A), suggesting that Mlph-ABD cannot 188 interact with F-actin and exon-G simultaneously. The exon-G/ABD interaction is essential for Myo5a-tail to induce dilute-like phenotype in 197 melanocytes. 198 While dilute-like phenotype was 208 presented in 48.78% of cells expressing the wild-type Mlph-RBD (n=194), only in 20.97% and 19.07% of 209 cells expressing E449A (n=208) and E542A (n=267), respectively (Figure 6). 210 To further determine the role of the exon-G/ABD interaction on the tethering of Myo5a with Mlph in 211 melanocyte, we expressed the GFP fusion protein of Myo5a-tail (residues 1106-1877), which contains all 212 three Mlph-binding regions, i.e., exon-F, exon-F, and GTD (Figure 7A). Overexpressing the tail region of 213 Myo5a in melanocyte was expected to compete with endogenous Myo5a in interacting with Mlph, thus 214 causing dilute-like phenotype (Wu et al 2002). Deletion analysis of the tail region of Myo5a have shown 215 that both exon-F and the GTD are required and that neither is sufficient for generating the dominant 216 negative phenotype in melanocytes (Wu et al 2002). Similar to the previous report, Myo5a-tail is well 217 co-localized with both melanosome and Mlph, and generated dilute-like phenotype in ~50% of transfected 218 melan-a melanocytes (n=233). In contrast, Myo5a-TailG generated dilute-like phenotype in only ~10% of 219 transfected cells (n=194) (Figure 7). Therefore, we concluded that, similar to exon-F, exon-G is also 220 required for Myo5a-Tail to disrupt endogenous Myo5a function in transportation of melanosomes. 221 The exon-G/ABD interaction is essential for Myo5a-tail to induce dilute-like phenotype in 197 melanocytes. 198 Myo5a associates with melanosome via its interaction with Mlph, which attaches to melanosome via 199 the interaction between its RBD and the melanosome-bound Rab27a. Overexpression of the GFP fusion 200 protein of Mlph-RBD in the melanocyte cell line, melan-a, caused dilute-like phenotype, i.e., the 201 perinuclear distribution of melanosomes in melanocyte (Figure 6A). Generation of dilute-like phenotype by 202 5 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint Mlph-RBD was attributed to the ability of Mlph-RBD to compete with the melanosome-bound Mlph in 203 interacting with Myo5a molecules, thereby dissociating Myo5a from melanosome. To investigate whether 204 the exon-G/ABD interaction is essential for the dominant negative effect of Mlph-RBD in melanocyte, we 205 introduced two single pointed mutations, E449A and E452A, in Mlph-RBD, both of which are essential 206 for Mlph-ABD to interact with the exon-G (Figure 3C). We found that both two mutations substantially 207 decreased the number of the transfected cells with dilute-like phenotype. While dilute-like phenotype was 208 presented in 48.78% of cells expressing the wild-type Mlph-RBD (n=194), only in 20.97% and 19.07% of 209 cells expressing E449A (n=208) and E542A (n=267), respectively (Figure 6). 210 Mlph-RBD was attributed to the ability of Mlph-RBD to compete with the melanosome-bound Mlph in 203 interacting with Myo5a molecules, thereby dissociating Myo5a from melanosome. To investigate whether 204 the exon-G/ABD interaction is essential for the dominant negative effect of Mlph-RBD in melanocyte, we 205 introduced two single pointed mutations, E449A and E452A, in Mlph-RBD, both of which are essential 206 for Mlph-ABD to interact with the exon-G (Figure 3C). We found that both two mutations substantially 207 decreased the number of the transfected cells with dilute-like phenotype. DISCUSSION At physiological ionic strength, the GTBM-mediated 244 activation of Myo5a depends on the interaction between the GTD and RilpL2, which is regulated by Rab36 245 (Cao et al., 2019). Those findings are consistent with the three-dimensional structure of Myo5a-GTD and 246 the folded structure of full-length Myo5a (Niu et al., 2022; Pylypenko et al., 2013; Z. Wei et al., 2013), in 247 both of which the GTBM-binding sites are buried at the GTD-GTD interface. Upon binding to the RH1 248 domain of RilpL2, the GTD exposes the GTBM-binding site (Z. Wei et al., 2013). We therefore proposed 249 that the binding of Rab36/RilpL2 to the GTD exposes the GTBM-binding site, thus facilitating GTBM to 250 bind to the GTD and to activate the motor function of Myo5a (Cao et al., 2019). 251 The GTD/GTBM interaction is also required for the stable association between Myo5a and Mlph. The 252 Myo5a tail construct containing all three Mlph-binding sites (the exon-F, the exon-G, and the GTD) 253 produced dominant negative effect in melanocytes, and deleting the GTD abolished the dominant negative 254 effect (Figure 7 and 8, and reference (X. Wu et al., 2002)). 255 The exon-F/EFBD and the exon-G/ABD interactions play a major role in the binding of Myo5a with 256 Mlph. Although exon-F and exon-G are adjacent to each other, the exon-F/EFBD interaction and the 257 exon-G/ABD interaction do not interfere with each other. Rather, those two interactions act synergically. 258 We observed that Myo5a-MTD, containing both exon-F and exon-G but lacking the GTD, did not generate 259 a dilute-like phenotype in melanocytes, but was co-localized well with Mlph and partially with 260 melanosomes. Deletion of either exon-F or exon-G substantially weakened the interaction between 261 Myo5a-MTD and Mlph and decreased the colocalization of Myo5a-MTD with Mlph, indicating the both 262 exon-F and exon-G are required for the colocalization. 263 Our observation that Myo5a-MTD, containing both exon-F and exon-G, was co-localized partially 264 with melanosomes seems to contradict the early work from Hammer laboratory, which showed that MC 265 STK (a Myo5a fragment contains both exon-F and exon-G) did not exhibit any tendency to co-localize with 266 melanosomes (X. Wu et al., 2002). However, careful examining their image (Figure 5, in reference(X. Wu 267 et al., 2002)) reveals a partial colocalization of MC STK with melanosomes, particularly at the perinuclear 268 region. Myo5a-MTD colocalized with Mlph but was unable to induce dilute-like phenotype. Unlike Myo5a-Tail, overexpression of Myo5a-MTD, which contains both exon-F and exon-G but 223 lacks the GTD, did not generate dilute-like phenotype in melan-a cells. However, Myo5a-MTD is well 224 colocalized with endogenous Mlph and partially colocalized with melanosome (Figure 8A). We reasoned 225 that the presence of both exon-F and exon-G is insufficient for binding to the Mlph occupied by Myo5a, but 226 sufficient for binding to the unoccupied Mlph. 227 Deletion of exon-F almost eliminated the co-localization of Myo5a-MTD and Mlph (Figure 8B), 228 consistent with its essential role for the binding of Myo5a with Mlph. Deletion of exon-G substantially 229 decreased, but did not eliminate, the co-localization of Myo5a-MTD and Mlph (Figure 8C), indicating that 230 exon-G plays an auxiliary role for binding of Myo5a with Mlph. 231 232 6 6 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint DISCUSSION 258 We observed that Myo5a-MTD, containing both exon-F and exon-G but lacking the GTD, did not generate 259 a dilute-like phenotype in melanocytes, but was co-localized well with Mlph and partially with 260 melanosomes. Deletion of either exon-F or exon-G substantially weakened the interaction between 261 Myo5a-MTD and Mlph and decreased the colocalization of Myo5a-MTD with Mlph, indicating the both 262 exon-F and exon-G are required for the colocalization. 263 Our observation that Myo5a-MTD, containing both exon-F and exon-G, was co-localized partially 264 with melanosomes seems to contradict the early work from Hammer laboratory, which showed that MC 265 STK (a Myo5a fragment contains both exon-F and exon-G) did not exhibit any tendency to co-localize with 266 melanosomes (X. Wu et al., 2002). However, careful examining their image (Figure 5, in reference(X. Wu 267 et al., 2002)) reveals a partial colocalization of MC STK with melanosomes, particularly at the perinuclear 268 region. It is likely that some melanosome-bound Mlph molecules are unoccupied, some interact with 269 Myo5a via the exon-F/EFBD and the exon-G/ABD interactions but not the GTD/GTBM interaction, and 270 some interact with Myo5a via three interactions. In the former two cases, Myo5a-MTD would be able to 271 bind to the melanosome via Mlph. We therefore conclude that presence of both exon-F and exon-G is 272 sufficient for Myo5a to associate with Mlph, but insufficient for substituting the melanosome-bound Myo5a 273 molecules which bind to Mlph via three distinct interactions. 274 As discussed above, the GTBM-binding site is buried at the GTD-GTD interface in the folded state of 275 Myo5a, thus is inaccessible for Mlph, and the opening of GTBM-site depends on the interaction with 276 Rab36/RilpL2. On the other hand, the exon-F and the exon-G are likely exposed. We therefore propose that 277 th f ld d M 5 fi t tt h t Ml h i th F/EFBD d G/ABD i t ti th i t t 278 It is well-established that Myo5a associates with melanosome via two independent interactions with 235 Mlph, i.e., the interaction between exon-F of Myo5a and Mlph-EFBD and the interaction between the GTD 236 and Mlph-GTBM. In the current study, we identified the third interaction between Myo5a and Mlph, i.e., 237 the interaction between the exon-G of Myo5a and the Mlph-ABD. DISCUSSION 258 We observed that Myo5a-MTD, containing both exon-F and exon-G but lacking the GTD, did not generate 259 a dilute-like phenotype in melanocytes, but was co-localized well with Mlph and partially with 260 melanosomes. Deletion of either exon-F or exon-G substantially weakened the interaction between 261 Myo5a-MTD and Mlph and decreased the colocalization of Myo5a-MTD with Mlph, indicating the both 262 exon-F and exon-G are required for the colocalization. 263 Our observation that Myo5a-MTD, containing both exon-F and exon-G, was co-localized partially 264 with melanosomes seems to contradict the early work from Hammer laboratory, which showed that MC 265 STK (a Myo5a fragment contains both exon-F and exon-G) did not exhibit any tendency to co-localize with 266 melanosomes (X. Wu et al., 2002). However, careful examining their image (Figure 5, in reference(X. Wu 267 et al., 2002)) reveals a partial colocalization of MC STK with melanosomes, particularly at the perinuclear 268 region. It is likely that some melanosome-bound Mlph molecules are unoccupied, some interact with 269 Myo5a via the exon-F/EFBD and the exon-G/ABD interactions but not the GTD/GTBM interaction, and 270 some interact with Myo5a via three interactions. In the former two cases, Myo5a-MTD would be able to 271 bind to the melanosome via Mlph. We therefore conclude that presence of both exon-F and exon-G is 272 sufficient for Myo5a to associate with Mlph, but insufficient for substituting the melanosome-bound Myo5 273 molecules which bind to Mlph via three distinct interactions. 274 As discussed above, the GTBM-binding site is buried at the GTD-GTD interface in the folded state of 275 Myo5a, thus is inaccessible for Mlph, and the opening of GTBM-site depends on the interaction with 276 Rab36/RilpL2. On the other hand, the exon-F and the exon-G are likely exposed. We therefore propose tha 277 the folded Myo5a first attaches to Mlph via the exon-F/EFBD and exon-G/ABD interactions, then interacts 278 with Rab36/RilpL2 to open the GTBM-site, and finally interacts with the GTBM, forming the extended 279 conformation and being activated. 280 Among the three interactions between Myo5a and Mlph, the GTD/GTBM interaction majorly regulates 241 the motor function of Myo5a. At relative high ionic strength but not at physiological ionic strength, 242 Mlph-GTBM binds to the GTD, inducing Myo5a to form the extended conformation and activating Myo5a 243 motor activity (Li et al., 2005; Yao et al., 2015). DISCUSSION DISCUSSION 234 It is well-established that Myo5a associates with melanosome via two independent interactions with 235 Mlph, i.e., the interaction between exon-F of Myo5a and Mlph-EFBD and the interaction between the GTD 236 and Mlph-GTBM. In the current study, we identified the third interaction between Myo5a and Mlph, i.e., 237 the interaction between the exon-G of Myo5a and the Mlph-ABD. We demonstrated that, similar to the 238 exon-F/EFBD interaction, the exon-G/ABD interaction is also required for the strong binding of Myo5a and 239 Mlph. 240 Among the three interactions between Myo5a and Mlph, the GTD/GTBM interaction majorly regulate 241 the motor function of Myo5a. At relative high ionic strength but not at physiological ionic strength, 242 Mlph-GTBM binds to the GTD, inducing Myo5a to form the extended conformation and activating Myo5a 243 motor activity (Li et al., 2005; Yao et al., 2015). At physiological ionic strength, the GTBM-mediated 244 activation of Myo5a depends on the interaction between the GTD and RilpL2, which is regulated by Rab36 245 (Cao et al., 2019). Those findings are consistent with the three-dimensional structure of Myo5a-GTD and 246 the folded structure of full-length Myo5a (Niu et al., 2022; Pylypenko et al., 2013; Z. Wei et al., 2013), in 247 both of which the GTBM-binding sites are buried at the GTD-GTD interface. Upon binding to the RH1 248 domain of RilpL2, the GTD exposes the GTBM-binding site (Z. Wei et al., 2013). We therefore proposed 249 that the binding of Rab36/RilpL2 to the GTD exposes the GTBM-binding site, thus facilitating GTBM to 250 bind to the GTD and to activate the motor function of Myo5a (Cao et al., 2019). 251 The GTD/GTBM interaction is also required for the stable association between Myo5a and Mlph. The 252 Myo5a tail construct containing all three Mlph-binding sites (the exon-F, the exon-G, and the GTD) 253 produced dominant negative effect in melanocytes, and deleting the GTD abolished the dominant negative 254 effect (Figure 7 and 8, and reference (X. Wu et al., 2002)). 255 The exon-F/EFBD and the exon-G/ABD interactions play a major role in the binding of Myo5a with 256 Mlph. Although exon-F and exon-G are adjacent to each other, the exon-F/EFBD interaction and the 257 exon-G/ABD interaction do not interfere with each other. Rather, those two interactions act synergically. DISCUSSION We demonstrated that, similar to the 238 exon-F/EFBD interaction, the exon-G/ABD interaction is also required for the strong binding of Myo5a and 239 Mlph. 240 and Mlph GTBM. In the current study, we identified the third interaction between Myo5a and Mlph, i.e., 237 the interaction between the exon-G of Myo5a and the Mlph-ABD. We demonstrated that, similar to the 238 exon-F/EFBD interaction, the exon-G/ABD interaction is also required for the strong binding of Myo5a and 239 Mlph. 240 Among the three interactions between Myo5a and Mlph, the GTD/GTBM interaction majorly regulate 241 the motor function of Myo5a. At relative high ionic strength but not at physiological ionic strength, 242 Mlph-GTBM binds to the GTD, inducing Myo5a to form the extended conformation and activating Myo5a 243 motor activity (Li et al., 2005; Yao et al., 2015). At physiological ionic strength, the GTBM-mediated 244 activation of Myo5a depends on the interaction between the GTD and RilpL2, which is regulated by Rab36 245 (Cao et al., 2019). Those findings are consistent with the three-dimensional structure of Myo5a-GTD and 246 the folded structure of full-length Myo5a (Niu et al., 2022; Pylypenko et al., 2013; Z. Wei et al., 2013), in 247 both of which the GTBM-binding sites are buried at the GTD-GTD interface. Upon binding to the RH1 248 domain of RilpL2, the GTD exposes the GTBM-binding site (Z. Wei et al., 2013). We therefore proposed 249 that the binding of Rab36/RilpL2 to the GTD exposes the GTBM-binding site, thus facilitating GTBM to 250 bind to the GTD and to activate the motor function of Myo5a (Cao et al., 2019). 251 The GTD/GTBM interaction is also required for the stable association between Myo5a and Mlph. The 252 Myo5a tail construct containing all three Mlph-binding sites (the exon-F, the exon-G, and the GTD) 253 produced dominant negative effect in melanocytes, and deleting the GTD abolished the dominant negative 254 effect (Figure 7 and 8, and reference (X. Wu et al., 2002)). 255 The exon-F/EFBD and the exon-G/ABD interactions play a major role in the binding of Myo5a with 256 Mlph. Although exon-F and exon-G are adjacent to each other, the exon-F/EFBD interaction and the 257 exon-G/ABD interaction do not interfere with each other. Rather, those two interactions act synergically. DISCUSSION We expect that 309 Myo5a-MTD, containing both exon-F and exon-G, is able to bind to Mlph at stage 1 and 2, but not at stage 310 3 and 4, and that Myo5a-Tail, containing all three Mlph-binding sites, is able to bind to Mlph at all four 311 stages. 312 In conclusion, we demonstrate a direct interaction between the exon-G of Myo5a and the ABD of 313 Mlph, which is essential for the tight binding of Myo5a to Mlph both in vitro and in vivo. We expect that 314 the melanosomal recruitment and activation of Myo5a are a highly coordinated process mediated by three 315 interactions between Myo5a and Mlph, i.e., the exon-F/EFBD interaction, the exon-G/ABD interaction, and 316 the GTD/GTBM interactions. 317 318 One interesting finding of this study is that Mlph-ABD was able to separately interact with the exon-G 281 of Myo5a or actin filament, but unable to interact with both of them simultaneously. In other words, 282 Mlph-ABD binds to either exon-G or actin filament. This is consistent with the geometry of Myo5a, in 283 which exon-G is located far from the motor domain, which interacts with actin filament to produce motility. 284 Given the small size of Mlph-ABD, it is unlikely that Mlph-ABD is able to bridge the exon-G at one end of 285 Myo5a and actin filament which associates with the motor domain located at the other end of Myo5a. An 286 unsolved issue is how Mlph-ABD’s interactions with exon-G and actin filament are regulated. In vitro 287 studies suggest that the interaction between Mlph-ABD and actin filament might be regulated by 288 phosphorylation (Oberhofer et al., 2017). 289 Based on our current finding and previous studies on the interaction between Myo5a and its associated 290 proteins, we propose following model for the Mlph-mediated Myo5a transportation of melanosome (Figure 291 9). At stage 1, Mlph associates with melanosome via its interaction with Rab27a, which directly binds to the 292 membrane of melanosome; the unattached Myo5a is in a folded conformation, in which the GTD binds to 293 and inhibits the motor domain. At stage 2, Mlph interacts with the folded Myo5a via the interactions of 294 EFBD/exon-F and ABD/exon-G; the attached Myo5a is still in folded conformation, because the 295 GTBM-binding surface in the GTD is buried at the GTD-GTD interface. DISCUSSION It is likely that some melanosome-bound Mlph molecules are unoccupied, some interact with 269 Myo5a via the exon-F/EFBD and the exon-G/ABD interactions but not the GTD/GTBM interaction, and 270 some interact with Myo5a via three interactions. In the former two cases, Myo5a-MTD would be able to 271 bind to the melanosome via Mlph. We therefore conclude that presence of both exon-F and exon-G is 272 sufficient for Myo5a to associate with Mlph, but insufficient for substituting the melanosome-bound Myo5a 273 molecules which bind to Mlph via three distinct interactions. 274 As discussed above, the GTBM-binding site is buried at the GTD-GTD interface in the folded state of 275 Myo5a, thus is inaccessible for Mlph, and the opening of GTBM-site depends on the interaction with 276 Rab36/RilpL2. On the other hand, the exon-F and the exon-G are likely exposed. We therefore propose that 277 the folded Myo5a first attaches to Mlph via the exon-F/EFBD and exon-G/ABD interactions, then interacts 278 with Rab36/RilpL2 to open the GTBM-site, and finally interacts with the GTBM, forming the extended 279 conformation and being activated. 280 7 7 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint One interesting finding of this study is that Mlph-ABD was able to separately interact with the exon-G 281 of Myo5a or actin filament, but unable to interact with both of them simultaneously. In other words, 282 Mlph-ABD binds to either exon-G or actin filament. This is consistent with the geometry of Myo5a, in 283 which exon-G is located far from the motor domain, which interacts with actin filament to produce motility. 284 Given the small size of Mlph-ABD, it is unlikely that Mlph-ABD is able to bridge the exon-G at one end of 285 Myo5a and actin filament which associates with the motor domain located at the other end of Myo5a. DISCUSSION An 286 unsolved issue is how Mlph-ABD’s interactions with exon-G and actin filament are regulated. In vitro 287 studies suggest that the interaction between Mlph-ABD and actin filament might be regulated by 288 phosphorylation (Oberhofer et al., 2017). 289 Based on our current finding and previous studies on the interaction between Myo5a and its associated 290 proteins, we propose following model for the Mlph-mediated Myo5a transportation of melanosome (Figure 291 9). At stage 1, Mlph associates with melanosome via its interaction with Rab27a, which directly binds to the 292 membrane of melanosome; the unattached Myo5a is in a folded conformation, in which the GTD binds to 293 and inhibits the motor domain. At stage 2, Mlph interacts with the folded Myo5a via the interactions of 294 EFBD/exon-F and ABD/exon-G; the attached Myo5a is still in folded conformation, because the 295 GTBM-binding surface in the GTD is buried at the GTD-GTD interface. At stage 3, the buried 296 GTBM-binding surface between the GTD-GTD interface is exposed and thus facilitate the binding of 297 GTBM, causing the dissociation of the GTD from the motor domain and inducing the extended 298 conformation of Myo5a (This step is probably regulated by the binding of Rab36/RilpL2 to the GTD). At 299 stage 4, Mlph-ABD dissociates from exon-G and then binds to actin filament, thus enhancing the processive 300 movement of Myo5a (This step might be regulated by the phosphorylation of Mlph-ABD). The interaction 301 between Mlph-ABD and actin filament is regulated by phosphorylation (Oberhofer et al., 2017). 302 Melanosomes in melanocytes are matured through a serial of well-defined morphological steps, from 303 melanin-lacking premelanosomes to blackened melanosomes with melanin fully loaded (Raposo & Marks, 304 2007). During this maturation process, melanosomes undergo microtubule and actin-based transport 305 towards the cell periphery, mediated by different Rab proteins and their effectors (Fukuda, 2021). For 306 example, Rab27a mainly associates with intermediate and mature melanosomes (Jordens et al., 2006). The 307 melanosome-bound Rab27a in GTP-bound state binds to Mlph, which in turn recruits Myo5a (X. S. Wu et 308 al., 2002). We propose that the recruitment of Myo5a by Mlph consists of multiple stages. DISCUSSION CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint DISCUSSION At stage 3, the buried 296 GTBM-binding surface between the GTD-GTD interface is exposed and thus facilitate the binding of 297 GTBM, causing the dissociation of the GTD from the motor domain and inducing the extended 298 conformation of Myo5a (This step is probably regulated by the binding of Rab36/RilpL2 to the GTD). At 299 stage 4, Mlph-ABD dissociates from exon-G and then binds to actin filament, thus enhancing the processive 300 movement of Myo5a (This step might be regulated by the phosphorylation of Mlph-ABD). The interaction 301 between Mlph-ABD and actin filament is regulated by phosphorylation (Oberhofer et al., 2017). 302 Melanosomes in melanocytes are matured through a serial of well-defined morphological steps, from 303 melanin-lacking premelanosomes to blackened melanosomes with melanin fully loaded (Raposo & Marks, 304 2007). During this maturation process, melanosomes undergo microtubule and actin-based transport 305 towards the cell periphery, mediated by different Rab proteins and their effectors (Fukuda, 2021). For 306 example, Rab27a mainly associates with intermediate and mature melanosomes (Jordens et al., 2006). The 307 melanosome-bound Rab27a in GTP-bound state binds to Mlph, which in turn recruits Myo5a (X. S. Wu et 308 al., 2002). We propose that the recruitment of Myo5a by Mlph consists of multiple stages. We expect that 309 Myo5a-MTD, containing both exon-F and exon-G, is able to bind to Mlph at stage 1 and 2, but not at stage 310 3 and 4, and that Myo5a-Tail, containing all three Mlph-binding sites, is able to bind to Mlph at all four 311 stages. 312 In conclusion, we demonstrate a direct interaction between the exon-G of Myo5a and the ABD of 313 Mlph, which is essential for the tight binding of Myo5a to Mlph both in vitro and in vivo. We expect that 314 the melanosomal recruitment and activation of Myo5a are a highly coordinated process mediated by three 315 interactions between Myo5a and Mlph, i.e., the exon-F/EFBD interaction, the exon-G/ABD interaction, and 316 the GTD/GTBM interactions. 317 318 8 8 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint . Proteins 328 All Myo5a constructs in this study were created from a melanocyte type Myo5a (Li et al., 2005). The 329 recombinant proteins of truncated Myo5a containing an N-terminal His-tag and Flag-tag were expressed in 330 BL21(DE3) E. coli. The cDNA of truncated Myo5a were amplified by using high-fidelity FastPfu DNA 331 polymerase (TransGen) with Myo5a cDNA as template (Li et al., 2005), and subcloned into pET30HFa (a 332 modified pET30a vector encoding His-tag and Flag-tag). To improve the expression of short peptides of 333 truncated Myo5a, including Myo5a(1411-1467) and Myo5a(1436-1467), the cDNA of Trx-1 (Genbank ID: 334 P0AA25) was inserted between the Flag-tag sequence and the cDNA of truncated Myo5a in pET30HFa. 335 Myo5a-MTDF were created by using overlap extension PCR with Myo5a-MTD/pFast FTb as template 336 and inserted into pET30HFa. The recombinant proteins were expressed in BL21(DE3) E. coli as His-Flag 337 tagged proteins were purified by Ni-agarose affinity chromatography using standard procedures. We also 338 produced an N-terminal His-tagged and Flag-tagged Myo5a-MTD using baculovirus/Sf9 system. The 339 cDNA of Myo5a-MTD was subcloned into the baculovirus transfer vector pFastHFTb (a modified 340 pFastHTb vector containing the His-tag and Flag-tag sequence). Recombinant baculovirus was generated 341 using Bac-to-Bac system. The Myo5a-MTD expressed in Sf9 insect cells was purified by Anti-FLAG M2 342 affinity chromatography (Li et al., 2008). 343 Three truncated Mlph constructs containing an N-terminal GST-tag, including Mlph-ABD (residues 344 401-590), Mlph-RBD (residues 148-590), Mlph-RBDABD (residues 148-400), were created by 345 subcloning the cDNAs of truncated Mlph into pGEX4T1 vector using BamHI and XhoI sites. Mlph-EFBD 346 (residues 241-400) having an N-terminal His-tag and Flag-tag was created by the cDNA of Mlph-EFBD 347 subcloning into pET30HFa vector using EcoRI and HindIII sites. Point mutations were created using Fast 348 Mutagenesis System (TransGen) according to the manufacturer’s instructions. The recombinant proteins 349 were expressed in BL21(DE3) E. coli as His-Flag tagged proteins (in pET30HFa vector) or GST tagged 350 proteins (in pGEX4T1 vector), and purified by Ni-agarose affinity chromatography or GSH-Sepharose 351 affinity chromatography using standard procedures. 352 GST pulldown assays were performed as described previously (W. B. Zhang, Yao, & Li, 2016). EXPERIMENTAL PROCEDURES EXPERIMENTAL PROCEDURES 319 Materials 320 Restriction enzymes and modifying enzymes were purchased from New England Biolabs (Beverly, 321 MA), unless indicated otherwise. Actin was prepared from rabbit skeletal muscle acetone powder according 322 to Spudich and Watt (Spudich & Watt, 1971). Ni-NTA agarose was purchased from Qiagen (Hilden, 323 Germany). Anti-Flag M2 affinity gel and HPR conjugated anti-Flag M2 antibody were from Sigma Co. (St. 324 Louis, MO). Glutathione(GSH)-Sepharose 4 Fast Flow was from GE Healthcare. FLAG peptide 325 (DYKDDDDK) was synthesized by Augct Co. (Beijing, China). Oligonucleotides were synthesized by 326 Invitrogen Co. (Beijing, China). 327 Proteins 328 All Myo5a constructs in this study were created from a melanocyte type Myo5a (Li et al., 2005). The 329 recombinant proteins of truncated Myo5a containing an N-terminal His-tag and Flag-tag were expressed in 330 BL21(DE3) E. coli. The cDNA of truncated Myo5a were amplified by using high-fidelity FastPfu DNA 331 polymerase (TransGen) with Myo5a cDNA as template (Li et al., 2005), and subcloned into pET30HFa (a 332 modified pET30a vector encoding His-tag and Flag-tag). To improve the expression of short peptides of 333 truncated Myo5a, including Myo5a(1411-1467) and Myo5a(1436-1467), the cDNA of Trx-1 (Genbank ID: 334 P0AA25) was inserted between the Flag-tag sequence and the cDNA of truncated Myo5a in pET30HFa. 335 Myo5a-MTDF were created by using overlap extension PCR with Myo5a-MTD/pFast FTb as template 336 and inserted into pET30HFa. The recombinant proteins were expressed in BL21(DE3) E. coli as His-Flag 337 tagged proteins were purified by Ni-agarose affinity chromatography using standard procedures. We also 338 produced an N-terminal His-tagged and Flag-tagged Myo5a-MTD using baculovirus/Sf9 system. The 339 cDNA of Myo5a-MTD was subcloned into the baculovirus transfer vector pFastHFTb (a modified 340 pFastHTb vector containing the His-tag and Flag-tag sequence). Recombinant baculovirus was generated 341 using Bac-to-Bac system. The Myo5a-MTD expressed in Sf9 insect cells was purified by Anti-FLAG M2 342 affinity chromatography (Li et al., 2008). 343 Three truncated Mlph constructs containing an N-terminal GST-tag, including Mlph-ABD (residues 344 401-590), Mlph-RBD (residues 148-590), Mlph-RBDABD (residues 148-400), were created by 345 subcloning the cDNAs of truncated Mlph into pGEX4T1 vector using BamHI and XhoI sites. Mlph-EFBD 346 (residues 241-400) having an N-terminal His-tag and Flag-tag was created by the cDNA of Mlph-EFBD 347 subcloning into pET30HFa vector using EcoRI and HindIII sites. Point mutations were created using Fast 348 Mutagenesis System (TransGen) according to the manufacturer’s instructions. EXPERIMENTAL PROCEDURES The recombinant proteins 349 were expressed in BL21(DE3) E. coli as His-Flag tagged proteins (in pET30HFa vector) or GST tagged 350 proteins (in pGEX4T1 vector), and purified by Ni-agarose affinity chromatography or GSH-Sepharose 351 affinity chromatography using standard procedures. 352 Protein pulldown assay 353 GST pulldown assays were performed as described previously (W. B. Zhang, Yao, & Li, 2016). For 354 GST pulldown of Myo5a-MTD with GST-Mlph-ABD, GSH-Sepharose beads (10 μl) were mixed with 95 355 Materials 320 Restriction enzymes and modifying enzymes were purchased from New England Biolabs (Beverly, 321 MA), unless indicated otherwise. Actin was prepared from rabbit skeletal muscle acetone powder according 322 to Spudich and Watt (Spudich & Watt, 1971). Ni-NTA agarose was purchased from Qiagen (Hilden, 323 Germany). Anti-Flag M2 affinity gel and HPR conjugated anti-Flag M2 antibody were from Sigma Co. (St. 324 Louis, MO). Glutathione(GSH)-Sepharose 4 Fast Flow was from GE Healthcare. FLAG peptide 325 (DYKDDDDK) was synthesized by Augct Co. (Beijing, China). Oligonucleotides were synthesized by 326 Invitrogen Co. (Beijing, China). 327 Proteins 328 In addition, Myo5a-MTD (0-8 μM) was incubate 377 μM) in a 60 μL solution of 20 mM Tris-HCl (pH 7.5), 378 for 20 min. The mixtures were centrifuged at 85,000 rp 379 for 15 min at 4 °C. Equal portions of the pellet and the 380 Coomassie brilliant blue staining. 381 Melanophilin antibody 382 To generate a polyclonal antibody against melanophili 383 melanophilin (Mlph-147-428) was amplified by PCR u 384 template. The PCR product was cloned into pET30a v 385 His-tagged protein (His-Mlph-147-428) and GST-tagg 386 His-Mlph-147-428 was purified using Ni-NTA agrose 387 GST-Mlph-147-428 was purified using GSH-Sepharo 388 affinity-purified from immune sera over a column of G 389 bromide-activated Sepharose 4B (GE Healthcare) by s 390 Plasmid constructions for melanocyte transfection 391 Myo5a-Tail/pEGFP-C1 was produced as describe 392 Myo5a-TailΔF/pEGFP-C1 and Myo5a-TailΔG/pEGFP 393 Myo5a-Tail/pEGFP-C1 as template. Myo5a-MTD/pEG 394 produced by PCR amplification using Myo5a-Tail/pEG 395 created by subcloning from Myo5a-MTDΔF/pET30 396 Melanocyte culture and transfection 397 The immortal mouse melanocyte cell line melan- 398 cultured as described (Bennett, Cooper, & Hart, 1987) 399 medium (HyClone) supplemented with 10% fetal bovi 400 and penicillin/streptomycin (1% v/v) in humidified ch 401 were passaged at 3–4 day intervals. Hieff Trans® Univ 402 transfection of pEGFP-C1 plasmids into melan-a cells 403 Immunocytochemistry 404 For immunofluorescence staining, melan-a cells were 405 were transfected of pEGFP-C1 plasmids. Two days af 406 paraformaldehyde for 20 min, then permeabilized with 407 coverslips were blocked with 1% BSA for 1 h. Melan- 408 Mlph overnight. Coverslips were washed with PBS fo 409 temperature with secondary antibodies (anti-rabbit IgG 410 For Flag pulldown assay, Flag-Myo5a-MTD (0.5 μM) was incubated with 1 μM GST-tagged Mlph 366 truncations in Pulldown Buffer I and rotated at 4 °C for 2 h, mixed with 10 μl of anti-FLAG M2 gel. The 367 anti-FLAG M2 gel were washed three times with 200 μl of Wash Buffer I. The bound proteins were eluted 368 twice with 20 μl of 0.1 mg/ml FLAG peptide in Wash Buffer I. 369 The inputs and the eluted proteins were analyzed by SDS-PAGE and visualized by Coomassie Brilliant 370 Blue (CBB) staining or Western blotting using the indicated antibody. The amounts of pulldowned proteins 371 were quantified using ImageJ (version 1.42Q), and their molar ratios were calculated on the basis of their 372 molecular masses. Proteins 328 373 Actin cosedimentation assay 374 Rabbit skeletal actin (2 μM) and Mlph-ABD (4 μM) was incubated in a 60 μL solution of 20 mM Tris-HCl 375 (pH 7.5), 100 mM NaCl, 1 mM EGTA, and 1 mM DTT at 4 °C for 20 min with or without Myo5a-MTD (2 376 μM). In addition, Myo5a-MTD (0-8 μM) was incubated with rabbit skeletal actin (2 μM) and Mlph-ABD (1 377 μM) in a 60 μL solution of 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EGTA, and 1 mM DTT at 4 °C 378 for 20 min. The mixtures were centrifuged at 85,000 rpm (Beckman Optima MAX-XP, TLA-120.1 rotor) 379 for 15 min at 4 °C. Equal portions of the pellet and the supernatant were subjected to SDS−PAGE and 380 Coomassie brilliant blue staining. 381 Melanophilin antibody 382 To generate a polyclonal antibody against melanophilin, the sequence encoding residues 147-428 of 383 melanophilin (Mlph-147-428) was amplified by PCR using a full-length mouse melanophilin cDNA as 384 template. The PCR product was cloned into pET30a vector and pGEX4T2 vector and expressed in E. coli as 385 His-tagged protein (His-Mlph-147-428) and GST-tagged protein (GST-Mlph-147-428), respectively. 386 His-Mlph-147-428 was purified using Ni-NTA agrose chromatography and used for immunizing rabbit. 387 GST-Mlph-147-428 was purified using GSH-Sepharose chromatography. Mlph antibody was 388 affinity-purified from immune sera over a column of GST-Mlph-147-428 coupled to cyanogen 389 bromide-activated Sepharose 4B (GE Healthcare) by standard procedure. 390 Plasmid constructions for melanocyte transfection 391 Myo5a-Tail/pEGFP-C1 was produced as described previously(X. Wu et al., 2002). 392 Myo5a-TailΔF/pEGFP-C1 and Myo5a-TailΔG/pEGFP-C1 were created by overlapping PCR using 393 Myo5a-Tail/pEGFP-C1 as template. Myo5a-MTD/pEGFP-C1 and Myo5a-MTDΔG/pEGFP-C1 were 394 produced by PCR amplification using Myo5a-Tail/pEGFP-C1 as template. Myo5a-MTDΔF/pEGFP-C1 was 395 created by subcloning from Myo5a-MTDΔF/pET30a into pEGFP-C1. 396 Melanocyte culture and transfection 397 The immortal mouse melanocyte cell line melan-a, kindly provided by Dr. Dorothy Bennett, was 398 cultured as described (Bennett, Cooper, & Hart, 1987). Briefly, melan-a cells were cultured in RPMI 1640 399 medium (HyClone) supplemented with 10% fetal bovine serum, 200 nM phorbol 12-myristate 13-acetate 400 and penicillin/streptomycin (1% v/v) in humidified chamber (37 °C, 5% CO2 incubator). Melan-a cells 401 were passaged at 3–4 day intervals. Hieff Trans® Universal Transfection Reagent (Yeasen) was used for 402 transfection of pEGFP-C1 plasmids into melan-a cells according to the manufacturer’s instructions. 403 Immunocytochemistry 404 For immunofluorescence staining, melan-a cells were plated on the coverslips. Proteins 328 For 354 GST pulldown of Myo5a-MTD with GST-Mlph-ABD, GSH-Sepharose beads (10 μl) were mixed with 95 355 μl of 2 μM GST-Mlph-ABD, 4 μM Myo5a-MTD truncations in Pulldown Buffer-I (5 mM Tris-HCl (pH 356 7.5), 100 mM NaCl, 1 mM DTT, and 1 mM EGTA, 0.1% NP-40) with rotation at 4 °C for 2 h. The 357 GSH-Sepharose beads were then washed three times with 200 μl of Wash Buffer-I (10 mM Tris-HCl (pH 358 7.5), 100 mM NaCl, 1 mM DTT, and 1 mM EGTA, 0.1% NP-40), before eluted by Elution Buffer (10 mM 359 GSH, 50 mM Tris-HCl (pH 8.0), 1 mM DTT, and 200 mM NaCl). For GST pulldown of Myo5a-MTD with 360 GST-Mlph-RBD, GSH-Sepharose beads (10 μl) were mixed with 95 μl of 2 μM GST-Mlph-RBD, 4 μM 361 Myo5a-MTD in Pulldown Buffer-II (5 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM DTT, and 1 mM 362 EGTA) with rotation at 4 °C for 2 h. The GSH-Sepharose beads were washed three times with 200 μl of 363 Wash Buffer-II (10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM DTT, and 1 mM EGTA) and then eluted 364 by Elution Buffer. 365 9 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint For Flag pulldown assay, Flag-Myo5a-MTD (0.5 366 truncations in Pulldown Buffer I and rotated at 4 °C fo 367 anti-FLAG M2 gel were washed three times with 200 368 twice with 20 μl of 0.1 mg/ml FLAG peptide in Wash 369 The inputs and the eluted proteins were analyzed 370 Blue (CBB) staining or Western blotting using the ind 371 were quantified using ImageJ (version 1.42Q), and the 372 molecular masses. 373 Actin cosedimentation assay 374 Rabbit skeletal actin (2 μM) and Mlph-ABD (4 μM) w 375 (pH 7.5), 100 mM NaCl, 1 mM EGTA, and 1 mM DT 376 μM). Proteins 328 After 24 h culturing, cells 405 were transfected of pEGFP-C1 plasmids. Two days after transfection, the cells were fixed using 4% 406 paraformaldehyde for 20 min, then permeabilized with 0.4% Triton-X in PBS for 15 min. Subsequently, the 407 coverslips were blocked with 1% BSA for 1 h. Melan-a cells were incubated with rabbit antibody against 408 Mlph overnight. Coverslips were washed with PBS for 3 times and then incubated for 1 h at room 409 temperature with secondary antibodies (anti-rabbit IgG coupled with DyLight 549, Jackson). Nuclei were 410 For Flag pulldown assay, Flag-Myo5a-MTD (0.5 μM) was incubated with 1 μM GST-tagged Mlph 366 truncations in Pulldown Buffer I and rotated at 4 °C for 2 h, mixed with 10 μl of anti-FLAG M2 gel. The 367 anti-FLAG M2 gel were washed three times with 200 μl of Wash Buffer I. The bound proteins were eluted 368 twice with 20 μl of 0.1 mg/ml FLAG peptide in Wash Buffer I. 369 The inputs and the eluted proteins were analyzed by SDS-PAGE and visualized by Coomassie Brilliant 370 Blue (CBB) staining or Western blotting using the indicated antibody. The amounts of pulldowned proteins 371 were quantified using ImageJ (version 1.42Q), and their molar ratios were calculated on the basis of their 372 molecular masses. 373 Actin cosedimentation assay 374 Rabbit skeletal actin (2 μM) and Mlph-ABD (4 μM) was incubated in a 60 μL solution of 20 mM Tris-HCl 375 (pH 7.5), 100 mM NaCl, 1 mM EGTA, and 1 mM DTT at 4 °C for 20 min with or without Myo5a-MTD (2 376 μM). In addition, Myo5a-MTD (0-8 μM) was incubated with rabbit skeletal actin (2 μM) and Mlph-ABD (1 377 μM) in a 60 μL solution of 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EGTA, and 1 mM DTT at 4 °C 378 for 20 min. The mixtures were centrifuged at 85,000 rpm (Beckman Optima MAX-XP, TLA-120.1 rotor) 379 for 15 min at 4 °C. Equal portions of the pellet and the supernatant were subjected to SDS−PAGE and 380 Coomassie brilliant blue staining. 381 Melanophilin antibody 382 To generate a polyclonal antibody against melanophilin, the sequence encoding residues 147-428 of 383 melanophilin (Mlph-147-428) was amplified by PCR using a full-length mouse melanophilin cDNA as 384 template. The PCR product was cloned into pET30a vector and pGEX4T2 vector and expressed in E. Proteins 328 coli as 385 His-tagged protein (His-Mlph-147-428) and GST-tagged protein (GST-Mlph-147-428), respectively. 386 His-Mlph-147-428 was purified using Ni-NTA agrose chromatography and used for immunizing rabbit. 387 GST-Mlph-147-428 was purified using GSH-Sepharose chromatography. Mlph antibody was 388 affinity-purified from immune sera over a column of GST-Mlph-147-428 coupled to cyanogen 389 bromide-activated Sepharose 4B (GE Healthcare) by standard procedure. 390 Plasmid constructions for melanocyte transfection 391 Myo5a-Tail/pEGFP-C1 was produced as described previously(X. Wu et al., 2002). 392 Myo5a-TailΔF/pEGFP-C1 and Myo5a-TailΔG/pEGFP-C1 were created by overlapping PCR using 393 Myo5a-Tail/pEGFP-C1 as template. Myo5a-MTD/pEGFP-C1 and Myo5a-MTDΔG/pEGFP-C1 were 394 produced by PCR amplification using Myo5a-Tail/pEGFP-C1 as template. Myo5a-MTDΔF/pEGFP-C1 was 395 created by subcloning from Myo5a-MTDΔF/pET30a into pEGFP-C1. 396 Melanocyte culture and transfection 397 The immortal mouse melanocyte cell line melan-a, kindly provided by Dr. Dorothy Bennett, was 398 cultured as described (Bennett, Cooper, & Hart, 1987). Briefly, melan-a cells were cultured in RPMI 1640 399 medium (HyClone) supplemented with 10% fetal bovine serum, 200 nM phorbol 12-myristate 13-acetate 400 and penicillin/streptomycin (1% v/v) in humidified chamber (37 °C, 5% CO2 incubator). Melan-a cells 401 were passaged at 3–4 day intervals. Hieff Trans® Universal Transfection Reagent (Yeasen) was used for 402 transfection of pEGFP-C1 plasmids into melan-a cells according to the manufacturer’s instructions. 403 Immunocytochemistry 404 For immunofluorescence staining, melan-a cells were plated on the coverslips. After 24 h culturing, cells 405 were transfected of pEGFP-C1 plasmids. Two days after transfection, the cells were fixed using 4% 406 For Flag pulldown assay, Flag-Myo5a-MTD (0.5 μM) was incubated with 1 μM GST-tagged Mlph 366 truncations in Pulldown Buffer I and rotated at 4 °C for 2 h, mixed with 10 μl of anti-FLAG M2 gel. The 367 anti-FLAG M2 gel were washed three times with 200 μl of Wash Buffer I. The bound proteins were eluted 368 twice with 20 μl of 0.1 mg/ml FLAG peptide in Wash Buffer I. 369 The inputs and the eluted proteins were analyzed by SDS-PAGE and visualized by Coomassie Brilliant 370 Blue (CBB) staining or Western blotting using the indicated antibody. The amounts of pulldowned proteins 371 were quantified using ImageJ (version 1.42Q), and their molar ratios were calculated on the basis of their 372 molecular masses. 373 To generate a polyclonal antibody against melanophilin, the sequence encoding residues 147-428 of 383 melanophilin (Mlph-147-428) was amplified by PCR using a full-length mouse melanophilin cDNA as 384 template. Proteins 328 The PCR product was cloned into pET30a vector and pGEX4T2 vector and expressed in E. coli as 385 His-tagged protein (His-Mlph-147-428) and GST-tagged protein (GST-Mlph-147-428), respectively. 386 His-Mlph-147-428 was purified using Ni-NTA agrose chromatography and used for immunizing rabbit. 387 GST-Mlph-147-428 was purified using GSH-Sepharose chromatography. Mlph antibody was 388 affinity-purified from immune sera over a column of GST-Mlph-147-428 coupled to cyanogen 389 bromide-activated Sepharose 4B (GE Healthcare) by standard procedure. 390 Myo5a-Tail/pEGFP-C1 was produced as described previously(X. Wu et al., 2002). 392 Myo5a-TailΔF/pEGFP-C1 and Myo5a-TailΔG/pEGFP-C1 were created by overlapping PCR using 393 Myo5a-Tail/pEGFP-C1 as template. Myo5a-MTD/pEGFP-C1 and Myo5a-MTDΔG/pEGFP-C1 were 394 produced by PCR amplification using Myo5a-Tail/pEGFP-C1 as template. Myo5a-MTDΔF/pEGFP-C1 was 395 created by subcloning from Myo5a-MTDΔF/pET30a into pEGFP-C1. 396 10 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint counter-stained with DAPI. coverslips were fixed on glass slides with Fluoromount-GTM (Yeasen). The 411 images were captured by Leica STELLARIS 5 fluorescence microscope. 412 413 414 FOOTNOTES 415 The abbreviations used are: ABD, actin-binding domain of melanophilin; CaM, calmodulin; EFBD, 416 exon-F-binding domain of melanophilin; GSH, glutathione; GST, glutathione S-transferase; GTBM, 417 GTD-binding motif of melanophilin; GTD, globular tail domain of myosin-5a; Mlph, melanophilin; MTD, 418 middle tail domain of myosin-5a; Myo5a, myosin-5a. 419 420 ACKNOWLEDGEMENTS 421 This work was supported by the National Natural Science Foundation of China (31970657). 422 423 CONFLICT OF INTEREST 424 The authors declare that they have no conflicts of interest with the contents of this article. 425 426 AUTHOR CONTRIBUTIONS 427 XdL conceived the study. JP performed most experiments with help from RZ. LLY, NZ and JZ performed 428 the initial experiments demonstrating the interaction between Mlph-ABD and Myo5a tail. QJC prepared 429 melanophilin antibody. SP participated in preparation of recombinant proteins and pulldown assays. JP and 430 XdL designed the study, analyzed the data and wrote the manuscript. REFERENCES 434 Bennett, D. C., Cooper, P. J., & Hart, I. R. (1987). A line of non-tumorigenic mouse melanocytes, syngeneic with the 435 B16 melanoma and requiring a tumour promoter for growth. Int J Cancer, 39(3), 414-418. 436 Cao, Q. J., Zhang, N., Zhou, R., Yao, L. L., & Li, X. D. (2019). The cargo adaptor proteins RILPL2 and melanophilin 437 co-regulate myosin-5a motor activity. J Biol Chem, 294(29), 11333-11341. doi:10.1074/jbc.RA119.007384 438 Fukuda, M. (2021). Rab GTPases: Key players in melanosome biogenesis, transport, and transfer. Pigment Cell 439 Melanoma Res, 34(2), 222-235. doi:10.1111/pcmr.12931 440 Fukuda, M., & Itoh, T. (2004). Slac2-a/melanophilin contains multiple PEST-like sequences that are highly sensitive 441 to proteolysis. 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Ca2+-induced activation of ATPase activity of myosin Va is 484 accompanied with a large conformational change. Biochem Biophys Res Commun, 315(3), 538-545. 485 doi:10.1016/j.bbrc.2004.01.084 486 Lindsay, A. J., & McCaffrey, M. W. (2014). Myosin Va is required for the transport of fragile X mental retardation 487 protein (FMRP) granules. Biol Cell, 106(2), 57-71. doi:10.1111/boc.201200076 488 12 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. FIGURE LEGENDS 558 Figure 1. The actin-binding domain of melanophilin (Mlph-ABD) interacts with the middle tail 559 domain (MTD) of Myo5a. (A) Diagram of the melanocyte-spliced isoform of Myo5a. Myo5a-MTD, 560 Myo5a coiled-coils (residues 1106-1469). IQ, the CaM binding site; GTD, the C-terminal globular tail 561 domain. (B) Diagram of melanophilin (Mlph). RBD, Rab27a-binding domain; GTBM, globular tail 562 domain-binding motif; EFBD, exon-F binding domain; ABD, actin-binding domain. (C, D) GST pulldown 563 of GST-Mlph-ABD with the N-terminus truncated (C) or the C-terminus truncated (D) with 564 Flag-Myo5a-MTD. GST-Mlph-ABD constructs were bound to GSH-Sepharose and then incubated with 565 Flag-Myo5a-MTD. The GSH-Sepharose-bound proteins were eluted by GSH and analyzed by Western blot 566 using anti-Flag antibody. The inputs were analyzed with SDS-PAGE and visualized by Coomassie brilliant 567 blue (CBB) staining. GST was used as negative control. Note: The Sf9 cell-expressed Myo5a-MTD was 568 used in the GST pulldown assays shown in this figure. 569 Figure 1-Source data 1 Original and uncropped gels and blots for Figure 1C. 570 Figure 1-Source data 2 Original and uncropped gels and blots for Figure 1D. 571 Figure 1-Source data 2 Original and uncropped gels and blots for Figure 1D. 571 Figure 2. The exon-G of Myo5a interacts with Mlph-ABD. (A) Summary of the interactions between 572 the truncated Myo5a constructs and Mlph-ABD based on the GST pulldown assay shown in Figure S1. +, 573 strong interaction; +/-, weak interaction; -, no interaction. (B) Deletion of the C-terminal portion of exon-G 574 abolishes the interaction between Myo5a-MTD and Mlph-ABD. (C) Deletion of the C-terminal portion of 575 exon-G does not affect the interaction between Myo5a-MTD and Mlph-EFBD. GST pulldown assays were 576 performed using GST-Mlph-ABD and Flag-Myo5a-MTD variants. The GSH-Sepharose-bound proteins 577 were eluted by GSH and analyzed by Western blot using anti-FLAG antibody; the inputs were analyzed 578 with SDS-PAGE and visualized by Coomassie brilliant blue (CBB) staining. 579 Figure 2. The exon-G of Myo5a interacts with Mlph-ABD. (A) Summary of the interactions between 572 the truncated Myo5a constructs and Mlph-ABD based on the GST pulldown assay shown in Figure S1. +, 573 strong interaction; +/-, weak interaction; -, no interaction. (B) Deletion of the C-terminal portion of exon-G 574 abolishes the interaction between Myo5a-MTD and Mlph-ABD. (C) Deletion of the C-terminal portion of 575 exon-G does not affect the interaction between Myo5a-MTD and Mlph-EFBD. GST pulldown assays were 576 performed using GST-Mlph-ABD and Flag-Myo5a-MTD variants. REFERENCES 434 J Cell Biol, 143(7), 534 1899-1918. 535 Wu, X., & Hammer, J. A. (2014). Melanosome transfer: it is best to give and receive. Curr Opin Cell Biol, 29, 1-7. 536 doi:10.1016/j.ceb.2014.02.003 537 Wu, X., Rao, K., Bowers, M. B., Copeland, N. G., Jenkins, N. A., & Hammer, J. A., 3rd. (2001). Rab27a enables 538 myosin Va-dependent melanosome capture by recruiting the myosin to the organelle. Journal of Cell Science, 539 114(Pt 6), 1091-1100. 540 ( ), Wu, X., Wang, F., Rao, K., Sellers, J. R., & Hammer, J. A., 3rd. (2002). Rab27a is an essential component of 541 melanosome receptor for myosin Va. Mol Biol Cell, 13(5), 1735-1749. doi:10.1091/mbc.01-12-0595 542 Wu, X. S., Rao, K., Zhang, H., Wang, F., Sellers, J. R., Matesic, L. E., . . . Hammer, J. A., 3rd. (2002). Identification 543 of an organelle receptor for myosin-Va. Nat Cell Biol, 4(4), 271-278. doi:10.1038/ncb760 544 13 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint Wu, X. S., Tsan, G. L., & Hammer, J. A., 3rd. (2005). Melanophilin and myosin Va track the microtubule plus end on 545 EB1. J Cell Biol, 171(2), 201-207. doi:10.1083/jcb.200503028 546 Yao, L. L., Cao, Q. J., Zhang, H. M., Zhang, J., Cao, Y., & Li, X. D. (2015). Melanophilin Stimulates Myosin-5a 547 Motor Function by Allosterically Inhibiting the Interaction between the Head and Tail of Myosin-5a. Sci Rep, 548 5, 10874. doi:10.1038/srep10874 549 Yoshimura, A., Fujii, R., Watanabe, Y., Okabe, S., Fukui, K., & Takumi, T. (2006). Myosin-Va facilitates the 550 accumulation of mRNA/protein complex in dendritic spines. Curr Biol, 16(23), 2345-2351. 551 doi:10.1016/j.cub.2006.10.024 552 j Zhang, N., Yao, L. L., & Li, X. D. (2018). Regulation of class V myosin. Cell Mol Life Sci, 75(2), 261-273. 553 doi:10.1007/s00018-017-2599-5 554 Zhang, W. B., Yao, L. L., & Li, X. D. (2016). . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint REFERENCES 434 The Globular Tail Domain of Myosin-5a Functions as a Dimer in 555 Regulating the Motor Activity. J Biol Chem, 291(26), 13571-13579. doi:10.1074/jbc.M116.724328 556 FIGURE LEGENDS 558 614 Note: a Sf9 cell-expressed Myo5a-MTD was used in the actin cosedimentation assays shown in this figure. 615 Figure 5-Source data 1 Original and uncropped gels for Figure 5A. 616 Figure 5-Source data 1 Original and uncropped gels for Figure 5A. 616 Figure 5-Source data 2 Original and uncropped gels and statistical data for Figure 5 B. Figure 6. Both E449 and E452 of Mlph are essential for the perinuclear distribution of melanosomes 618 induced by Mlph-RBD overexpression. Melan-a melanocytes were transfected to express 619 EGFP-Mlph-RBD or EGFP. The distribution of melanosomes in the transfected melanocytes was imaged 620 and the number of melanocytes with perinuclear distribution of melanosomes was counted. (A) Typical 621 images of melanocytes expressing EGFP-Mlph-RBD, its mutants, or EGFP. Cells are outlined with a 622 dashed line. Scale bars = 10 μm. (B) The percentage of melanocytes exhibiting perinuclear melanosome 623 aggregation among the transfected melanocytes. Results are presented as the mean ± SD of three 624 independent experiments with one-way ANOVA with post-hoc Bonferroni test. **p<0.0001. 625 Figure 6-Source data 1 Original and statistical data for Figure 6 B. 626 Figure 6. Both E449 and E452 of Mlph are essential for the perinuclear distribution of melanosomes 618 induced by Mlph-RBD overexpression. Melan-a melanocytes were transfected to express 619 EGFP-Mlph-RBD or EGFP. The distribution of melanosomes in the transfected melanocytes was imaged 620 and the number of melanocytes with perinuclear distribution of melanosomes was counted. (A) Typical 621 images of melanocytes expressing EGFP-Mlph-RBD, its mutants, or EGFP. Cells are outlined with a 622 dashed line. Scale bars = 10 μm. (B) The percentage of melanocytes exhibiting perinuclear melanosome 623 aggregation among the transfected melanocytes. Results are presented as the mean ± SD of three 624 independent experiments with one-way ANOVA with post-hoc Bonferroni test. p<0.0001. 625 Figure 6-Source data 1 Original and statistical data for Figure 6 B. 626 Figure 7. Exon-G region of Myo5a is essential for the perinuclear distribution of melanosomes 627 induced by Myo5a-Tail overexpression. (A) Diagram of Myo5a-Tail. (B) Melan-a melanocytes were 628 transfected to express EGFP-Myo5a-Tail and the mutant. The distribution of melanosomes in the 629 transfected melanocytes was imaged and the number of melanocytes with perinuclear distribution of 630 melanosomes was counted. (B) Typical images of melanocytes expressing EGFP-Myo5a-Tail, its mutants, 631 or EGFP. Cells are outlined with a dashed line. Scale bars = 10 μm. FIGURE LEGENDS 558 (B) Both exon-F and exon-G of Myo5a-MTD are required for the strong 598 interaction with Mlph-RBD. GST pulldown of GST-Mlph-RBD with Flag-Myo5a-MTD variants. (C) 599 ABD is essential for the strong interaction between Mlph-RBD and Myo5a-MTD. The input samples were 600 analyzed by SDS-PAGE and visualized by CBB staining. The pulled down samples were analyzed by 601 Western blot using anti-Flag antibody (A and B) or by SDS-PAGE with CBB staining (C). 602 Fi 4 S d t 1 O i i l d d l d bl f Fi 4A 603 Figure 4-Source data 3 Original and uncropped gels for Figure 4C. 605 Figure 5. Myo5a-MTD antagonizes the interaction between Mlph-ABD and actin. (A) 606 GST-Mlph-ABD and/or Flag-Myo5a-MTD were incubated with actin and then subjected to 607 ultracentrifugation. The supernatants (S) and the pellets (P) were analyzed by SDS-PAGE (10%) with CBB 608 staining. (B) GST-Mlph-ABD was incubated with actin in the presence of different concentrations of 609 Flag-Myo5a-MTD and then subjected to to ultracentrifugation. Upper panel, the supernatants (S) and the 610 pellets (P) were analyzed by SDS-PAGE (10%) with CBB staining. Lower panel, the amounts of 611 GST-Mlph-ABD co-sedimentated with actin in the presence of different concentration of 612 Flag-Myo5a-MTD were quantified based on the density in the SDS-PAGE. Data are the mean ± SD of three 613 independent experiments with one-way ANOVA with post-hoc Bonferroni test. p<0.01, **p<0.0001. 614 Note: a Sf9 cell-expressed Myo5a-MTD was used in the actin cosedimentation assays shown in this figure. 615 Figure 5-Source data 1 Original and uncropped gels for Figure 5A. 616 Figure 5. Myo5a-MTD antagonizes the interaction between Mlph-ABD and actin. (A) 606 GST-Mlph-ABD and/or Flag-Myo5a-MTD were incubated with actin and then subjected to 607 ultracentrifugation. The supernatants (S) and the pellets (P) were analyzed by SDS-PAGE (10%) with CBB 608 staining. (B) GST-Mlph-ABD was incubated with actin in the presence of different concentrations of 609 Flag-Myo5a-MTD and then subjected to to ultracentrifugation. Upper panel, the supernatants (S) and the 610 pellets (P) were analyzed by SDS-PAGE (10%) with CBB staining. Lower panel, the amounts of 611 GST-Mlph-ABD co-sedimentated with actin in the presence of different concentration of 612 Flag-Myo5a-MTD were quantified based on the density in the SDS-PAGE. Data are the mean ± SD of three 613 independent experiments with one-way ANOVA with post-hoc Bonferroni test. p<0.01, **p<0.0001. FIGURE LEGENDS 558 The GSH-Sepharose-bound proteins 577 were eluted by GSH and analyzed by Western blot using anti-FLAG antibody; the inputs were analyzed 578 with SDS-PAGE and visualized by Coomassie brilliant blue (CBB) staining. 579 Figure 2-Source data 1 Original and uncropped gels and blots for Figure 2B. 580 Figure 2-Source data 2 Original and uncropped gels and blots for Figure 2C. 581 Figure 3. Identification of the key residues for the exon-G/ABD interaction. GST pulldown was 582 performed using GST-Mlph-ABD variants and Flag-Myo5a-MTD variants. (A) Ionic strength dependence 583 of the interaction between Myo5a-MTD and Mlph-ABD. GST pulldown was performed using 584 GST-Mlph-ABD and Flag-Myo5a-MTD in the presence of different concentrations of NaCl. (B) Sequence 585 alignments of the regions in Mlph-ABD essential for binding to Myo5a-MTD. Conserved charged residues 586 are indicated. (C) Effects of alanine mutations of the conserved charged residues in Mlph-ABD on the 587 interaction with Myo5a-MTD. E449A and E452A mutations in Mlph abolished the interaction between 588 Mlph-ABD and Myo5a-MTD. (D) Sequence alignments of the C-terminal portion of exon-G, an essential 589 region for binding to Mlph-ABD. Two conserved basic residues (K1456 and K1460) are indicated. (E) 590 Effects of K1456A and K1460A mutations of Myo5a-MTD on the interaction with Mlph-ABD. 591 14 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint g g pp g g Figure 3-Source data 2 Original and uncropped gels and blots for Figure 3C. 593 g pp g g Figure 3-Source data 3 Original and uncropped gels and blots for Figure 3E. 594 Figure 4. The exon-F/EFBD and the exon-G/ABD interactions act synergistically. (A) Myo5a-MTD 595 bridges between Mlph-ABD and Mlph-EFBD. Upper, diagram shows Myo5a-MTD binds to both the EFBD 596 and the ABD of Mlph. Lower, GST pulldown assays of GST-Mlph-ABD and Flag-Mlph-EFBD with or 597 without Flag-Myo5a-MTD. FIGURE LEGENDS 558 GST pulldown assays were performed using GST-Mlph-ABD and FLAG-Myo5a-MTD variants. 652 The GSH-Sepharose-bound proteins were eluted by GSH and analyzed by Western blot using anti-FLAG 653 antibody; the inputs were analyzed with SDS-PAGE and visualized by Coomassie brilliant blue (CBB) 654 staining. (A) GST pulldown assays of GST-Mlph-ABD with Flag-Myo5a-MTD expressed in E. coli or Sf9 655 cells. (B and C) GST pulldown assay of GST-Mlph-ABD with Flag-tagged, truncated Myo5a tail. 656 Figure 2-figure supplement Source data 1 Original and uncropped gels and blots for Figure 2-figure 657 supplement A. 658 Figure 2-figure supplement Source data 2 Original and uncropped gels and blots for Figure 2-figure 659 supplement B. 660 Figure 2-figure supplement Source data 3 Original and uncropped gels and blots for Figure 2-figure 661 supplement C. 662 663 Figure 1-Source data 1 Original and uncropped gels and blots. 664 Figure 1-Source data 2 Original and uncropped gels and blots. 665 Figure 2-Source data 1 Original and uncropped gels and blots. 666 Figure 2-Source data 2 Original and uncropped gels and blots. 667 Figure 3-Source data 1 Original and uncropped gels and blots. 668 Figure 3-Source data 2 Original and uncropped gels and blots. 669 Figure 3-Source data 3 Original and uncropped gels and blots. 670 Figure 4-Source data 1 Original and uncropped gels and blots. 671 Figure 4-Source data 2 Original and uncropped gels and blots. 672 Figure 4-Source data 3 Original and uncropped gels. 673 Figure 5-Source data 1 Original and uncropped gels. 674 Figure 8. Effects of deletion of exon-F or exon-G on the localization of Myo5a-MTD in melan-a cells. 636 Melan-a melanocytes were transfected to express EGFP-Myo5a-MTD (A), EGFP-Myo5a-MTDF (B), or 637 EGFP-Myo5a-MTDG (C) and stained for endogenous Mlph. Scale bars = 10 μm. 638 Figure 9. A model for the Mlph-mediated Myo5a transportation of melanosome. The Mlph-mediated 639 Myo5a transportation of melanosome is comprised of four stages. At stage 1, Mlph associates with 640 melanosome via its interaction with Rab27a, which directly binds to the membrane of melanosome; the 641 unattached Myo5a is in a folded conformation, in which the GTD binds to and inhibits the motor domain. 642 At stage 2, Mlph interacts with the folded Myo5a via the interactions of EFBD/exon-F and ABD/exon-G; 643 the attached Myo5a is still in folded conformation, because the GTBM-binding surface in the GTD is buried 644 at the GTD-GTD interface. FIGURE LEGENDS 558 (C) The percentages of melanocytes 632 exhibiting perinuclear melanosome aggregation. Data are the mean ± SD of three independent experiments 633 with one-way ANOVA with post-hoc Bonferroni test. p<0.0001. 634 Figure 7-Source data 1 Original and statistical data for Figure 7 C. 635 15 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint Figure 8. Effects of deletion of exon-F or exon-G on the localization of Myo5a-MTD in melan-a cell 636 Melan-a melanocytes were transfected to express EGFP-Myo5a-MTD (A), EGFP-Myo5a-MTDF (B), or 637 EGFP-Myo5a-MTDG (C) and stained for endogenous Mlph. Scale bars = 10 μm. 638 Figure 9. A model for the Mlph-mediated Myo5a transportation of melanosome. The Mlph-mediated 639 Myo5a transportation of melanosome is comprised of four stages. At stage 1, Mlph associates with 640 melanosome via its interaction with Rab27a, which directly binds to the membrane of melanosome; the 641 unattached Myo5a is in a folded conformation, in which the GTD binds to and inhibits the motor domain. 642 At stage 2, Mlph interacts with the folded Myo5a via the interactions of EFBD/exon-F and ABD/exon-G; 643 the attached Myo5a is still in folded conformation, because the GTBM-binding surface in the GTD is buried 644 at the GTD-GTD interface. At stage 3, the buried GTBM-binding surface between the GTD-GTD interface 645 is exposed and thus facilitate the binding of GTBM, causing the dissociation of the GTD from the motor 646 domain and inducing the extended conformation of Myo5a (This step is probably regulated by the binding 647 of Rab36/RilpL2 to the GTD). At stage 4, Mlph-ABD dissociates from exon-G and then binds to actin 648 filament, thus enhancing the processive movement of Myo5a (This step might be regulated by the 649 phosphorylation of Mlph-ABD). 650 Figure 2-figure supplement. Identification of the Mlph-ABD-binding site in the middle tail of 651 Myo5a. FIGURE LEGENDS 558 At stage 3, the buried GTBM-binding surface between the GTD-GTD interface 645 is exposed and thus facilitate the binding of GTBM, causing the dissociation of the GTD from the motor 646 domain and inducing the extended conformation of Myo5a (This step is probably regulated by the binding 647 of Rab36/RilpL2 to the GTD). At stage 4, Mlph-ABD dissociates from exon-G and then binds to actin 648 filament, thus enhancing the processive movement of Myo5a (This step might be regulated by the 649 phosphorylation of Mlph-ABD). 650 p ) g , p filament, thus enhancing the processive movement of Myo5a (This step might be regulated by the 649 phosphorylation of Mlph-ABD). 650 Figure 2-figure supplement. Identification of the Mlph-ABD-binding site in the middle tail of 651 Myo5a. GST pulldown assays were performed using GST-Mlph-ABD and FLAG-Myo5a-MTD variants. 652 The GSH-Sepharose-bound proteins were eluted by GSH and analyzed by Western blot using anti-FLAG 653 antibody; the inputs were analyzed with SDS-PAGE and visualized by Coomassie brilliant blue (CBB) 654 staining. (A) GST pulldown assays of GST-Mlph-ABD with Flag-Myo5a-MTD expressed in E. coli or Sf9 655 cells. (B and C) GST pulldown assay of GST-Mlph-ABD with Flag-tagged, truncated Myo5a tail. 656 Figure 2-figure supplement Source data 1 Original and uncropped gels and blots for Figure 2-figure 657 supplement A. 658 Figure 2-figure supplement Source data 2 Original and uncropped gels and blots for Figure 2-figure 659 supplement B. 660 Figure 2-figure supplement Source data 3 Original and uncropped gels and blots for Figure 2-figure 661 supplement C. 662 663 Figure 1-Source data 1 Original and uncropped gels and blots. 664 Figure 1-Source data 2 Original and uncropped gels and blots. 665 Figure 2-Source data 1 Original and uncropped gels and blots. 666 Figure 2-Source data 2 Original and uncropped gels and blots. 667 Figure 3-Source data 1 Original and uncropped gels and blots. 668 Figure 3-Source data 2 Original and uncropped gels and blots. 669 Figure 3-Source data 3 Original and uncropped gels and blots. 670 Figure 4-Source data 1 Original and uncropped gels and blots. 671 Figure 4-Source data 2 Original and uncropped gels and blots. 672 Figure 4-Source data 3 Original and uncropped gels. 673 Figure 5-Source data 1 Original and uncropped gels. 674 Figure 5-Source data 2 Original and uncropped gels and data. 675 Figure 6-Source data 1 Original and statistical data. 676 16 16 . Figure 7-Source data 1 Original and statistical data. 677 Figure 2-figure supplement Source data 1 Original and uncropped gels and blots. 678 Figure 2-figure supplement Source data 2 Original and uncropped gels and blots. 679 Figure 2-figure supplement Source data 3 Original and uncropped gels and blots. 680 681 FIGURE LEGENDS 558 CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint Figure 7-Source data 1 Original and statistical data. 677 Figure 2-figure supplement Source data 1 Original and uncropped gels and blots. 678 Figure 2-figure supplement Source data 2 Original and uncropped gels and blots. 679 Figure 2-figure supplement Source data 3 Original and uncropped gels and blots. 680 681 17 17 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint FIGURE LEGENDS 558 ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint A B C D Figure 1 43 29 Flag-Myo5a-MTD GST-Mlph-ABD GST-Mlph-ABD (401-578) GST-Mlph-ABD (401-571) GST-Mlph-ABD (401-560) GST-Mlph-ABD (401-525) GST Flag-Myo5a-MTD GST GST-Mlph (401-560) GST-Mlph (401-571) GST-Mlph (401-578) GST-Mlph-ABD + + + + + + GST-Mlph (401-525) Input CBB staining Flag-Myo5a-MTD 70 55 1 2 3 4 5 6 GST-pulldown WB: anti-Flag 29 43 GST-Mlph-ABD GST-Mlph (431-590) GST-Mlph (437-590) GST-Mlph (446-590) GST-Mlph (455-590) Flag-Myo5a-MTD GST Flag-Myo5a-MTD GST GST-Mlph (431-590) GST-Mlph (437-590) GST-Mlph (446-590) GST-Mlph (455-590) + + + + + + GST-Mlph-ABD Input CBB staining Flag-Myo5a-MTD 70 55 1 2 3 4 5 6 GST-pulldown WB: anti-Flag Motor Domain 1- -1877 GTD 6X IQ C1 C2 C3 C4 C5 Myo5a 1106- -1469 C2 C3 C4 C5 Myo5a-MTD 1152 1234 1344 1467 Exon A C D E F G 1198 1284 1318 1345 1411 1436 1467 Length 86 34 27 66 25 32 aa Head Neck Tail Mlph RBD EFBD ABD 1 147 241 401 590 GTBM (176-201) Rab27a Myo5a -GTD Myo5a exon-F actin EFBD ABD EFBD Mlph-ABD Mlph-∆RBD Mlph-EFBD 241 400 EFBD Mlph-∆RBD∆ABD 400 148 148 590 ABD 401 590 Figure 1 A B Motor Domain 1- -1877 GTD 6X IQ C1 C2 C3 C4 C5 Myo5a 1106- -1469 C2 C3 C4 C5 Myo5a-MTD 1152 1234 1344 1467 Exon A C D E F G 1198 1284 1318 1345 1411 1436 1467 Length 86 34 27 66 25 32 aa Head Neck Tail B Mlph RBD EFBD ABD 1 147 241 401 590 GTBM (176-201) Rab27a Myo5a -GTD Myo5a exon-F actin EFBD ABD EFBD Mlph-ABD Mlph-∆RBD Mlph-EFBD 241 400 EFBD Mlph-∆RBD∆ABD 400 148 148 590 ABD 401 590 B A A C 29 43 GST-Mlph-ABD GST-Mlph (431-590) GST-Mlph (437-590) GST-Mlph (446-590) GST-Mlph (455-590) Flag-Myo5a-MTD GST Flag-Myo5a-MTD GST GST-Mlph (431-590) GST-Mlph (437-590) GST-Mlph (446-590) GST-Mlph (455-590) + + + + + + GST-Mlph-ABD Input CBB staining Flag-Myo5a-MTD 70 55 1 2 3 4 5 6 GST-pulldown WB: anti-Flag D 43 29 Flag-Myo5a-MTD GST-Mlph-ABD GST-Mlph-ABD (401-578) GST-Mlph-ABD (401-571) GST-Mlph-ABD (401-560) GST-Mlph-ABD (401-525) GST Flag-Myo5a-MTD GST GST-Mlph (401-560) GST-Mlph (401-571) GST-Mlph (401-578) GST-Mlph-ABD + + + + + + GST-Mlph (401-525) Input CBB staining Flag-Myo5a-MTD 70 55 1 2 3 4 5 6 GST-pulldown WB: anti-Flag D C . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. FIGURE LEGENDS 558 CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. FIGURE LEGENDS 558 It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint Figure 2 Figure 2 A B C F G -1467 Trx G -1467 Trx 1411 1436 A C D E F G 1106- -1467 1198 A C D E F G 1193- -1467 A C D E 1106- -1410 A C D E F 1106- -1453 A C D E F 1193- -1453 E F G 1345- -1467 A C D E G 1106- -1467 Myo5a-MTDΔF Myo5a(1106-1410) Myo5a(1193-1453) Myo5a(1193-1467) Myo5a-MTD Myo5a(1345-1467) Trx-Myo5a(1411-1467) Trx-Myo5a(1436-1467) Myo5a-MTDΔG Binding to Mlph-ABD + + - - + + +/- + - GST pulldown WB: anti-Flag 55 Flag-Myo5a-MTD Flag-Myo5a-MTDΔF 1 2 3 Flag-Myo5a-MTD Flag-Myo5a-MTDΔF Flag-Myo5a-MTDΔG GST-Mlph-ABD Input CBB staining 43 29 66 Flag-Myo5a-MTDΔG Flag-Myo5a-MTDΔG Flag-Myo5a-MTDΔF Flag-Myo5a-MTD -GST-Mlph-ABD + + + GST-Mlph-EFBD GST - - - - + + + + Flag-Myo5a-MTD Flag-Myo5a-MTD∆G 1 2 3 4 GST GST-Mlph-EFBD 29 66 43 Input CBB staining Flag-Myo5a-MTD -Flag-Myo5a-MTDΔG 55 GST pulldown WB: anti-Flag Flag-Myo5a-MTD -Flag-Myo5a-MTDΔG A F G -1467 Trx G -1467 Trx 1411 1436 A C D E F G 1106- -1467 1198 A C D E F G 1193- -1467 A C D E 1106- -1410 A C D E F 1106- -1453 A C D E F 1193- -1453 E F G 1345- -1467 A C D E G 1106- -1467 Myo5a-MTDΔF Myo5a(1106-1410) Myo5a(1193-1453) Myo5a(1193-1467) Myo5a-MTD Myo5a(1345-1467) Trx-Myo5a(1411-1467) Trx-Myo5a(1436-1467) Myo5a-MTDΔG Binding to Mlph-ABD + + - - + + +/- + - A B C GST pulldown WB: anti-Flag 55 Flag-Myo5a-MTD Flag-Myo5a-MTDΔF 1 2 3 Flag-Myo5a-MTD Flag-Myo5a-MTDΔF Flag-Myo5a-MTDΔG GST-Mlph-ABD Input CBB staining 43 29 66 Flag-Myo5a-MTDΔG Flag-Myo5a-MTDΔG Flag-Myo5a-MTDΔF Flag-Myo5a-MTD -GST-Mlph-ABD + + + GST-Mlph-EFBD GST - - - - + + + + Flag-Myo5a-MTD Flag-Myo5a-MTD∆G 1 2 3 4 GST GST-Mlph-EFBD 29 66 43 Input CBB staining Flag-Myo5a-MTD -Flag-Myo5a-MTDΔG 55 GST pulldown WB: anti-Flag Flag-Myo5a-MTD -Flag-Myo5a-MTDΔG B GST pulldown WB: anti-Flag 55 Flag-Myo5a-MTD Flag-Myo5a-MTDΔF 1 2 3 Flag-Myo5a-MTD Flag-Myo5a-MTDΔF Flag-Myo5a-MTDΔG GST-Mlph-ABD Input CBB staining 43 29 66 Flag-Myo5a-MTDΔG Flag-Myo5a-MTDΔG Flag-Myo5a-MTDΔF Flag-Myo5a-MTD -GST-Mlph-ABD + + + B C . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint . FIGURE LEGENDS 558 CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. FIGURE LEGENDS 558 ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint Figure 3 A B C Figure 3 D T T T SPGNPARPTKS DEELSEMEDR SPGNPAQPTKS DEELSEMEDR ELEEGAQHCGT DLELSELEDK Mlph-Mm (430) Mlph-Rn (429) Mlph-Gg (525) T DPGDPVQYNRT DEELSELEDR Mlph-Hs (414) 449 447 452454 VYR VYR PYR VYR Mlph-Mm Mlph-Rn Mlph-Rabbit Mlph-Hs (560) (558) (532) (542) GSLTQRNPN GSLTQRNPN GSLTQRNPS GSLTQRNPN 562 568 T SPGDPEQPSRT EAELAELEGQ Mlph-Goat (430) MYR Mlph-Goat (558) GSLTQRNPN D KK L VFA KIGELE Myo5a-Mouse (1451) KK L VFA KIGELE Myo5a-Human (1437) KK L VFA KIGELE Myo5a-Chick (1411) KK L IYM KVQDLE Myo5b-Mouse (1401) KK L IYM KVQDLE Myo5b-Rat (1429) QD V TLT TTEKAN Myo5c-Mouse (1343) QD V TLS TIGKAN Myo5c-Human (1342) ED I TLT TTEKDG Myo5c-Chick (1337) L L L L L L L L Q Q Q Q Q Q Q Q K K K K K K K K K K K K K K K K 1456 1460 -Flag-Myo5a-MTD 43 29 -GST Flag-Myo5a-MTD WT K1456A GST + + + + + + - - - - - - GST-Mlph-ABD K1460A Input CBB staining 55 -Flag-Myo5a-MTD 1 2 3 4 5 6 GST pulldown WB: anti-Flag -Flag-Myo5a-MTD -GST-Mlph-ABD 43 29 GST-Mlph-ABD + + + + + + + + Flag-Myo5a-MTD GST WT E449A E452A D447A E454A R562A R568A 66 Input CBB staining -Flag-Myo5a-MTD GST-pulldown 55 WB: anti-Flag 1 2 3 4 5 6 7 8 E GST-Mlph-ABD 1 2 3 4 GST pulldown WB: anti-Flag GST + + + + + + + + 100 100 300 500 NaCl (mM) - - - - GST-Mlph-ABD GST-Mlph-ABD Flag-Myo5a-MTD 66 43 29 55 -Flag-Myo5a-MTD -Flag-Myo5a-MTD Input CBB staining -GST -GST A A 1 2 3 4 GST pulldown WB: anti-Flag GST + + + + + + + + 100 100 300 500 NaCl (mM) - - - - GST-Mlph-ABD GST-Mlph-ABD Flag-Myo5a-MTD 66 43 29 55 -Flag-Myo5a-MTD -Flag-Myo5a-MTD Input CBB staining -GST B C D T T T SPGNPARPTKS DEELSEMEDR SPGNPAQPTKS DEELSEMEDR ELEEGAQHCGT DLELSELEDK Mlph-Mm (430) Mlph-Rn (429) Mlph-Gg (525) T DPGDPVQYNRT DEELSELEDR Mlph-Hs (414) 449 447 452454 VYR VYR PYR VYR Mlph-Mm Mlph-Rn Mlph-Rabbit Mlph-Hs (560) (558) (532) (542) GSLTQRNPN GSLTQRNPN GSLTQRNPS GSLTQRNPN 562 568 T SPGDPEQPSRT EAELAELEGQ Mlph-Goat (430) MYR Mlph-Goat (558) GSLTQRNPN D KK L VFA KIGELE Myo5a-Mouse (1451) KK L VFA KIGELE Myo5a-Human (1437) KK L VFA KIGELE Myo5a-Chick (1411) KK L IYM KVQDLE Myo5b-Mouse (1401) KK L IYM KVQDLE Myo5b-Rat (1429) QD V TLT TTEKAN Myo5c-Mouse (1343) QD V TLS TIGKAN Myo5c-Human (1342) ED I TLT TTEKDG Myo5c-Chick (1337) L L L L L L L L Q Q Q Q Q Q Q Q K K K K K K K K K K K K K K K K 1456 1460 -Flag-Myo5a-MTD 43 29 -GST Flag-Myo5a-MTD WT K1456A GST + + + + + + - - - - - - GST-Mlph-ABD K1460A Input CBB staining 55 Flag Myo5a MTD GST pulldown -Flag-Myo5a-MTD -GST-Mlph-ABD 43 29 GST-Mlph-ABD + + + + + + + + Flag-Myo5a-MTD GST WT E449A E452A D447A E454A R562A R568A 66 Input CBB staining E GST-Mlph-ABD 1 2 3 4 GST pulldown WB: anti-Flag 55 -Flag-Myo5a-MTD D D KK L VFA KIGELE Myo5a-Mouse (1451) KK L VFA KIGELE Myo5a-Human (1437) KK L VFA KIGELE Myo5a-Chick (1411) KK L IYM KVQDLE Myo5b-Mouse (1401) KK L IYM KVQDLE Myo5b-Rat (1429) QD V TLT TTEKAN Myo5c-Mouse (1343) QD V TLS TIGKAN Myo5c-Human (1342) ED I TLT TTEKDG Myo5c-Chick (1337) L L L L L L L L Q Q Q Q Q Q Q Q K K K K K K K K K K K K K K K K 1456 1460 D B T T T SPGNPARPTKS DEELSEMEDR SPGNPAQPTKS DEELSEMEDR ELEEGAQHCGT DLELSELEDK Mlph-Mm (430) Mlph-Rn (429) Mlph-Gg (525) T DPGDPVQYNRT DEELSELEDR Mlph-Hs (414) 449 447 452454 VYR VYR PYR VYR Mlph-Mm Mlph-Rn Mlph-Rabbit Mlph-Hs (560) (558) (532) (542) GSLTQRNPN GSLTQRNPN GSLTQRNPS GSLTQRNPN 562 568 T SPGDPEQPSRT EAELAELEGQ Mlph-Goat (430) MYR Mlph-Goat (558) GSLTQRNPN B C -Flag-Myo5a-MTD 43 29 -GST Flag-Myo5a-MTD WT K1456A GST + + + + + + - - - - - - GST-Mlph-ABD K1460A Input CBB staining 55 -Flag-Myo5a-MTD 1 2 3 4 5 6 GST pulldown WB: anti-Flag -Flag-Myo5a-MTD -GST-Mlph-ABD 43 29 GST-Mlph-ABD + + + + + + + + Flag-Myo5a-MTD GST WT E449A E452A D447A E454A R562A R568A 66 Input CBB staining -Flag-Myo5a-MTD GST-pulldown 55 WB: anti-Flag 1 2 3 4 5 6 7 8 E GST-Mlph-ABD -GST C -Flag-Myo5a-MTD -GST-Mlph-ABD 43 29 GST-Mlph-ABD + + + + + + + + Flag-Myo5a-MTD GST WT E449A E452A D447A E454A R562A R568A 66 Input CBB staining -Flag-Myo5a-MTD GST-pulldown 55 WB: anti-Flag 1 2 3 4 5 6 7 8 -GST E C E C . . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint FIGURE LEGENDS 558 ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint Figure 4 Figure 4 A C B Myo5a-MTD A C D E F G EFBD ABD -Flag-Mlph-EFBD -Flag-Myo5a-MTD 35 55 1 2 3 4 GST pulldown WB: anti-Flag -Flag-Mlph-EFBD GST-Mlph-ABD 43 66 GST-Mlph-ABD + + + - Flag-Mlph-EFBD + + + - + + + - Flag-Myo5a-MTD Flag-Myo5a-MTD 29 Input CBB staining 1106 1469 A C B Myo5a-MTD A C D E F G EFBD ABD CBB staining -GST-Mlph-∆RBD -Flag-Myo5a-MTD 66 120 43 Flag pulldown Input Flag-Myo5a-MTD GST-Mlph-∆RBD GST-Mlph-∆RBDΔABD -GST-Mlph-∆RBD ΔABD + + + + + + + + + + - - - - - - - - 1 2 3 1 2 3 GST-Mlph-∆RBD Flag-Myo5a-MTDΔG Flag-Myo5a-MTDΔG Flag-Myo5a-MTDΔF Flag-Myo5a-MTD GST-Mlph-∆RBD + + + Flag-Myo5a-MTDΔF + - - Flag-Myo5a-MTDΔG Flag-Myo5a-MTDΔG + - - Flag-Myo5a-MTD + - - 100 70 55 Input CBB staining Flag-Myo5a-MTDΔG Flag-Myo5a-MTDΔG Flag-Myo5a-MTDΔF Flag-Myo5a-MTDΔF Flag-Myo5a-MTD 55 1 2 3 GST pulldown WB: anti-Flag -Flag-Mlph-EFBD -Flag-Myo5a-MTD 35 55 1 2 3 4 GST pulldown WB: anti-Flag -Flag-Mlph-EFBD GST-Mlph-ABD 43 66 GST-Mlph-ABD + + + - Flag-Mlph-EFBD + + + - + + + - Flag-Myo5a-MTD Flag-Myo5a-MTD 29 Input CBB staining 1106 1469 A C B Myo5a-MTD A C D E F G EFBD ABD CBB staining -GST-Mlph-∆RBD -Flag-Myo5a-MTD 66 120 43 Flag pulldown Input Flag-Myo5a-MTD GST-Mlph-∆RBD GST-Mlph-∆RBDΔABD -GST-Mlph-∆RBD ΔABD + + + + + + + + + + - - - - - - - - 1 2 3 1 2 3 GST-Mlph-∆RBD Flag-Myo5a-MTDΔG Flag-Myo5a-MTDΔG Flag-Myo5a-MTDΔF Flag-Myo5a-MTD GST-Mlph-∆RBD + + + Flag-Myo5a-MTDΔF + - - Flag-Myo5a-MTDΔG Flag-Myo5a-MTDΔG + - - Flag-Myo5a-MTD + - - 100 70 55 Input CBB staining Flag-Myo5a-MTDΔG Flag-Myo5a-MTDΔG Flag-Myo5a-MTDΔF Flag-Myo5a-MTDΔF Flag-Myo5a-MTD 55 1 2 3 GST pulldown WB: anti-Flag -Flag-Mlph-EFBD -Flag-Myo5a-MTD 35 55 1 2 3 4 GST pulldown WB: anti-Flag -Flag-Mlph-EFBD GST-Mlph-ABD 43 66 GST-Mlph-ABD + + + - Flag-Mlph-EFBD + + + - + + + - Flag-Myo5a-MTD Flag-Myo5a-MTD 29 Input CBB staining 1106 1469 GST-Mlph-∆RBD Flag-Myo5a-MTDΔG Flag-Myo5a-MTDΔG Flag-Myo5a-MTDΔF Flag-Myo5a-MTD GST-Mlph-∆RBD + + + Flag-Myo5a-MTDΔF + - - Flag-Myo5a-MTDΔG Flag-Myo5a-MTDΔG + - - Flag-Myo5a-MTD + - - 100 70 55 Input CBB staining Flag-Myo5a-MTDΔG Flag-Myo5a-MTDΔG Flag-Myo5a-MTDΔF Flag-Myo5a-MTDΔF Flag-Myo5a-MTD 55 1 2 3 GST pulldown WB: anti-Flag A B 3 C CBB staining -GST-Mlph-∆RBD -Flag-Myo5a-MTD 66 120 43 Flag pulldown Input Flag-Myo5a-MTD GST-Mlph-∆RBD GST-Mlph-∆RBDΔABD -GST-Mlph-∆RBD ΔABD + + + + + + + + + + - - - - - - - - 1 2 3 1 2 3 C CBB staining . FIGURE LEGENDS 558 CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint A B Figure 5 GST-Mlph-ABD - + + S P S P S P Flag-Myo5a-MTD + - + GST-Mlph-ABD Actin Flag-Myo5a-MTD 55 40 55 40 CBB staining 1 2 3 4 5 6 S P S P S P S P Flag-Myo5a-MTD (µM) 0 2 4 8 GST-Mlph-ABD Actin Flag-Myo5a-MTD CBB staining 1 2 3 4 5 6 7 8 Flag-Myo5a-MTD (µM) Relative amount of Mlph-ABD bound to Actin 0 2 4 6 8 10 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Figure 5 A GST-Mlph-ABD - + + S P S P S P Flag-Myo5a-MTD + - + GST-Mlph-ABD Actin Flag-Myo5a-MTD 55 40 CBB staining 1 2 3 4 5 6 A B 55 40 S P S P S P S P Flag-Myo5a-MTD (µM) 0 2 4 8 GST-Mlph-ABD Actin Flag-Myo5a-MTD CBB staining 1 2 3 4 5 6 7 8 Flag-Myo5a-MTD (µM) Relative amount of Mlph-ABD bound to Actin 0 2 4 6 8 10 0.0 0.2 0.4 0.6 0.8 1.0 1.2 ** B Figure 6 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint DAPI EGFP Bright Field EGFP EGFP-Mlph-∆RBD E449A E452A A B WT 80 ng ** DAPI EGFP Bright Field EGFP EGFP-Mlph-∆RBD E449A E452A A B WT 80 A A EGFP EGFP-Mlph-∆RBD E449A E452A B WT 0 20 40 60 80 EGFP WT E449A E452A EGFP-Mlph-∆RBD Percentage of the EGFP-expressing cells with perinucler aggregation of melanosomes EGFP-Mlph-∆RBD B 0 20 40 60 80 EGFP WT E449A E452A EGFP-Mlph-∆RBD Percentage of the EGFP-expressing cells with perinucler aggregation of melanosomes B 0 20 40 60 80 EGFP WT E449A E452A EGFP-Mlph-∆RBD Percentage of the EGFP-expressing cells with perinucler aggregation of melanosomes B Figure 7 . FIGURE LEGENDS 558 CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint Fi 7 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint Figure 7 A 1106- -1877 GTD A C D E F G Myo5a-Tail Myo5a-Tail-ΔG GTD A C D E F 1453 1467 1106- -1877 A DAPI EGFP Bright Field EGFP EGFP-Myo5a-Tail EGFP-Myo5a-Tail-ΔG B B DAPI EGFP Bright Field EGFP EGFP-Myo5a-Tail EGFP-Myo5a-Tail-ΔG B C 0 20 40 60 80 EGFP WT ∆G EGFP-Myo5a-Tail Percentage of the EGFP-expressing cells with perinucler aggregation of melanosomes EGFP-Myo5a-Tail C 0 20 40 60 80 EGFP WT ∆G EGFP-Myo5a-Tail Percentage of the EGFP-expressing cells with perinucler aggregation of melanosomes C 0 20 40 60 80 ** EGFP WT ∆G EGFP-Myo5a-Tail Percentage of the EGFP-expressing cells with perinucler aggregation of melanosomes C EGFP-Myo5a-Tail . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint EGFP- Myo5a-MTD EGFP- Myo5a-MTDΔF EGFP- Myo5a-MTDΔG Mlph Bright Field DAPI/EGFP/Mlph A B C Mlph Bright Field DAPI/EGFP/Mlph Mlph Bright Field DAPI/EGFP/Mlph Figure 8 Figure 8 EGFP- Myo5a-MTD A Mlph Bright Field DAPI/EGFP/Mlph EGFP- Myo5a-MTD EGFP- Myo5a-MTDΔF EGFP- Myo5a-MTDΔG Mlph Bright Field DAPI/EGFP/Mlph A B C Mlph Bright Field DAPI/EGFP/Mlph Mlph Bright Field DAPI/EGFP/Mlph A Mlph DAPI/EGFP/Mlph A Bright Field B Bright Field B EGFP- o5a-MTDΔF Mlph DAPI/EGFP/Mlph Bright Field C EGFP- Myo5a-MTDΔG Mlph Bright Field DAPI/EGFP/Mlph C Bright Field C Mlph DAPI/EGFP/Mlph Bright Field . FIGURE LEGENDS 558 CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint Figure 9 Rab27 EFBD ABD RBD GTBM Rab27 ABD EFBD RBD GTBM Rab27 EFBD ABD RBD GTBM Rab27 EFBD ABD RBD GTBM Mlph Myo5a Melanosome Melanosome Melanosome Melanosome Actin filament Stage 1: Mlph associates with melanosome via Rab27, and the unattached Myo5a is in the folded conformation. Stage 2: Myo5a in folded conformation binds to Mlph via exon-F/EFBD and exon-G/ABD. Stage 3: Mlph-GTBM binds to the GTD and induces Myo5a to form the extended conformation. Stage 4: Mlph-ABD dissociates from exon-G, and then binds to actin filament, enhancing the processibility of Myo5a. Exon-G Exon-F GTD Motor domain Rab36/RilpL2 Protein kinase A? ? Motor domain IQx6 GTD GTD Rab27 EFBD ABD RBD GTBM Rab27 EFBD ABD RBD GTBM Mlph Myo5a Melanosome Melanosome Stage 1: Mlph associates with melanosome via Rab27, and the unattached Myo5a is in the folded conformation. Stage 2: Myo5a in folded conformation binds to Mlph via exon-F/EFBD and exon-G/ABD. Exon-G Exon-F Rab36/RilpL2 ? Motor domain IQx6 GTD GTD Melanosome Myo5a Stage 2: Myo5a in folded conformation binds to Mlph via exon-F/EFBD and exon-G/ABD. Stage 1: Mlph associates with melanosome via Rab27, and the unattached Myo5a is in the folded conformation. Rab27 EFBD ABD RBD GTBM Rab27 ABD EFBD RBD GTBM Melanosome Melanosome GTD Motor domain Protein kinase A? Protein kinase A? Motor domain Actin filament Stage 4: Mlph-ABD dissociates from exon-G, and then binds to actin filament, enhancing the processibility of Myo5a. Stage 3: Mlph-GTBM binds to the GTD and induces Myo5a to form the extended conformation. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 4, 2023. FIGURE LEGENDS 558 ; https://doi.org/10.1101/2023.11.01.565146 doi: bioRxiv preprint Figure 2-figure supplement Figure 2-figure supplement Figure 2-figure supplement C A B Flag-Myo5a(1106-1410) Flag-Myo5a(1193-1453) Flag-Myo5a(1193-1467) GST-MlphABD GST + + + - - - GST-MlphABD + + + - - - Flag-Myo5a(1106-1410) Flag-Myo5a(1193-1453) Flag-Myo5a(1193-1467) 66 43 29 Input CBB staining 35 GST pulldown WB: Flag Flag-Myo5a(1193-1467) GST 1 2 3 4 5 6 Input CBB staining -GST-Mlph-ABD Flag-Myo5a-MTD -Flag-Myo5a-MTD Flag-Myo5a(1345-1467) -Flag-Myo5a(1345-1467) Flag-Trx-Myo5a(1411-1467) -Flag-Trx-Myo5a(1411-1467) Flag-Trx-Myo5a(1436-1467) -Flag-Trx Flag-Trx 66 43 29 66 43 29 55 20 GST-Mlph-ABD + + + + + -Flag-Trx-Myo5a(1436-1467) 1 2 3 4 5 WB: anti-Flag GST pulldown -Flag-Myo5a-MTD -Flag-Myo5a(1345-1467) -Flag-Trx-Myo5a(1411-1467) -Flag-Trx-Myo5a(1436-1467) 55 40 25 GST GST-Mlph-ABD + - + - + - + - Flag-Myo5a-MTD (E.Coli) Flag-Myo5a-MTD (Sf9) Input CBB staining GST pulldown WB: anti-Flag 1 2 3 4 Figure 2-figure supplement C ) Input CBB staining -GST-Mlph-ABD Flag-Myo5a-MTD -Flag-Myo5a-MTD Flag-Myo5a(1345-1467) -Flag-Myo5a(1345-1467) Flag-Trx-Myo5a(1411-1467) -Flag-Trx-Myo5a(1411-1467) Flag-Trx-Myo5a(1436-1467) -Flag-Trx Flag-Trx 66 43 29 20 GST-Mlph-ABD + + + + + -Flag-Trx-Myo5a(1436-1467) 1 2 3 4 5 WB: anti-Flag GST pulldown -Flag-Myo5a-MTD -Flag-Myo5a(1345-1467) -Flag-Trx-Myo5a(1411-1467) -Flag-Trx-Myo5a(1436-1467) 55 40 25 ) Figure 2-figure supplement A 66 43 29 55 GST GST-Mlph-ABD + - + - + - + - Flag-Myo5a-MTD (E.Coli) Flag-Myo5a-MTD (S Input CBB staining GST pulldown WB: anti-Flag 1 2 3 4 C A B Flag-Myo5a(1106-1410) Flag-Myo5a(1193-1453) Flag-Myo5a(1193-1467) GST-MlphABD GST + + + - - - GST-MlphABD + + + - - - Flag-Myo5a(1106-1410) Flag-Myo5a(1193-1453) Flag-Myo5a(1193-1467) 66 43 29 Input CBB staining 35 GST pulldown WB: Flag Flag-Myo5a(1193-1467) GST 1 2 3 4 5 6 B
https://openalex.org/W4242430272	https://hal-meteofrance.archives-ouvertes.fr/meteo-03657896/file/tc-13-1147-2019.pdf	English	null	Saharan dust events in the European Alps: role on snowmelt and geochemical characterization	null	2,018	cc-by	17,717	To cite this version: Biagio Di Mauro, Roberto Garzonio, Micol Rossini, Gianluca Filippa, Paolo Pogliotti, et al.. Saharan dust events in the European Alps: role in snowmelt and geochemical characterization. The Cryosphere, 2019, 13 (4), pp.1147-1165. 10.5194/tc-13-1147-2019. meteo-03657896 Saharan dust events in the European Alps: role in snowmelt and geochemical characterization Biagio Di Mauro, Roberto Garzonio, Micol Rossini, Gianluca Filippa, Paolo Pogliotti, Marta Galvagno, Umberto Morra Di Cella, Mirco Migliavacca, Giovanni Baccolo, Massimiliano Clemenza, et al. snowmelt and geochemical characterization Biagio Di Mauro, Roberto Garzonio, Micol Rossini, Gianluca Filippa, Paolo Pogliotti, Marta Galvagno, Umberto Morra Di Cella, Mirco Migliavacca, Giovanni Baccolo, Massimiliano Clemenza, et al. Saharan dust events in the European Alps: role in snowmelt and geochemical characterization Biagio Di Mauro, Roberto Garzonio, Micol Rossini, Gianluca Filippa, Paolo Pogliotti, Marta Galvagno, Umberto Morra Di Cella, Mirco Migliavacca, Giovanni Baccolo, Massimiliano Clemenza, et al. Distributed under a Creative Commons Attribution 4.0 International License Correspondence: Biagio Di Mauro (biagio.dimauro@unimib.it) Correspondence: Biagio Di Mauro (biagio.dimauro@unimib.it) Correspondence: Biagio Di Mauro (biagio.dimauro@unimib.it) Received: 7 November 2018 – Discussion started: 15 November 2018 Revised: 5 March 2019 – Accepted: 19 March 2019 – Published: 8 April 2019 origin and compared with Saharan sources. A strong enrich- ment in Fe was observed in snow containing Saharan dust. In our case study, the comparison between modelling results and observations showed that impurities deposited in snow anticipated the disappearance of snow up to 38 d a out of a to- tal 7 months of typical snow duration. This happened for the season 2015–2016 that was characterized by a strong dust deposition event. During the other seasons considered here (2013–2014 and 2014–2015), the snow melt-out date was 18 and 11 d earlier, respectively. We conclude that the effect of the Saharan dust is expected to reduce snow cover duration through the snow-albedo feedback. This process is known to have a series of further hydrological and phenological feed- back effects that should be characterized in future research. Abstract. The input of mineral dust from arid regions im- pacts snow optical properties. The induced albedo reduc- tion generally alters the melting dynamics of the snowpack, resulting in earlier snowmelt. In this paper, we evaluate the impact of dust depositions on the melting dynamics of snowpack at a high-elevation site (2160 m) in the European Alps (Torgnon, Aosta Valley, Italy) during three hydrological years (2013–2016). These years were characterized by sev- eral Saharan dust events that deposited signiﬁcant amounts of mineral dust in the European Alps. We quantify the short- ening of the snow season due to dust deposition by com- paring observed snow depths and those simulated with the Crocus model accounting, or not, for the impact of impu- rities. The model was run and tested using meteorological data from an automated weather station. We propose the use of repeated digital images for tracking dust deposition and resurfacing in the snowpack. The good agreement between model prediction and digital images allowed us to propose the use of an RGB index (i.e. snow darkening index – SDI) for monitoring dust on snow using images from a digital camera. We also present a geochemical characterization of dust reaching the Alpine chain during spring in 2014. El- ements found in dust were classiﬁed as a function of their 1 Introduction Mineral dust (hereafter referred as dust) plays an important role in Earth’s climate and in biogeochemical cycles (Ma- howald et al., 2010, 2013; Thornton et al., 2009). It provides nutrients such as iron, nitrogen, and phosphorous to ma- rine and terrestrial ecosystems (Aciego et al., 2017; Jickells, HAL Id: meteo-03657896 https://meteofrance.hal.science/meteo-03657896v1 Submitted on 3 May 2022 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License The Cryosphere, 13, 1147–1165, 2019 https://doi.org/10.5194/tc-13-1147-2019 © Author(s) 2019. This work is distributed under the Creative Commons Attribution 4.0 License. Saharan dust events in the European Alps: role in snowmelt and geochemical characterization Biagio Di Mauro1, Roberto Garzonio1, Micol Rossini1, Gianluca Filippa2, Paolo Pogliotti2, Marta Galvagno2, Umberto Morra di Cella2, Mirco Migliavacca3, Giovanni Baccolo1,4, Massimiliano Clemenza4,5, Barbara Delmonte1, Valter Maggi1, Marie Dumont6, François Tuzet6,7, Matthieu Lafaysse6, Samuel Morin6, Edoardo Cremonese2, and Roberto Colombo1 1Earth and Environmental Sciences Department, University of Milano-Bicocca, 20126 Milan, Italy 2Environmental Protection Agency of Aosta Valley, Aosta, Italy 3 3Max Planck Institute for Biogeochemistry, Jena, Germany 4National Institute of Nuclear Physics (INFN), University of Milano-Bicocca, 20126 Milan, Italy 5Department of Physics “Giuseppe Occhialini”, University of Milano-Bicocca, 20126 Milan, Italy 6Université Grenoble Alpes, Université de Toulouse, Météo-France, CNRS, CNRM, Centre d’Etudes de la Neige, Grenoble, France 7UGA/CNRS, Institut des Géosciences de l’Environnement (IGE), Saint Martin d’Hères, France A/CNRS, Institut des Géosciences de l’Environnement (IGE), Saint Martin d’Hères, France B. Di Mauro et al.: Saharan dust events in the European Alps (2010) presented a comprehensive character- ization of the mineralogical and geochemical properties of dust deposited from the atmosphere in the San Juan Moun- tains (Colorado, US). In this area, dust is dominated by silt and clay particles, indicating a regional source area. In the European Alps, a large fraction of dust reaching high moun- tains and glaciers originate from the Sahara (Haeberli, 1977; Kandler et al., 2007; Krueger et al., 2004; Schwikowski et al., 1995; Thevenon et al., 2009), but inputs from local sources cannot be excluded. Even though the Alps are located at a distance of about 3000 km from the largest desert of the planet, they are frequently affected by dust depositions. Due to their considerable elevation, the Alps act as an orographic barrier, enhancing cloud formation, precipitation, and hence dust scavenging from the atmosphere to the ground (De An- gelis and Gaudichet, 1991; Prodi and Fea, 1979). Dust de- position in the Alps is a well-known process, and its fre- quency is studied using ice cores from mountain glaciers (De Angelis and Gaudichet, 1991; Thevenon et al., 2009). Each year, the Sahara provides up to 760 million tons of dust to the atmosphere (Callot et al., 2000). Dust reaching Eu- rope is dominated by silicates and aluminium oxide (Goudie and Middleton, 2001); other contributions come from quartz, calcium-rich particles, sulfates, hematite, and soot (Kandler et al., 2007). The optical properties of particles are directly related to dust composition (Linke et al., 2006), and hence the latter is expected to modify the dust radiative effect on snow (Reynolds et al., 2013). Saharan dust can provide nutrients to many alpine ecosys- tems (Field et al., 2010; Okin et al., 2004). Aciego et al. (2017) recently showed that dust transported from Asia to the western US provides nutrients to montane forest ecosys- tems. This aspect has never been evaluated for mountain ecosystems in the European Alps, where dust may com- pete with ﬁne debris from local rocks in providing nutrients to soils. Conversely, the direct deposition of dust on plants can limit the photosynthetic capacity (Neves et al., 2009). Steltzer et al. (2009) reported results from a manipulation experiment conducted in the western US to study the depen- dence of vegetation phenology on snowmelt. They measured an advancement of 7 d in snowmelt when dust was manu- ally added to the snowpack. B. Di Mauro et al.: Saharan dust events in the European Alps Because of its peculiar optical properties, dust ef- ﬁciently scatters incoming solar radiation and exerts a direct climate forcing in the atmosphere (Tegen and Lacis, 1996). As a function of key variables (e.g. imaginary part of the refractive index, height of the dust layer, dust particle size, and dust optical depth), the net radiative forcing of dust can be either negative or positive at the top of the atmosphere (Liao and Seinfeld, 1998; Tegen et al., 1996), representing a signiﬁcant uncertainty in current climate models (Potenza et al., 2016). The main sources of dust are arid and hyper- arid regions of the planet. Under speciﬁc atmospheric condi- tions, ﬁne and coarse particles of dust can be suspended in the troposphere, generating characteristic dust storms (Fran- cis et al., 2018; Goudie and Middleton, 2001). Finer dust (< 5 µm) has a prolonged atmospheric lifetime, of the order of days, allowing for its long-range transport (Mahowald et al., 2013; Tegen and Lacis, 1996). When dust is deposited on snow- and ice-covered regions, its radiative impact at the surface results in a positive radiative forcing (Painter et al., 2012; Skiles et al., 2018). Snow optical properties largely de- pend on its microstructure and on the presence of impurities (also referred as light-absorbing particles – LAPs), such as carbonaceous or mineral particles (Warren and Wiscombe, 1980). Indeed, dust lowers the snow albedo in the visible wavelengths, enhancing the absorption of solar radiation (Di Mauro et al., 2015; Painter et al., 2007) and thus triggering the snow-albedo feedback (Hansen and Nazarenko, 2004). The alterations of the optical properties of snow are known to accelerate the melting processes (Drake, 1981; Painter et al., 2012). First estimations of the impact of dust on snow date back to the beginning of the last century; Jones (1913) estimated 1 month of earlier snowmelt due to dust deposition in the US. Drake (1981) estimated 4 d of advancement in the snowmelt. cal properties and snowpack dynamics. Impacts on glaciers optical properties and mass balance were also reported in the literature (Gabbi et al., 2015; Di Mauro et al., 2017; Oerle- mans et al., 2009). ) The composition of dust varies as a function of its ori- gin (Krueger et al., 2004) and timing (Kumar et al., 2018), with an effect on its optical properties (Caponi et al., 2017). Lawrence et al. B. Di Mauro et al.: Saharan dust events in the European Alps B. Di Mauro et al.: Saharan dust events in the European Alps 1148 2005; Yu et al., 2015), and it inﬂuences the shortwave radia- tion balance of the atmosphere (Ginoux, 2017; Mahowald et al., 2013). Because of its peculiar optical properties, dust ef- ﬁciently scatters incoming solar radiation and exerts a direct climate forcing in the atmosphere (Tegen and Lacis, 1996). As a function of key variables (e.g. imaginary part of the refractive index, height of the dust layer, dust particle size, and dust optical depth), the net radiative forcing of dust can be either negative or positive at the top of the atmosphere (Liao and Seinfeld, 1998; Tegen et al., 1996), representing a signiﬁcant uncertainty in current climate models (Potenza et al., 2016). The main sources of dust are arid and hyper- arid regions of the planet. Under speciﬁc atmospheric condi- tions, ﬁne and coarse particles of dust can be suspended in the troposphere, generating characteristic dust storms (Fran- cis et al., 2018; Goudie and Middleton, 2001). Finer dust (< 5 µm) has a prolonged atmospheric lifetime, of the order of days, allowing for its long-range transport (Mahowald et al., 2013; Tegen and Lacis, 1996). When dust is deposited on snow- and ice-covered regions, its radiative impact at the surface results in a positive radiative forcing (Painter et al., 2012; Skiles et al., 2018). Snow optical properties largely de- pend on its microstructure and on the presence of impurities (also referred as light-absorbing particles – LAPs), such as carbonaceous or mineral particles (Warren and Wiscombe, 1980). Indeed, dust lowers the snow albedo in the visible wavelengths, enhancing the absorption of solar radiation (Di Mauro et al., 2015; Painter et al., 2007) and thus triggering the snow-albedo feedback (Hansen and Nazarenko, 2004). The alterations of the optical properties of snow are known to accelerate the melting processes (Drake, 1981; Painter et al., 2012). First estimations of the impact of dust on snow date back to the beginning of the last century; Jones (1913) estimated 1 month of earlier snowmelt due to dust deposition in the US. Drake (1981) estimated 4 d of advancement in the snowmelt 2005; Yu et al., 2015), and it inﬂuences the shortwave radia- tion balance of the atmosphere (Ginoux, 2017; Mahowald et al., 2013). 2.2 Digital image analysis In recent years, digital images analysis was applied to mon- itor vegetation phenology (Julitta et al., 2014; Migliavacca et al., 2011; Richardson et al., 2007), landslides, glaciers (Jung et al., 2010), and snow (Corripio, 2004; Dumont et al., 2011; Hinkler et al., 2002; Parajka et al., 2012). Regarding the latter two, snow albedo and snow cover were success- fully estimated using digital cameras in alpine areas. For this study, digital RGB images were collected using a Nikon dig- ital camera (model d5000, also referred as “Phenocam”) in- stalled at the experimental site in 2013 in the vicinity of the AWS. Following Richardson et al. (2007), the camera was pointed north and set at an angle of about 20◦below horizon- tal. The camera focal length is 33 mm, and the ﬁeld of view is 79.8◦. The camera was ﬁxed at 2.5 m above the ground, and the same scene was repeatedly photographed. Digital im- ages were collected in the Joint Photographic Experts Group (JPEG) format with a resolution of 12 megapixels and three- colour channels (namely red, green, and blue) featuring eight bits of radiometric resolution. The images were collected from 10:00 to 17:00 LT (local time: UTC+1), with an hourly temporal resolution. Exposure mode and white balance were set to automatic. p g In this paper, we quantitatively estimate the impact of dust from the Sahara on snow dynamics. As a test area, we use the experimental site in Torgnon (Aosta Valley, western Ital- ian Alps) equipped with several sensors for measuring snow properties. Snow dynamics were simulated with a multilayer, physically based energy balance model (Crocus, Vionnet et al., 2012), which can incorporate the effect of LAPs (min- eral dust and black carbon) in snow and estimate their im- pact on snowmelt (Tuzet et al., 2017). The timing and inten- sity of Saharan dust depositions were simulated using two independent models (ALADIN-Climate and NMMB/BSC- Dust). Observed and simulated snow variables are compared, and the role of impurities on snowmelt is discussed. We also made use of repeated images from a digital camera to track the deposition and resurfacing of impurities. Finally, we present a geochemical characterization of dust reaching the Alps, and thus we discuss the possible biogeochemical and hydrological role of dust in the Alps. A region of interest (ROI) was ﬁrstly identiﬁed in an ap- proximately ﬂat area to analyse snow evolution. B. Di Mauro et al.: Saharan dust events in the European Alps (45◦50′40′′ N, 7◦34′41′′ E). The experimental site belongs to the Phenocam (Torgnon-nd, https://phenocam.sr.unh. edu/webcam/, last access: 2 April 2019), ICOS (IT-Tor; https://www.icos-ri.eu/, last access: 2 April 2019), and LTER (lter_eu_it_077; https://deims.org/site, last access: 2 April 2019) networks. The area is a subalpine unmanaged pasture classiﬁed as intra-alpine with a semi-continental cli- mate. The site is generally covered by snow from the end of October to late May. Further information regarding the site can be found in Galvagno et al. (2013). An AWS was in- stalled in 2009 at the experimental site of Torgnon. Air tem- perature is measured by a HMP45 (Vaisala Inc.); snow depth is measured with a SR50A sonic sensor (Campbell Scientiﬁc, Inc.). Albedo is measured with a Kipp & Zonen CNR4 net radiometer. The snow water equivalent (SWE) is measured with a gamma monitor (GMON; Campbell Scientiﬁc, Inc.) sensor. Solid and liquid precipitations were measured with a Pluvio2 OTT instrument. Wind speed and direction were measured with a CSAT3 three-dimensional sonic anemome- ter (Campbell Scientiﬁc, Inc.). Data are available at hourly time resolution. (45◦50′40′′ N, 7◦34′41′′ E). The experimental site belongs to the Phenocam (Torgnon-nd, https://phenocam.sr.unh. edu/webcam/, last access: 2 April 2019), ICOS (IT-Tor; https://www.icos-ri.eu/, last access: 2 April 2019), and LTER (lter_eu_it_077; https://deims.org/site, last access: 2 April 2019) networks. The area is a subalpine unmanaged pasture classiﬁed as intra-alpine with a semi-continental cli- mate. The site is generally covered by snow from the end of October to late May. Further information regarding the site can be found in Galvagno et al. (2013). An AWS was in- stalled in 2009 at the experimental site of Torgnon. Air tem- perature is measured by a HMP45 (Vaisala Inc.); snow depth is measured with a SR50A sonic sensor (Campbell Scientiﬁc, Inc.). Albedo is measured with a Kipp & Zonen CNR4 net radiometer. The snow water equivalent (SWE) is measured with a gamma monitor (GMON; Campbell Scientiﬁc, Inc.) sensor. Solid and liquid precipitations were measured with a Pluvio2 OTT instrument. Wind speed and direction were measured with a CSAT3 three-dimensional sonic anemome- ter (Campbell Scientiﬁc, Inc.). Data are available at hourly time resolution. dust may exert a stronger effect with respect to dry depo- sitions. Shifts in vegetation phenology also affect the timing of migration, breeding, and asynchronies between interacting animal species (Cohen et al., 2018; Thackeray et al., 2016). B. Di Mauro et al.: Saharan dust events in the European Alps Dust-induced snowmelt can anticipate the beginning of the growing season, and this can result in an earlier start of the seasonal cycle of both animals and plants. Changes in snow- falls and dust depositions are likely to occur more frequently in a warming climate. At the moment, the impact of Saharan dust events on the biogeochemistry of ecosystems in the Eu- ropean Alps has been poorly analysed (Avila and Peñuelas, 1999). Seasonal snow represents an important reservoir of fresh water in mountain ranges and polar regions. Recent climate changes showed that this exerts a strong impact on the du- ration of snow cover (Vaughan et al., 2013), in particular in the European Alps (Beniston, 2005; Beniston et al., 2018). It has been observed that, especially in spring, snow cover extent has decreased in the Northern Hemisphere (Brown and Robinson, 2011; Brown et al., 2009). Earlier snowmelt can have an impact on vegetation phenology (Steltzer et al., 2009) and water availability (Beniston et al., 2003), and it is expected to alter hydrologic regimes in the future. Accel- erated snowmelt due to dust can alter also surface hydrol- ogy in large mountain chains like the European Alps. In the Po Plain, for example, the most important renewable energy source is represented by hydropower. Meltwater from sea- sonal snow is a fundamental resource for agriculture during spring and summer (Huss et al., 2017). 2.2 Digital image analysis Images were acquired during the hydrological years 2013–2016. Red, green, and blue chromatic coordinates were extracted from the selected ROI using the Phenopix R package (Filippa et al., 2016). Then, the snow darkening index (SDI; Di Mauro et al., 2015) was calculated from a red and green digital num- bers (DNs) as follows: 2.1 Torgnon experimental site The study area is located in the north-western Italian Alps (Aosta Valley, Italy) at an elevation of 2160 m a.s.l. The Cryosphere, 13, 1147–1165, 2019 B. Di Mauro et al.: Saharan dust events in the European Alps This process can simulate a dry deposition from the atmosphere. In the Alps, most dust de- positions occur by wet deposition (mainly snowfalls; Sode- mann et al., 2006), so dust is expected to be included within ice grains. Flanner et al. (2012) showed that when black car- bon is internally mixed in ice grains, its radiative effect is stronger. If this also holds true for dust, wet deposition of The impact of dust on snow melting has been largely in- vestigated in the western US, where both radiative and hy- drological effects have been assessed using aerial, satellite, and automatic weather station (AWS) data (Painter et al., 2012a, b, 2013a, 2018; Reynolds et al., 2013; Skiles et al., 2012). In this area, the proximity of arid regions to the moun- tain ranges determines massive dust depositions on snow- covered mountain ranges. Dust depositions caused an earlier snowmelt that ranged from 35 d (Painter et al., 2007) to a maximum of 51 d (Skiles et al., 2012), strongly impacting water supplies around the area (Painter et al., 2012, 2018). Increases in dust deposition have been recently observed in this area, and they were linked to human activity and climate change (Neff et al., 2008). Other studies, conducted in Ice- land (Dagsson-Waldhauserova et al., 2015; Wittmann et al., 2017), in the Himalayas (Gautam et al., 2013), in Norway (Matt et al., 2018), and in the European Alps (Dumont et al., 2017; Di Mauro et al., 2015; Tuzet et al., 2017; Greilinger et al., 2018) reported signiﬁcant impacts of dust on snow opti- The Cryosphere, 13, 1147–1165, 2019 www.the-cryosphere.net/13/1147/2019/ 1149 B. Di Mauro et al.: Saharan dust events in the European Alps B. Di Mauro et al.: Saharan dust events in the European Alps B. Di Mauro et al.: Saharan dust events in the European Alps Figure 1. (a) Location of Torgnon (Aosta) and Artavaggio plains (Lecco) in the European Alps. (b) A picture of the experimental site of Torgnon (2160 m a.s.l.). (c) Aerial view of the site in Torgnon with the location of different instruments installed. The ﬁeld of view of the Phenocam is represented with a blue shaded area. Figure 1. (a) Location of Torgnon (Aosta) and Artavaggio plains (Lecco) in the European Alps. (b) A picture of the experimental site of Torgnon (2160 m a.s.l.). (c) Aerial view of the site in Torgnon with the location of different instruments installed. The ﬁeld of view of the Phenocam is represented with a blue shaded area. obtain simulations of snow spectral albedo as a function of snow properties, LAP concentrations, and LAP optical properties. The TARTES model is based on the asymp- totic approximation of the radiative transfer theory (AART; Kokhanovsky and Zege, 2004) and accounts for the effect of snow microstructure and impurities, such as dust and black carbon. The snow spectral albedo simulated with TARTES was used to calculate the SDI (using the formulation pro- posed in Di Mauro et al., 2015), and it was compared with the SDI calculated from the digital camera. A complete de- scription of this speciﬁc Crocus model version can be found in Tuzet et al. (2017). Crocus is embedded in the SURFEX surface scheme and is permanently coupled with the ISBA- DIF soil model. SDI = DNRed −DNGreen DNRed + DNGreen . (1) SDI = DNRed −DNGreen DNRed + DNGreen . (1) The SDI was correlated with the concentration of dust in snow (Di Mauro et al., 2015) and was used to represent the spatial distribution of impurities from space (Ganey et al., 2017; Di Mauro et al., 2017) and from hyperspectral images of ice cores (Garzonio et al., 2018). The SDI calculated from RGB data collected from an unmanned aerial vehicle (UAV) was found to be correlated with the SDI calculated from ﬁeld spectroscopy data (Di Mauro et al., 2015). This motivated the idea to monitor dust deposition and resurfacing dynam- ics using repeated digital images from the camera installed in Torgnon. In this work, the SDI was calculated for each avail- able image, then a daily average was calculated. B. Di Mauro et al.: Saharan dust events in the European Alps Days with an SDI > 0 were considered to be markers of the presence of dust on snow. Variables needed for running Crocus simulations are the following: air temperature, direct and diffuse shortwave in- coming radiation, longwave radiation, wind speed and direc- tion, speciﬁc humidity, surface pressure, and solid and liq- uid precipitation. The model was forced using meteorolog- ical data from the station in Torgnon for the seasons 2013 and 2016 at an hourly time step. All variables were mea- sured at the station of Torgnon, except for diffuse short- wave incoming radiation, which was measured (with a BF3 sensor; Delta-T Devices Ltd., Cambridge, UK) in another station located 2 km from Torgnon. The instrument used for precipitation measurements (Pluvio2 OTT) does not fea- ture a windshield. This can be problematic, since underesti- mations of snowfall can occur during intense wind events. For this reason, we corrected the data following the pre- scriptions proposed in Kochendorfer et al. (2017). Some manual adjustments to solid precipitations were needed in the case of strong wind events. In addition to the above- mentioned meteorological data, the Crocus version of Tuzet et al. (2017) needs dust and black carbon deposition ﬂuxes. www.the-cryosphere.net/13/1147/2019/ 1150 www.the-cryosphere.net/13/1147/2019/ B. Di Mauro et al.: Saharan dust events in the European Alps retrieve 202 ± 11 µg of particulate matter. For both samples, in addition to absolute concentration (mass fraction), normal- ized ones were also calculated. The average upper continen- tal crust composition (UCC; Rudnick and Gao, 2003) was selected as a normalizing reference to highlight the inﬂu- ence played by crust-derived material and the possible role of non-crustal sources for speciﬁc elements. Neutron irradi- ation was performed at the LENA laboratories at the Univer- sity of Pavia (Borio di Tigliole et al., 2010), where a TRIGA Mark II research nuclear reactor is installed (250 kW). Acti- vated samples were successively analysed using high-purity germanium detector available at the Radioactivity laboratory of the Milano-Bicocca University. Two irradiations and sev- eral acquisitions of the γ spectra were necessary to detect the largest number of radionuclides, ranging from the short-lived species to the long-lived ones. For a complete description of the method, including the estimation of errors, see Baccolo et al. (2015, 2016). In this study, these ﬂuxes were taken from the atmospheric model ALADIN-Climate (Nabat et al., 2015). For evaluat- ing the impact of dust depositions on the snowpack dynam- ics, key variables (e.g. albedo, snow depth, and snow wa- ter equivalent) measured from the AWS were compared with Crocus simulations with and without impurities (dust and black carbon) in snow. In addition, soil temperature was ini- tialized using a spin-up simulation of 4 years. 2.4 Dust concentration, size distribution, and geochemistry On 6 April 2016, a ﬁeld campaign was organized to col- lect snow samples at the experimental site of Torgnon. Six snow pits were dug in different locations placed a few me- tres away from the AWS. For each snow pit, we collected a surface sample at 0 cm and three samples at depths equal to 20, 40, and 60 cm from the surface. Samples were collected using sterilized Corning tubes (50 mL) and kept frozen until measurements. Dust concentration and size distribution were measured using a Coulter counter technique. Samples were melted in a clean room (class 1000 clean room at EuroCold Laboratory Facilities, University of Milano-Bicocca) and analysed with a Multisizer™4e COULTER COUNTER®. The instrument was set with a 100 µm oriﬁce, allowing for the detection of particles with a diameter (equivalent spheri- cal) between 2 and 60 µm, divided into 400 size channels. To obtain dust mass from particle volume, a crustal density of 2.5 g cm−3 was adopted. Total dust concentration was calcu- lated considering the integral of the concentration between 2 and 60 µm. Details about the technique can be found in Ruth et al. (2008). 2.5 Dust transport and deposition modelling In addition to the ALADIN-Climate model, dust transport and deposition were monitored using the NMMB/BSC-Dust model. This is an online multi-scale atmospheric dust model (Pérez et al., 2011); it was used here to provide dust fore- casts from the Sahara to the European Alps. NMMB/BSC- Dust provides both atmospheric concentration and depo- sition ﬂuxes of dust with a 0.3◦× 0.3◦horizontal resolu- tion. During the three seasons considered here, we classi- ﬁed dust events as “strong” events with dust deposition ﬂuxes larger than 800 mg m−2 and “weak” events with lower con- centrations. The timings of the events simulated with the NMMB/BSC-Dust model were qualitatively compared to those simulated with the ALADIN-Climate model during the period analysed here (2013–2016). In addition, dust samples collected in the Alps at 150 km from Torgnon (Artavaggio, Lecco, Italy, 1650 m a.s.l.) in March 2014 are used here to characterize the bulk com- position of dust events and the elemental input to alpine ecosystems. Snow samples were transported before melting in a cold facility, where they were stored until the prepara- tion for the successive analyses. At ﬁrst, they were melted, and an aliquot (5–10 mL) was measured through Coulter counter technique (CC) for the determination of dust size distribution. These data were already published (Di Mauro et al., 2015). A second aliquot consisting in few millilitres of melted snow was dedicated to instrumental neutron acti- vation analysis (INAA) for the analysis of elemental com- position (Greenberg et al., 2011). To this aim, dust was ex- tracted and separated using a ﬁltration system equipped with polycarbonate membranes (pore size 0.4 µm, well below the typical volume mode grain size of Saharan dust deposited on the Alps). Two distinct samples were prepared. One sample (SH1) was extracted from the reddish snow corresponding to the snow deposited during the Saharan event; it consisted of 7.2 ± 0.2 mg of dust. A second sample (SH2) was pre- pared for comparison, ﬁltering clean white snow. In this case, given the low concentration of impurities, it was possible to 2.3 Snowpack modelling Snow dynamics in Torgnon were simulated using the SUR- FEX and ISBA-Crocus model, hereinafter referred as Cro- cus. Crocus is a snow model initially developed for avalanche forecasting and used for hydrological estimation as well as numerical prediction (Brun et al., 1989). Crocus is a one- dimensional multilayer model that simulates the evolution of the snowpack based on input meteorological driving con- ditions (Brun et al., 1989, 1992). Snow dynamics are rep- resented as a function of energy and mass transfer within the snowpack, between both the snowpack and the atmo- sphere, and the snowpack and the ground below (Vionnet et al., 2012). In this study, we used a speciﬁc Crocus version using the Two-stream Analytical Radiative TransfEr in Snow (TARTES) radiative transfer model (Libois et al., 2013) to www.the-cryosphere.net/13/1147/2019/ The Cryosphere, 13, 1147–1165, 2019 1151 3.1 Modelled dust deposition events The period between 2013 and 2016 was characterized by two strong events (dust ﬂuxes > 800 mg m−2) and several weak events (dust ﬂuxes < 800 mg m−2) distributed during the sea- sons. The strong events that occurred on February 2014 and on April 2016. The event of February 2014 was already analysed in the scientiﬁc literature (Di Mauro et al., 2015; Tuzet et al., 2017; Dumont et al., 2017). The event of April 2016 lasted several days and transported a considerable dust amount to the western sector of the European Alps (Fig. 2; Greilinger et al., 2018). According to the NMMB/BSC-Dust model, during these two strong events, dust was primarily deposited in the Alpine chain by wet deposition. In Fig. 2, we show an example (5 April 2016) of the concentration of dust deposited according to NMMB/BSC-Dust and a longi- tudinal and latitudinal transect. NMMB/BSC-Dust predicted 3.2 Observed and simulated snow dynamics In Fig. 3, a comparison of variables observed at the Torgnon station and simulated with the Crocus model using impu- rity ﬂuxes is presented. We show time series of the snow albedo, SWE, and snow depth (SD). In general, the Crocus model represented snow dynamics well during the hydro- logical seasons 2013–2016. In Fig. 4, we present a quanti- tative comparison (coefﬁcient of determination – R2 – and root-mean-square error – RMSE) between snow variables observed and simulated, including and excluding the effect of LAPs. Crocus simulations accounting for the impact of LAPs showed a better agreement with observations than Cro- cus simulations that not account for the effect of LAPs. The snow albedo was underestimated from Crocus during the ac- cumulation period (see Fig. 3a). Instead, during the melt- ing period, the decreasing trend observed in snow albedo was reproduced well by the Crocus model accounting for the role of impurities. During the accumulation period, the albedo modelled by both Crocus simulations was always lower than the observed albedo. During the melting season, a clear divergence is observed between the Crocus simula- tion with LAPs and that without LAPs. Instead, the Cro- cus simulation with LAPs is more correlated with the ob- served snow depth. The observed and simulated SWE and snow depth show a large interannual variability. The SWE is strongly overestimated in the season 2013–2014; while dur- ing the accumulation period snow depth is represented well in the model, the melting rate is higher in the observed snow depth. This results in a delay of snow melt-out dates in both Crocus simulations (with and without impurities). A similar pattern in snow depth is also observed in the season 2014– 2015. Unfortunately, the measured SWE was not available for this season. During the season 2015–2016, the corre- lation between the observed and simulated snow depth ac- counting for the impact of impurities was very high, both for snow depth (R2 = 0.96; RMSE = 0.05 m) and the SWE (R2 = 0.97; RMSE = 13 mm). The difference in snow melt- out dates between observed and simulated data accounting for LAPs was 12, 10, and 11 d, respectively, for the sea- sons 2013–2014, 2014–2015, and 2015–2016. Instead, the comparison between snow melt-out dates simulated with and without impurities was 18, 11, and 38 d for the seasons 2013– 2014, 2014–2015, and 2015–2016, respectively. For snow depth (Fig. B. Di Mauro et al.: Saharan dust events in the European Alps 1152 Figure 2. NMMB/BSC-Dust forecast for the event of April 2016. In the top panel is the estimated surface concentration by wet deposition. The lower panels represent, respectively, a latitudinal and longitudinal transect centred on the city of Aosta (45.74◦N; 7.36◦E). Images are from the NMMB/BSC-Dust model, operated by the Barcelona Supercomputing Center (http://www.bsc.es/ess/ bsc-dust-daily-forecast/, last access: 2 April 2019). www.the-cryosphere.net/13/1147/2019/ The Cryosphere, 13, 1147–1165, 2019 1152 B. Di Mauro et al.: Saharan dust events in the European Alps This can be due to the longer duration of the dust event in April 2016 and may also explain the large change (38 d) in the snow melt-out dates observed in the data. Results from samples collected in Torgnon showed that signiﬁcant concentrations of dust were present in the snow- pack in April 2016 (Fig. 5). It is interesting to note that the mode of the dust size distribution is 7.9 µm for surface snow and 8.5 µm for snow samples collected at 20 and 40 cm depth. Instead, dust particles found in snow at the bottom of the snowpack (60 cm depth) feature a mode of 3.2 µm. This deeper layer can be probably due to the scavenging of small dust particles by meltwater or to other undetected pro- cesses. The ﬁrst three distributions can be due to the weak depositions that happened in February and March and were then buried by new snow. Dust size distributions are com- patible with other measurements of dust enclosed in snow and ice in the Alps (3–5 µm; Maggi et al., 2006) and in the Caucasus (1.98–4.16 µm; Kutuzov et al., 2013). Differences between our samples and these studies may be ascribed to the different elevation of the samplings. Samples shown in Fig. 5 feature a signiﬁcant noise in the tail of the distribution. This can be ascribed to the aggregation of ﬁne particles or to an input of local particles with larger diameters. A contri- bution of large particles of local origin cannot be excluded, and it may have a strong inﬂuence on snowmelt. At the mo- ment, we do not have enough data to decouple the effect of large and small particles on the snow albedo. Total concentra- tion of dust in Torgnon was estimated by adding up different channels from the size distributions. Among the six differ- ent snow proﬁles measured, surface concentrations reached a maximum of 65 µgdust g−1 snow, with a mean of 45.6 µgdust g−1 snow and a standard deviation of 15.8 µgdust g−1 snow. Hereafter we focus on the season 2015–2016, since Cro- cus simulations with impurities resulted in a 38 d advance- ment of the snow melt-out date compared to the correspond- ing simulations without impurities. This season was charac- terized by dust surface concentration in snow being almost double with respect to the other two seasons considered in this study (see Fig. 3f). B. Di Mauro et al.: Saharan dust events in the European Alps the most concentrated surface sample and 65.1 µgdust g−1 snow for the mean of the six snow pits. This variability can be ex- plained by the strong spatial mismatch between the spatial resolution of ALADIN-Climate model (50 km) and the point measurement of dust concentration. Differences can also de- pend on snow sampling, vertical resolution, and Crocus layer thickness. Model improvements are needed to downscale the spatial resolution of LAPs ﬂuxes. The installation of a wet and dry sampler (e.g. deposimeter) at experimental sites may help to drive the Crocus model with measured deposition ﬂuxes. It is important to notice that ALADIN-Climate pre- dicted also depositions of black carbon. At the moment, we do not have measurements to validate this estimation, but the presence of black carbon in snow may have ampliﬁed the snow-albedo feedback in the snowpack. The role of black carbon in Alpine snow still represents a great uncertainty in snow modelling and climate prediction in the Alps. While the role of industrial black carbon on post-industrial glacier retreat has been debated (Painter et al., 2013b; Sigl et al., 2018), its role in seasonal snow melting has not been studied in the European Alps. In Fig. 3d, we show also a qualitative comparison between the dust ﬂuxes simulated with ALADIN-Climate and with the NMMB/BSC-Dust model. In general, a good agreement between the two models was observed. The two most intense events (February 2014 and April 2016) are identiﬁed by both models. Smaller events are also reproduced, whereas some- times small events are seen only by ALADIN-Climate. Once dust ﬂuxes are deposited on the snowpack, they are buried by subsequent snowfalls. In Fig. 3e, we show the mul- tilayer concentration of dust in snow simulated with Crocus. It is clear that dust is resurfacing towards the end of the sea- son, when the snow albedo feedback intensiﬁes and promotes the melting. The surface concentration of dust (average of the ﬁrst 10 cm of snow) in the three seasons considered in this study show an important interannual variability (Fig. 3f). In fact, whereas the ﬁrst two seasons show surface concentra- tions of dust lower than 150 µg g−1, the last season (2015– 2016) shows concentrations up to 350 µg g−1 at the end of the season. 3.2 Observed and simulated snow dynamics 4a) and the SWE (Fig. 4b), Crocus simulations with LAPs generally resulted in a lower RMSE and higher R2 with respect to Crocus simulations without LAPs. In- stead, for snow albedo (Fig. 4c), the resulting RMSE was smaller for Crocus simulations without LAPs. We under- line that these RMSE values are associated with very low explained variance (R2 ∼0.2 for the seasons 2014–2015, 2015–2016, and all years, and R2 = 0.43 for the season 2013–2014). Thus, Crocus simulations with LAPs perform Figure 2. NMMB/BSC-Dust forecast for the event of April 2016. In the top panel is the estimated surface concentration by wet deposition. The lower panels represent, respectively, a latitudinal and longitudinal transect centred on the city of Aosta (45.74◦N; 7.36◦E). Images are from the NMMB/BSC-Dust model, operated by the Barcelona Supercomputing Center (http://www.bsc.es/ess/ bsc-dust-daily-forecast/, last access: 2 April 2019). up to 1600 mg m−2 of dust deposition in the western Alps. In the latitudinal and longitudinal proﬁles, it is clearly visible that the plume reached almost 6 km in altitude. The highest concentrations in the atmosphere were reached in southern France and in the north-west of Italy. The experimental site of Torgnon is located in the Italian western Alps, and it repre- sents a good candidate for analysing the effect of this strong dust event on snow dynamics. The Cryosphere, 13, 1147–1165, 2019 www.the-cryosphere.net/13/1147/2019/ 1153 B. Di Mauro et al.: Saharan dust events in the European Alps In Fig. 6, we show the comparison of the snow depth simulated with Crocus, including and exclud- ing the impact of impurities. During the season 2015–2016, about 1 m of snow was on the ground in Torgnon. The model without impurities predicted a longer duration of snow on the ground than the model with impurities. Two late snow- falls occurred in May, and this probably increased the differ- ence between the simulations. Since air temperatures were still close to 0◦(data not shown), snow was preserved at the ground in the simulations without impurities, and this fur- ther prolonged the snow season duration. Considering that the ﬁrst signiﬁcant snowfalls occurred in January, the snow season was shortened by about 20 % of the total because of impurities. In Fig. 6, we plot the SDI calculated from the radiative transfer model (TARTES) included in Crocus (SDI-Crocus hereafter) and from the RGB camera (SDI-Phenocam here- after). Regarding the digital camera data, days with SDI > 0 are represented as shaded green bands. We observed an agreement between the two datasets. SDI-Crocus increased during April. In particular, at the beginning of April two peaks in SDI-Crocus are also seen by SDI-Phenocam. A peak then is not clearly seen by the digital camera. This could be due to the occurrence of two small snowfalls during the resur- facing of dust layers. At the end of April, the concentration of dust on the surface of snow is represented well, both by Crocus and digital images. During this last period, a marked change in snowmelt rate is observed from the snow depth Measurements of dust concentrations are available only for 6 April 2016. On this day, the dust concentration pro- ﬁle simulated by Crocus spans from 11 (bottom) to 108.7 (top) µgdust g−1 snow. Modelled and measured surface concen- trations of dust showed some difference: 43.7 µgdust g−1 snow for www.the-cryosphere.net/13/1147/2019/ The Cryosphere, 13, 1147–1165, 2019 B. Di Mauro et al.: Saharan dust events in the European Alps 1154 B. Di Mauro et al.: Saharan dust events in the European Alps Figure 3. (a, b, c) Time series of albedo, snow water equivalent (SWE), and snow depth (SD) measured with the AWS and simulated with Crocus model including and excluding the impact of LAPs. SWE data are missing in December 2013 because of problems with the power supply. (d) Dust ﬂuxes simulated with ALADIN (maroon bars, note that the scale is inverted), and strong (large stars) and weak (small stars) dust events simulated with NMMB/BSC-Dust. (e) Dust concentration (µg g−1) in the snowpack (yellow to black palette) simulated with Crocus and superimposed on the snow depth proﬁle (grey shaded area). (f) Surface concentration (averaged over the ﬁrst 10 cm) of dust simulated with Crocus. Figure 3. (a, b, c) Time series of albedo, snow water equivalent (SWE), and snow depth (SD) measured with the AWS and simulated with Crocus model including and excluding the impact of LAPs. SWE data are missing in December 2013 because of problems with the power supply. (d) Dust ﬂuxes simulated with ALADIN (maroon bars, note that the scale is inverted), and strong (large stars) and weak (small stars) dust events simulated with NMMB/BSC-Dust. (e) Dust concentration (µg g−1) in the snowpack (yellow to black palette) simulated with Crocus and superimposed on the snow depth proﬁle (grey shaded area). (f) Surface concentration (averaged over the ﬁrst 10 cm) of dust simulated with Crocus. Figure 4. Comparison between (a) snow depth, (b) snow water equivalent (SWE), and (c) albedo observed from the AWS in Torgnon and simulated with Crocus accounting (red dots) and not accounting (cyan dots) for the impact of LAPs on snow. Figure 4. Comparison between (a) snow depth, (b) snow water equivalent (SWE), and (c) albedo observed from the AWS in Torgnon and simulated with Crocus accounting (red dots) and not accounting (cyan dots) for the impact of LAPs on snow. series around the 20 April (Fig. 6). The agreement between SDI-Crocus and SDI-Phenocam suggests that low-cost digi- tal RGB data can be used for monitoring the resurfacing of dust in snowﬁelds, useful for satellite and model validation. In order to use these RGB data quantitatively, further com- parisons with ﬁeld spectroscopy and ground data are needed. posure. B. Di Mauro et al.: Saharan dust events in the European Alps 1155 Figure 5. Dust particle distribution (expressed in µgdust kg−1 snow) for a snow proﬁle sampled at Torgnon on 6 April at different depths (0, 20, 40, and 60 cm). Blue lines are experimental data, and black lines are moving averages (kernel: 25 points). Numbers in the plots represent the peak of the size distributions. Please note that the scale changes within different plots. Figure 6. Comparison between snow depth simulated using Cro- cus with impurities (grey area) and without impurities (purple line). Observed data are also shown (black line). Dust concentration in snow is represented. Shaded green bands represent days with SDI- Phenocam > 0. SDI-Crocus is represented as a continuous green line. Figure 6. Comparison between snow depth simulated using Cro- cus with impurities (grey area) and without impurities (purple line). Observed data are also shown (black line). Dust concentration in snow is represented. Shaded green bands represent days with SDI- Phenocam > 0. SDI-Crocus is represented as a continuous green line. we inverted the non-linear (rational) model developed in Di Mauro et al. (2015) that links mineral dust concentration and SDI values, and we obtained an estimated dust concentra- tion equal to 56 µgdust g−1 snow. This value is very close to the concentrations measured with the Coulter counter integrating particles smaller than 60 µm, which reached a maximum of 65 µgdust g−1 snow. snow Our estimations of shifts in the melt-out day are compa- rable to previous ﬁndings in the western US estimating a reduction of snow cover up to 51 days due to the presence of mineral dust in snow (Painter et al., 2007; Skiles et al., 2012). Despite the different deposition rates in the Alps, the advancement of the snowmelt due to dust is comparable to published results regarding the western US. This is true at least for one season (2015–2016) characterized by a major Saharan dust deposition. Tuzet et al. (2017) estimated up to 9 d of advanced snowmelt during 2014 in a lower eleva- tion site located in the European Alps as well. In this pa- per, we estimate an advance in snow melt-out days of 18, 11, and 38 d for the three seasons considered. The estima- tion for the season 2015–2016 is very high, also considering that snow cover normally lasts about 7 months at this ele- vation (2160 m a.s.l.). www.the-cryosphere.net/13/1147/2019/ Surface runoff may also represent an important pro- cess in shaping snow surface and in distributing dust in snow- ﬁelds. This may explain the variability observed in SDI and also the differences between the measured dust concentration in snow samples and Crocus modelled concentrations. Dust redistribution on snowﬁelds might strongly affect its radia- tive impact. In Fig. 7c and d, we present two SDI maps ac- quired before (10 April) and after (20 April) the resurfacing of dust layers. The transition from cold to warm colours re- ﬂects the increase in the values of the index. Positive values of the SDI are associated here with the presence of dust on snow (Di Mauro et al., 2015). On the right of both images, a snow pit is visible. It is interesting to note that in the SDI map series around the 20 April (Fig. 6). The agreement between SDI-Crocus and SDI-Phenocam suggests that low-cost digi- tal RGB data can be used for monitoring the resurfacing of dust in snowﬁelds, useful for satellite and model validation. In order to use these RGB data quantitatively, further com- parisons with ﬁeld spectroscopy and ground data are needed. In Fig. 7a and b, we show two examples of digital im- ages collected from the digital camera. The spatial variabil- ity of SDI can be explained by local topography. The exper- imental site is located in a plain area, with a gentle slope (∼5◦). Microtopography created by snow melting and re- freezing cycles can locally concentrate and dilute impurities in the snowﬁeld, also in relation to the differential sun ex- In Fig. 7a and b, we show two examples of digital im- ages collected from the digital camera. The spatial variabil- ity of SDI can be explained by local topography. The exper- imental site is located in a plain area, with a gentle slope (∼5◦). Microtopography created by snow melting and re- freezing cycles can locally concentrate and dilute impurities in the snowﬁeld, also in relation to the differential sun ex- The Cryosphere, 13, 1147–1165, 2019 www.the-cryosphere.net/13/1147/2019/ 3.3 Geochemical characterization of dust in snow Dust composition is strictly tied to its optical characteris- tics and hence to its radiative effect on snow (Caponi et al., 2017; Reynolds et al., 2013). Iron oxides contained in dust are particularly absorptive in the visible wavelengths (Alfaro et al., 2004; Linke et al., 2006), and this further enhances the albedo feedback when dust is deposited on snow. The com- position of dust is also important for the correct representa- tion of dust in radiative transfer models and global climate models (Albani et al., 2014). Saharan dust events provide an input of nutrients to alpine ecosystems (Goudie and Middle- ton, 2001), and this has been poorly studied in the scientiﬁc literature (Arvin et al., 2017). Figure 7. (a, b) Examples of digital images acquired from the Phe- nocam installed at Torgnon before and after the resurfacing of dust layers. (c, d) Snow darkening index (SDI) calculated using the red and green channels of the images. A region of interest (ROI, a) was used to create SDI time series. The asterisk in (a) indicates the po- sition of the snow pit. The main source area of Saharan dust events reaching the Alps is represented by northern Algeria (potential source area in Northern Africa 1 – PSANAF-1 – in Formenti et al., 2011). For this reason, the dataset presented in this study can be considered representative for the main composition of long-range dust deposition on snow in the Alps. Saharan dust events are regional episodes that move large quantities of mineral dust from arid regions to different latitudes and longitudes. There are two main pathways for the transport of dust: it can reach Europe overpassing the Mediterranean and also by looping back over the Atlantic (Israelevich et al., 2012; Sodemann et al., 2006). For this reason, we can assume that the bulk geochemical composition of dust events that oc- curred in different locations in the Alps and at different times is comparable. Although no trends were found in the annual number of Saharan dust days since 1997 (Flentje et al., 2015), further re- search is needed to assess the role of impurities on snow dy- namics in the Alps. Measurements of surface concentrations of dust and black carbon in snow are very scarce in the whole Alpine chain. At the Jungfraujoch station (3454 m a.s.l.), dust concentration in the atmosphere is measured continuously (Collaud Coen et al., 2004). 3.3 Geochemical characterization of dust in snow A comparison with these data will be fundamental in validating Saharan dust ﬂuxes in the Alps and quantifying their effect on snow dynamics. Between 18 and 20 February 2014 a relevant event was observed, involving not only southern Europe and the Alps but also a large fraction of Europe. It was described as one of the most intense events of this kind that was observed in previous years. The event was associated to a particularly favourable atmospheric setting which could uplift a massive amount of Saharan dust from northern Africa and transport it toward Europe in association to south-westerly winds driven by an anticyclonic structure located on the central Mediter- ranean. Given the magnitude of the event, many studies re- ported it, spanning from microbiology (Meola et al., 2015; Weil et al., 2017) to remote and proximal sensing (Dumont et al., 2017; Di Mauro et al., 2015; Tuzet et al., 2017) and at- mospheric chemistry and physics (Belosi et al., 2017; Telloli et al., 2018). p q y g y Snow duration was very short during the 2015–2016 hy- drologic year. Usually, grassland in Torgnon is covered by a thick snow cover from the end of October to late May (aver- age 1928–2010). During 2015–2016 snow arrived in January and disappeared at the beginning of May. It is known that ear- lier snowmelt impacts the carbon uptake period (Galvagno et al., 2013), altering carbon exchange with the atmosphere during spring. Shifts in phenological dates, such as the be- ginning and end of season, may impact ecosystem function- ing related to net and gross ecosystem productivity in alpine grasslands and might lead to early depletion of soil mois- ture and early senescence related to summer water stress. Extreme events like heatwaves have impacts on phenology of mountain grasslands (Cremonese et al., 2017). With future climate change, these extreme events are likely to increase. With the intensiﬁcation of climate change, snow is expected to occur later in autumn and to be depleted earlier in spring (Frei et al., 2018; Verfaillie et al., 2018), with signiﬁcant con- sequences for the hydrological cycle. The effect of Saharan dust in the European Alps is to accelerate the melt via the di- rect and indirect effect on snow albedo, thus enhancing snow season shortening. Hereafter, we provide results from a geochemical charac- terization of dust sampled in snow in the Alps (Artavaggio, Lecco, Italy). B. Di Mauro et al.: Saharan dust events in the European Alps 1156 Figure 7. (a, b) Examples of digital images acquired from the Phe- nocam installed at Torgnon before and after the resurfacing of dust layers. (c, d) Snow darkening index (SDI) calculated using the red and green channels of the images. A region of interest (ROI, a) was used to create SDI time series. The asterisk in (a) indicates the po- sition of the snow pit. B. Di Mauro et al.: Saharan dust events in the European Alps In the future, impurity concentration estimated with the atmospheric model should be evaluated using ground observations. In this sense, data from in situ spectrometers (e.g. Dumont et al., 2017; Picard et al., 2016) and repeated digital images can be very helpful. In fact, the concentration of different impurities may be retrieved from spectral reﬂectance using both inversion of radiative transfer models and spectral indices. Figure 5. Dust particle distribution (expressed in µgdust kg−1 snow) for a snow proﬁle sampled at Torgnon on 6 April at different depths (0, 20, 40, and 60 cm). Blue lines are experimental data, and black lines are moving averages (kernel: 25 points). Numbers in the plots represent the peak of the size distributions. Please note that the scale changes within different plots. from 20 April, a red layer is visible in the snow pit. This can be possibly associated with the precedent weak depositions from February and March, which were concentrated in a thin snow layer by melting during early spring. At the end of the season, weak and strong depositions are concentrated by sur- face melting. This process ampliﬁes the feedback mechanism of dust on snow. In fact, while the melting of snow concen- trates the dust on the surface, higher concentrations of dust intensify the melting. This feedback is expected to act for each day with sufﬁcient solar radiation during the melting period. The feedback is also expected to be enhanced until the total disappearing of the snow cover. The SDI is also sensitive to other impurities, such as black carbon (Di Mauro et al., 2017) and organic material (Ganey et al., 2017). We cannot exclude that other impu- rities were present on snow surface, but at present we do not have enough data to evaluate these aspects. We interpret SDI variability only in relation to dust deposition and resur- facing. In the selected ROI (Fig. 7a), frequency distribution of the SDI shows a peak at 0.005. Using this information, Dust transport is a natural phenomenon, but it can be inten- siﬁed by anthropogenic activities (Neff et al., 2008). Further research is needed to assess possible inputs of local dust to mountain environments. Recently, dust was found to be more important than temperature in determining snowmelt in the western US (Painter et al., 2018). The Cryosphere, 13, 1147–1165, 2019 www.the-cryosphere.net/13/1147/2019/ www.the-cryosphere.net/13/1147/2019/ B. Di Mauro et al.: Saharan dust events in the European Alps (a) Major elements, (b) anthropogenic elements, and (c) incompatible elements (with respect to Fe), listed following the order proposed by Sun and McDonough (1989). Figure 8. The elemental composition of the Saharan dust extracted from the snow precipitated on the Alps in February 2014 (red line, SH1), and the composition of the particulate matter retrieved from the clean snow deposited few days later (blue line, SH2). Grey lines refer to samples collected from the Sahara–Sahel dust corridor (Moreno et al., 2006). Data are expressed in terms of concentration normalized to the average upper continental crust (UCC; Rudnick and Gao, 2003). (a) Major elements, (b) anthropogenic elements, and (c) incompatible elements (with respect to Fe), listed following the order proposed by Sun and McDonough (1989). One of the main differences between SH1 and SH2 is re- garding iron (Fe). With respect to this element, SH1 presents absolute concentrations that are more than 2 orders of mag- nitude higher than in SH2. This suggests that Saharan dust could be important for supplying this essential element to high-elevation alpine ecosystems where other nutrient sources could be limited, as already pointed out to in rela- tion to other species and to other environments (Avila et al., 1998; Greilinger et al., 2018; Rizzolo et al., 2017). Another issue related to Fe concentration in atmospheric dust is re- lated to its optical properties, since iron oxide concentration and mineralogy strongly inﬂuence them (Alfaro et al., 2004; Caponi et al., 2017; Formenti et al., 2014; Linke et al., 2006). The large abundance of Fe is thus expected to affect the ra- diative effects of dust on snow (Reynolds et al., 2013). mobilization of dust from the central sector of the Sahara– Sahel dust corridor, i.e. the Hoggar, Chad, and Niger basins (Moreno et al., 2006). The elemental composition of dust might also have an im- portant effect on the biogeochemical cycles of the alpine grasslands. Among the elements listed in Table 1 there are elements such as K and Ca that are known to be rele- vant to ecosystem functioning (Sardans and Peñuelas, 2015; Schaffner et al., 2012). For both elements, SH1 shows no- tably higher concentrations (see Table 1). This requires more attention and further studies to understand the feedback of Saharan dust deposition on the biogeochemistry of high- elevation ecosystems. B. Di Mauro et al.: Saharan dust events in the European Alps 1157 Figure 8. The elemental composition of the Saharan dust extracted from the snow precipitated on the Alps in February 2014 (red line, SH1), and the composition of the particulate matter retrieved from the clean snow deposited few days later (blue line, SH2). Grey lines refer to samples collected from the Sahara–Sahel dust corridor (Moreno et al., 2006). Data are expressed in terms of concentration normalized to the average upper continental crust (UCC; Rudnick and Gao, 2003). (a) Major elements, (b) anthropogenic elements, and (c) incompatible elements (with respect to Fe), listed following the order proposed by Sun and McDonough (1989). may pave the way for a more exhaustive characterization of dust composition in the future. The concentrations of major elements normalized to the upper continental crust composition are shown in Fig. 8a. It can be easily appreciated that SH1 and SH2 display a very different composition. SH1, corresponding to the dusty snow deposited during the Saharan advection episode of Febru- ary 2014, presents a typical crustal signature, with UCC nor- malized values close to 1. On the contrary, SH2 shows very low normalized concentrations, suggesting that in this case, the crustal fraction is not the dominant one. Since all the considered major elements are strongly depleted (normal- ized concentrations span from 0.17 in the case of Na to 0.38 for Fe), it can be inferred that its composition is probably dominated by the only major element which is not consid- ered here: carbon. Unfortunately INAA is not suited for its detection, but it is known that the carbonaceous fraction is an important component of snow impurities (Li et al., 2016; Wang et al., 2015). Comparing SH1 to Sahel and Saharan dust source composition, a substantial correspondence can be appreciated, as illustrated in Fig. 8. This is not unexpected, but direct observations linking the geochemical properties of Saharan dust to the dust deposited in the Alps are quite scarce. Figure 8. The elemental composition of the Saharan dust extracted from the snow precipitated on the Alps in February 2014 (red line, SH1), and the composition of the particulate matter retrieved from the clean snow deposited few days later (blue line, SH2). Grey lines refer to samples collected from the Sahara–Sahel dust corridor (Moreno et al., 2006). Data are expressed in terms of concentration normalized to the average upper continental crust (UCC; Rudnick and Gao, 2003). 3.3 Geochemical characterization of dust in snow The analysis of the elemental composition al- lowed detecting 36 elements, spanning from the so-called major elements (the ones whose oxides constitute more than 1 % of the average composition of Earth’s crust) to many mi- nor and trace ones. Data of interest are shown in Fig. 8, the full list of elemental concentrations is reported in Table 1. We acknowledge that only one snow sample containing dust is not enough to provide a complete overview on the compo- sition of Saharan dust in snow in the Alps, but our analysis The Cryosphere, 13, 1147–1165, 2019 www.the-cryosphere.net/13/1147/2019/ www.the-cryosphere.net/13/1147/2019/ B. Di Mauro et al.: Saharan dust events in the European Alps Looking at Ca and Ti, further information can be inferred about the most likely provenance of SH1. Northern African sources (grey lines in Fig. 8a) can be clearly distinguished in relation to the content of these two elements, and indeed two groups are recognized. A ﬁrst one is characterized by high Ca concentration and low Ti content, and the second group shows an opposite composition, with a larger amount of Ti and a lower amount of Ca (see Fig. 8a). Carbonates are rich in Ca (a constituent of these rocks) and poor in Ti, in relation to their limited content in accessory and heavy minerals. The ﬁrst group (high Ca and low Ti) corresponds to the samples collected in Western Sahara, where carbonate rocks are com- mon. On the contrary, samples from northern Africa display an opposed composition. Comparing SH1 to these groups, it is clear that its composition is in accordance with the sec- ond group. Its provenance is more probably related to the Anthropogenic elements are presented in Fig. 8b. They are W, Hg, Sb, Se, and Zn. This group of elements con- cerns those elements presenting important positive deviations with respect to UCC composition. They are deﬁned “anthro- pogenic” to highlight that the their biogeochemical cycles have been strongly impacted by human activities in the last decades and that their mobilization in the environment re- lated to anthropogenic activities exceeds the natural one (Sen and Peucker-Ehrenbrink, 2012). Again, the signature of SH1 is completely different from that of SH2. Unlike the case of major and incompatible elements, for anthropogenic el- ements the sample presenting the higher relative concentra- B. Di Mauro et al.: Saharan dust events in the European Alps Table 1. The elemental composition of SH1 (snow sample containing mineral dust) and SH2 (clean snow sample). Data are expressed (in terms of µg g−1) and are referred to the mass of the extracted material, not to the considered snow volume. Values in brackets are measurement uncertainties. Normalized concentrations were calculated considering the upper continental crust as a reference (Rudnick and Gao, 2003). Element SH1 SH2 Conc. Conc. Conc. Conc. (µg g−1) (UCC norm.) (µg g−1) (UCC norm.) Na 5600(500) 0.47(0.04) 2000(300) 0.17(0.03) Mg 12 000(2000) 0.8(0.1) 3600(900) 0.24(0.06) Al 63 000(14 000) 0.8(0.2) 13 500(3000) 0.17(0.04) Si 200 000(35 000) 0.6(0.1) 48 000(25 000) 0.15(0.08) K 17 000(2000) 0.75(0.09) 4500(400) 0.19(0.02) Ca 16 000(5000) 0.6(0.2) 6000(2000) 0.25(0.09) Ti 6000(500) 1.6(0.1) 900(200) 0.25(0.06) Mn 470(50) 0.61(0.07) 180(33) 0.23(0.04) Fe 40 000(4000) 1.0(0.1) 180(30) 0.38(0.05) Sc 13(1) 0.91(0.08) 3.1(0.4) 0.22(0.03) V 100(10) 1.0(0.1) 26(5) 0.27(0.05) Cr 123(27) 1.3(0.03) 84(22) 0.9(0.2) Co 14(1) 0.82(0.06) 6.6(0.7) 0.38(0.05) Ni 35(8) 0.7(0.2) 38(11) 0.8(0.2) Zn 132(13) 2.0(0.2) 233(30) 3.5(0.4) As 6(1) 1.3(0.2) 4(1) 0.9(0.2) Se < 0.002 – 0.5(0.1) 5(1) Rb 82(8) 0.98(0.09) 28(5) 0.34(0.06) Sr 118(19) 0.37(0.06) 86(23) 0.27(0.07) Sb 1.3(0.2) 3.1(0.4) 12(2) 30(5) Cs 3.7(0.4) 0.75(0.08) 1.5(0.2) 0.30(0.05) Ba 500(100) 0.8(0.2) 300(200) 0.5(0.4) La 43(5) 1.4(0.1) 9(2) 0.29(0.05) Ce 87(4) 1.39(0.06) 29(2) 0.46(0.04) Nd 38(8) 1.4(0.3) 10(6) 0.4(0.2) Sm 7.2(0.9) 1.5(0.2) 1.5(0.2) 0.32(0.05) Eu 1.5(0.2) 1.5(0.2) 0.24(0.07) 0.24(0.07) Tb 1.05(0.08) 1.5(0.1) 0.22(0.05) 0.31(0.06) Ho 1.05(0.08) 1.3(0.1) 0.24(0.05) 0.29(0.07) Yb 3.9(0.6) 1.9(0.3) 0.7(0.2) 0.3(0.1) Hf 7.6(0.7) 1.4(0.1) 1.5(0.2) 0.28(0.04) Ta 1.8(0.5) 2.0(0.6) 0.4(0.1) 0.4(0.1) W 3.1(0.7) 16(3) 19(3) 100(15) Hg 0.4(0.1) 8(2) 2.9(0.9) 60(20) Th 12(1) 1.2(0.1) 2.6(0.6) 0.24(0.05) U 2.5(0.6) 0.9(0.2) 2.2(0.9) 0.8(0.3) tion is SH2. SH1 shows values near 1, suggesting that its composition is also mainly crustal for these elements. On the contrary, sample SH2 presents extremely high enrichments, near 100 in the case of W. Such values are not compatible with a crustal origin. Contributions from other sources must be involved. Atmospheric emissions related to human activ- ities are the best candidate for explaining the enrichment of almost all of these elements. Hg, Sb, Se, and Zn are all quite volatile elements, are easily mobilized in the atmosphere, and are related to industrial processes. Indeed, the sampling site is located less than 100 km far from the Po Valley, one of the most industrialized and densely inhabited regions of Eu- rope. www.the-cryosphere.net/13/1147/2019/ www.the-cryosphere.net/13/1147/2019/ The Cryosphere, 13, 1147–1165, 2019 1158 www.the-cryosphere.net/13/1147/2019/ B. Di Mauro et al.: Saharan dust events in the European Alps depth) simulated with Crocus model were compared with ob- served variables from an AWS in the Aosta Valley (western Alps). Good agreement between observations and simula- tions accounting for the role of impurities was observed. The size distribution of dust found in snow conﬁrms the Saha- ran origin of the event during April 2016 (Baumann-Stanzer et al., 2018). The geochemical characterization of dust and particulate matter samples distinguished the snow associated with Saharan dust from clean snow. Dusty snow showed a composition compatible with the geochemistry of the dust sources located in the central sector of the Sahara–Sahel dust corridor, i.e. the Hoggar, Chad, and Niger basin northern African sources. On the contrary, clean snow was character- ized by strong contaminations related to anthropogenic ele- ments. These results demonstrate that an accurate geochemi- cal characterization of dust deposited on the Alps allows the identiﬁcation of the different Saharan sources involved in the single transport events, but the ﬁngerprint of the local sources may also play an important role. al., 2008), refractory and non-volatile elements are instead more easily transported directly as airborne particles gener- ated by industrial processes (Sheppard et al., 2007). y p ( pp , ) The composition of a suite of elements found in trace is presented in Fig. 8c. The elements displayed there were or- dered following their incompatibility degree with respect to Fe (Sun and McDonough, 1989). This is a useful geochem- ical feature for understanding the provenance and the geo- chemical signature of rock-related samples. Focussing on SH1, it can be appreciated that there is a slight enrichment of poorly incompatible elements (the ones on the right side of Fig. 8c). The same feature is also recognized in the African dust sources, as it was extensively discussed by Moreno et al. (2006), which related the point to the geochemical and mineralogical properties of the sources. Sr and Ta are the two elements presenting the most evident anomalies: a depletion in the ﬁrst case and an enrichment in the second case. The concentration of Sr is generally related to the presence or ab- sence of carbonates, since Sr is a well-known substituent for Ca in carbonate lattice. In Fig. 8c, it is possible to appreciate that SH1 and most of the African sources are signiﬁcantly de- pleted in Sr, conﬁrming what was already suggested by ma- jor elements. B. Di Mauro et al.: Saharan dust events in the European Alps Indeed, the samples with low Sr content are the same samples presenting low Ca concentrations, pointing to a limited presence of carbonates and conﬁrming that sources from Western Sahara were not involved in this episode. In the paper, we also made use of repeated digital images for monitoring dust deposition and resurfacing in the snow- pack of Torgnon. Dust deposition and resurfacing agreed well with modelling predictions. This allowed us to propose the use of an RGB index (i.e. snow darkening index – SDI) for tracking dust on snow using repeated digital images from digital cameras. The good agreement between dust deposi- tion and the SDI suggests that data from this experimen- tal site can be used as a possible calibration and validation for satellite imagery (e.g. MODIS, Landsat, and Sentinel) and for regional and global climate model (WFR-Chem and CLM) validation. p The case of Ta is completely different, given the analyt- ical difﬁculties related to its detection; its behaviour in the environment is not yet well constrained, but it seems quite common to deal with samples that present an enrichment, in particular when atmosphere-related samples are considered (Filella, 2017). Looking at Fig. 8c, it can be seen that both the African sources, and to a lesser extent SH1, present a positive anomaly for Ta. Recent studies suggested that the Ta enrichment in rocks, sediments, and atmospheric particulate matter could be attributed to the effect of chemical weather- ing. Being extremely stable from a chemical and geochem- ical perspective, the loss of mobile fractions during weath- ering, enhanced by atmospheric transport, could explain the enrichment of Ta (Baccolo et al., 2016; Vlastelic et al., 2015). Several questions are still open regarding the role of dust in the Alps. For example, the spatial distribution of dust concen- tration on snow at alpine scale has never been quantitatively estimated. Possible differences between eastern and western Alps may arise as a function of distance from the sources. Another unresolved issue is the input from local sources: coarser dust particles can be suspended from snow-free ar- eas and deposited on snow. Regarding the geochemical and mineralogical characteristics of dust, future research should explore, in detail, the relation between dust characteristics and their radiative effect on snow. In addition to the well- known snow-albedo feedback, other complex mechanisms can inﬂuence the impact of dust on snow. B. Di Mauro et al.: Saharan dust events in the European Alps For example, the presence of dissolved carbonates may accelerate the melt of snow lowering the melting point of snow and ice crystals. The role of carbonaceous particles on snow optical properties in the Alps is also an open question. Measurements of black carbon, brown carbon, organic carbon, and elemental carbon concentration in snow are virtually absent in surface snow in the Alps. The Po Plain is one of the most polluted areas of the planet. At lower elevations, black carbon emissions from fossil fuel combustion and biomass burning may reach snow- covered areas and exert an impact on snow optical proper- ties. Future research efforts should aim at providing spatially B. Di Mauro et al.: Saharan dust events in the European Alps The same interpretation is not sufﬁcient for explain- ing the considerably high amount of W in SH2. There is no previous available information about its occurrence in snow, and in general its behaviour in the environment is quite ob- scure (Koutsospyros et al., 2006). It is traditionally consid- ered a non-volatile element, given its refractory properties. The high concentration found in SH2 could be related to the anthropogenic activities, since W is used in many indus- trial and manufacturing activities (Koutsospyros et al., 2006). Thus, a different transport mechanism is probably involved. Volatile elements can easily be scavenged from the atmo- sphere after being adsorbed on particulate matter (Marx et The Cryosphere, 13, 1147–1165, 2019 www.the-cryosphere.net/13/1147/2019/ www.the-cryosphere.net/13/1147/2019/ 1159 4 Conclusions In this paper, we investigated the role of impurity depositions in snow dynamics. In particular, we analysed the role of Sa- haran dust events on snowmelt in a high-elevation site of the European Alps. We estimated that impurities induced an ad- vance in snow melt-out dates of 38 d for the season 2015– 2016. During the other seasons considered here (2013–2014 and 2014–2015), the advancement in snow melt-out dates was 18 and 11 d, respectively. The season 2015–2016 was characterized by dust depositions that were almost double with respect to the other years considered in this study. Snow key variables (snow water equivalent, snow albedo, and snow The Cryosphere, 13, 1147–1165, 2019 The Cryosphere, 13, 1147–1165, 2019 Competing interests. The authors declare that they have no conﬂict of interest. Competing interests. The authors declare that they have no conﬂict of interest. Acknowledgements. We acknowledge the ARPA (Environmental Protection Agency) of the Aosta Valley region for maintaining the AWS in Torgnon and for providing the dataset. RGB im- ages were analysed using the Phenopix R package (https://r-forge. r-project.org/projects/phenopix/, last access: 2 April 2019). The complete set of Phenocam images is available at the following web- site: https://phenocam.sr.unh.edu/webcam/sites/torgnon-nd/ (last access: 2 April 2019). Edoardo Cremonese acknowledges the sup- port of the NextData Data-LTER-Mountain project CNRM. CEN and IGE are part of Labex OSUG@2020. The modelling work was funded by the ANRJCJC EBONI grant no. 16-CE01-0006. We thank the editor and the two reviewers for the constructive com- ments on a previous version of the paper. 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https://openalex.org/W4388683871	https://www.frontiersin.org/articles/10.3389/fpubh.2023.1165728/pdf?isPublishedV2=False	English	null	Design-redesign, implementation, and evaluation of effectiveness of maternal nutrition and responsive parenting program on child development at 2 years of age from rural India: a cluster RCT	Frontiers in public health	2,023	cc-by	11,113	Design-redesign, implementation, and evaluation of effectiveness of maternal nutrition and responsive parenting program on child development at 2 years of age from rural India: a cluster RCT OPEN ACCESS EDITED BY Satinder Aneja, Sharda University, India REVIEWED BY Rosnah Sutan, National University of Malaysia, Malaysia Nandita Chattopadhyay, MGM Medical College Kishanganj, India CORRESPONDENCE Abhay Gaidhane abhaygaidhane@gmail.com Zahiruddin Quazi Syed zahirquazi@gmail.com †These authors have contributed equally to this work and share senior authorship RECEIVED 21 March 2023 ACCEPTED 17 October 2023 PUBLISHED 14 November 2023 CITATION Gaidhane A, Khatib MN, Telrandhe S, Patil M, Kogade P, Gaidhane S, Choudhari SG, Holding PA, Saxena D and Syed ZQ (2023) Design-redesign, implementation, and evaluation of effectiveness of maternal nutrition and responsive parenting program on child development at 2 years of age from rural India: a cluster RCT. Front. Public Health 11:1165728. d i 10 3389/f bh 2023 1165728 OPEN ACCESS EDITED BY Satinder Aneja, Sharda University, India REVIEWED BY Rosnah Sutan, National University of Malaysia, Malaysia Nandita Chattopadhyay, MGM Medical College Kishanganj, India Abhay Gaidhane 1†, Mahalaqua Nazli Khatib 2, Shital Telrandhe 3,4, Manoj Patil 5, Priti Kogade 3,4, Shilpa Gaidhane 6, Sonali G. Choudhari 6, Penny A. Holding 6, Deepak Saxena 7 and Zahiruddin Quazi Syed 6,8† Abhay Gaidhane 1†, Mahalaqua Nazli Khatib 2, Shital Telrandhe 3,4, Manoj Patil 5, Priti Kogade 3,4, Shilpa Gaidhane 6, Sonali G. Choudhari 6, Penny A. Holding 6, Deepak Saxena 7 and Zahiruddin Quazi Syed 6,8*† †These authors have contributed equally to this work and share senior authorship RECEIVED 21 March 2023 ACCEPTED 17 October 2023 PUBLISHED 14 November 2023 CITATION Gaidhane A, Khatib MN, Telrandhe S, Patil M, Kogade P, Gaidhane S, Choudhari SG, Holding PA, Saxena D and Syed ZQ (2023) Design-redesign, implementation, and evaluation of effectiveness of maternal nutrition and responsive parenting program on child development at 2 years of age from rural India: a cluster RCT. TYPE Original Research PUBLISHED 14 November 2023 DOI 10.3389/fpubh.2023.1165728 TYPE Original Research PUBLISHED 14 November 2023 DOI 10.3389/fpubh.2023.1165728 TYPE Original Research PUBLISHED 14 November 2023 DOI 10.3389/fpubh.2023.1165728 Design-redesign, implementation, and evaluation of effectiveness of maternal nutrition and responsive parenting program on child development at 2 years of age from rural India: a cluster RCT F t P bli H lth 11 1165728 1 Centre of One Health, School of Epidemiology and Public Health, Jawaharlal Nehru Medical College, Datta Meghe Institute of Higher Education and Research, Wardha, Maharashtra, India, 2 Global Evidence Synthesis Initiative, Division of Evidence Synthesis, Jawaharlal Nehru Medical College, Datta Meghe Institute of Higher Education and Research, Wardha, Maharashtra, India, 3 Global Health Academy, Centre of Early Childhood Development - Stepping Stones Project, Wardha, India, 4 Datta Meghe Institute of Higher Education and Research, Wardha, Maharashtra, India, 5 School of Epidemiology and Public Health, Jawaharlal Nehru Medical College, Datta Meghe Institute of Higher Education and Research, Wardha, Maharashtra, India, 6 Jawaharlal Nehru Medical College, Datta Meghe Institute of Higher Education and Research, Wardha, Maharashtra, India, 7 i Health Consortium, Department of Epidemiology, Indian Institute of Public Health, Gandhinagar, Gujarat, India, 8 South Asia Infant Feeding Research Network (SAIFRN), School of Epidemiology and Public Health, Wardha, Maharashtra, India Gaidhane A, Khatib MN, Telrandhe S, Patil M, Kogade P, Gaidhane S, Choudhari SG, Holding PA, Saxena D and Syed ZQ (2023) Design-redesign, implementation, and evaluation of effectiveness of maternal nutrition and responsive parenting program on child development at 2 years of age from rural India: a cluster RCT. Front. Public Health 11:1165728. doi: 10.3389/fpubh.2023.1165728 Front. Public Health 11:1165728. doi: 10.3389/fpubh.2023.1165728 Introduction suggested an adapted curriculum and a framework to oversee the program implementation. Despite the primary emphasis of ICDS being on the early years of life, its efforts primarily revolve around nutritional supplementation and children’s healthcare needs. Unfortunately, the responsive parenting program, vital for fostering early childhood development, is considerably underrepresented within ICDS (8). The early years are crucial to ensure that each child reaches their productive and creative potential in adulthood (1, 2). To provide adequate nurturing care, families must address multiple needs for psychosocial stimulation, health care, nutrition, and environmental and economic security (3–7). Evidence of the effectiveness of single- target interventions in the early years of life is available and encouraging. However, information that adequately guides implementing complex programs that address holistic child development is limited. In India, 55% of children under 6 months are exclusively breastfed. Although breastfeeding is nearly universal in Maharashtra, only 57% of children under 6 months are exclusively breastfed, as the World Health Organization (WHO) recommends. Encouragingly, 87% of infants are introduced to breastfeeding within the first day of their lives, but it drops to 57% for those who begin breastfeeding within the recommended first hour of life. In the context of child health, the infant mortality rate in Maharashtra in NFHS-4 is estimated at 24 deaths before the age of 1 year per 1,000 live births (9). A Holistic Early Childhood Development (ECD) fosters the overall growth of a child, with the various domains of child development collectively shaping a child’s development and growth. This includes their physical development (i.e., gross and fine motor skills), brain or cognitive development, language development, socio- emotional development and behavioral development. India’s national Integrated Child Development Service (ICDS) program was initiated in 1975 to tackle child malnutrition and illnesses. ICDS is one of the government’s most extensive and prominent initiatives that offer nutritional supplementation, immunization, height and weight monitoring, and non-formal education to children under six through Anganwadi Centers (AWCs). An Anganwadi Worker (AWW), operating at the grassroots level, is responsible for catering to a thousand population through an Anganwadi Center (AWC), with assistance from the Anganwadi Helper (AWH). However, the recent assessment of the ICDS programs recommended reinforcing the infrastructure, training, and support systems for AWC and staff. The report Integrating parenting with nutrition interventions blended with traditional community-focused child-rearing approaches for Early Child Development (ECD) are evidence-based practices proven effective for ECD (6, 10–12). COPYRIGHT 96% of women initiated breastfeed within one-hour of delivery, and exclusive- breastfeeding rate of 89.80%. Interpretations: The study provides an evidence-based community centered ECD curriculum and implementation strategies to enhance service providers, and caregivers’ knowledge and skills for promoting ECD in low-resource settings with the potential to scale within existing Government Program. Funding: The trial was funded by the Saving Brains Round 5 Initiative of Grand Challenges Canada (Grant no. SB-1707-05084), and we are grateful for their ongoing support through online sessions and orientation workshops. The trial was also supported by the Indian Council of Medical Research (File No: 5/7/1693/ CH/Adhoc/RBMCH-2020). KEYWORDS early child development, integrated intervention, nutrition program, responsive parenting, rural India Gaidhane et al. 10.3389/fpubh.2023.1165728 on community and stakeholder feedback. Post-re-designing, session quality improved, with program coverage rising from 32 to 98%. Male participation in home visits increased from 4.3 to 32.65%, and data errors reduced from 270 to 140 per month on average. At 24 months, program showed moderate–mild impact on ECD – cognitive (0.31, 95%CI: 0.13–0.48), language (0.2, 95%CI: 0.01–0.39), and socioemotional-development (0.19, 95%CI: 0.01–0.37), moderate effect on home-environment and mother–child interaction. 96% of women initiated breastfeed within one-hour of delivery, and exclusive- breastfeeding rate of 89.80%. on community and stakeholder feedback. Post-re-designing, session quality improved, with program coverage rising from 32 to 98%. Male participation in home visits increased from 4.3 to 32.65%, and data errors reduced from 270 to 140 per month on average. At 24 months, program showed moderate–mild impact on ECD – cognitive (0.31, 95%CI: 0.13–0.48), language (0.2, 95%CI: 0.01–0.39), and socioemotional-development (0.19, 95%CI: 0.01–0.37), moderate effect on home-environment and mother–child interaction. 96% of women initiated breastfeed within one-hour of delivery, and exclusive- breastfeeding rate of 89.80%. Interpretations: The study provides an evidence-based community centered ECD curriculum and implementation strategies to enhance service providers, and caregivers’ knowledge and skills for promoting ECD in low-resource settings with the potential to scale within existing Government Program. Funding: The trial was funded by the Saving Brains Round 5 Initiative of Grand Challenges Canada (Grant no. SB-1707-05084), and we are grateful for their ongoing support through online sessions and orientation workshops. The trial was also supported by the Indian Council of Medical Research (File No: 5/7/1693/ CH/Adhoc/RBMCH-2020). early child development, integrated intervention, nutrition program, responsive parenting, rural India Frontiers in Public Health early child development, integrated intervention, nutrition program, responsive parenting, rural India COPYRIGHT COPYRIGHT © 2023 Gaidhane, Khatib, Telrandhe, Patil, Kogade, Gaidhane, Choudhari, Holding, Saxena and Syed. 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. Background: To promote early childhood development (ECD), we require information not only on what needs to be addressed and on what effects can be achieved but also on effective delivery methods that can be adapted to local context. We describe design, implementation, and evaluation of a complex intervention to strengthen nurturing environment for young children. Methods: Study participants were pregnant women and their children from birth to 2 years. We used design and redesign, implementation, and evaluation approaches for the study. We co-created curriculum and delivery plan with stakeholders, based on the theoretical framework, findings from formative research, and our preliminary work. We recruited 656 pregnant women and newborns, 326 (49.69%) from intervention and 330 (50.30%) from the control group. We conducted a cluster randomized controlled trial to evaluate the program’s effectiveness. The outcomes of children were assessed at 12 and 24 months. Findings: At recruitment, study participants from both the study arms were similar in sociodemographic characteristics. We conducted 6,665 home visits, 25 toy-making workshops, and 65 caregiver-meetings. The initial examination of program data revealed gaps in quality and coverage of interventions. The intervention was redesigned based on feedback from stakeholders in community meetings. At recruitment, participants in both study groups had similar socio-demographics. We conducted 6,665 home visits, 25 toy workshops, and 65 caregiver meetings. Initial program data showed intervention quality and coverage gaps, leading to a redesign program based Frontiers in Public Health 01 frontiersin.org 10.3389/fpubh.2023.1165728 on community and stakeholder feedback. Post-re-designing, session quality improved, with program coverage rising from 32 to 98%. Male participation in home visits increased from 4.3 to 32.65%, and data errors reduced from 270 to 140 per month on average. At 24 months, program showed moderate–mild impact on ECD – cognitive (0.31, 95%CI: 0.13–0.48), language (0.2, 95%CI: 0.01–0.39), and socioemotional-development (0.19, 95%CI: 0.01–0.37), moderate effect on home-environment and mother–child interaction. The context and the target population The study area was a hard-to-reach rural setting, remotely located in two Blocks of the Wardha and the Nagpur districts in central India. The study villages were in the Forest Buffer Zone, a Tiger Sanctuary. People in the study area had an average annual per-capita income below the state average and worked as unskilled daily wage laborers in forests, farms, or cattle rearing. The traditional socio-cultural customs greatly influence childcare practices in these regions. Availability and accessibility of education, health, and social services for people from these villages are challenging, and access worsens during the rains and summer. Women in these villages are overburdened; as they are traditionally responsible for childcare, they work for income and face gender- specific risks and vulnerabilities. Wages for women are lower than for men. This social and economic distress contributes to challenges to adequate nutrition and caregiving. From design to implementation to evaluation Design & Re- Design Implementaon Evaluaon Intervention design and development From design to implementation to evaluation Randomization and masking programs is well established; however, a critical design issue for such complex interventions is adequately addressing the sociocultural context and current childcare practices (17). Our proposed family- centered, locally developed intervention aimed to enhance the ICDS services targeted at the 0–2 years of age. The theory of change assumes that the additional components of this study shall enhance responsive parenting competencies and improve children’s developmental trajectory (18, 19).hf The unit of randomization were sub centers. We randomly selected four PHCs comprising 21 sub-centers (clusters) and 106 villages from the study area. The 21 subcenters were randomly allocated using a random number sequence in the intervention and the control group. The random allocation of clusters were masked for the study team. The intervention group had 11 sub-centers (clusters) comprising 58 villages, and the control had ten sub-centers (clusters), including 48 villages. The intervention clusters received study intervention in addition to the routine ICDS services, while the participants from the control clusters received the routine ICDS services as part of the study (Figure 1). This study aims to determine the effectiveness of the integrated responsive parenting and nutrition program on child development outcomes in children under 2 years from rural India. The study also reflects upon the cycle of design, implementation, and evaluation using the lens of the Measurement for Change (10, 20) to develop an insight into the path to generating sustainability at scale. Introduction To take these complex interventions to scale requires a commitment of resources, often scarce and constantly competing with other demands. To achieve sustainability at scale, detailed evidence is necessary that convinces parents, service providers, policymakers, and the political system of the feasibility and value of the intervention in context, and thus to take on these costs (12–16). In this paper, we describe the design, implementation, and evaluation of a complex intervention to strengthen the nurturing environment for young children. The evidence for ECD intervention 02 frontiersin.org Gaidhane et al. 10.3389/fpubh.2023.1165728 Methodology The participants were pregnant women and their children from birth to 2 years residing in selected villages. We recruited pregnant women in the first or second trimester of pregnancy, permanently residing in study areas after informed written consent. High-risk pregnancies have the potential to introduce confounding factors into the study, making it challenging to determine the true impact of the intervention. By excluding high-risk cases, we aimed to enhance the quality and reliability of the data, thereby reducing the likelihood of bias that could affect the child outcome assessment. Additionally, this exclusion not only prioritized the safety of high-risk pregnant women by referring them to specialized healthcare facilities but also enhanced the generalizability of our findings, increasing their relevance to a wider range of expectant mothers. We recruited the participants over a period of 6 months, during which we provided counseling to pregnant women and their families, explaining the nature and purpose of the study. Frontiers in Public Health Study design Design & Re- Design Evaluaon We evaluated an integrated community-based intervention using a Cluster-Randomized Control Trial (C-RCT) design. We ensured all cluster members received similar interventions. Administratively, districts are subdivided into talukas or blocks in India. One taluka has around five Primary Health Centers (PHCs), and PHCs comprise five to six sub-centers (SCs). Sub-center caters to nearly five to six villages with a population of around 5,000. Each village has an Anganwadi center (AWC) catering to a population of 1,000 in a rural area. Intervention design and development We conducted formative research and collected data on the household, community, and environmental factors related to caregiving practices that influence the growth and development of children under 3 years through three group discussions at village levels and six interviews of Anganwadi workers, caregivers and other stakeholders. The formative research also explored the underlying beliefs and attitudes around childcare practices, nutrition practices, breastfeeding and complementary feeding practices, household decision-making, income levels, cultural traditions, and social norms around childcare. Additionally, the study team conducted three We selected two adjacent blocks in central India, the Seloo and the Hingna blocks. The study team has strong community linkages in these areas, so delivering intervention and data collection in selected blocks was convenient. Since the community volunteers administered the intervention at the village level (Anganwadi center area) and the villages are situated very close to each other, we defined the sub-center as a unit of randomization to minimize the risk of contamination across the intervention and control groups. We used a stratified randomization approach. 03 frontiersin.org Gaidhane et al. 10.3389/fpubh.2023.1165728 FIGURE 1 Consort flow chart. The development of a responsive parenting package drew its theoretical framework from Vygotsky’s Zone of Proximal Development (ZPD). The ZPD is a theoretical framework that focuses on the difference between a learner’s actual developmental level and their potential developmental level when provided with appropriate guidance and support. This concept involves nurturing independent actions, skills, and knowledge by offering the necessary scaffolding (11–13). Based on the theoretical framework, the findings from formative research, and our preliminary work (17, 21), we identified six areas of parental competencies, namely (1) shelter and nurturing care, (2) food and nutrition, (3) protection and discipline, (4) socio- emotional learning, (5) health, and (6) early childhood education. community meetings to understand the sociocultural and childcare practices and specific challenges to access health and childcare services. Frontiers in Public Health Community group meetings We conducted monthly group meetings to create an enabling environment for ECD at the village level by intentionally building partnerships to support this process. The research assistants and Balsakhi coordinated these community meetings, inviting all stakeholders: Anganwadi workers, caregivers/parents, panchayat leaders, schoolteachers, volunteers, and the research team. We used a collaborative learning and sharing approach by inviting caregivers and parents to share their experiences around ECD and asking the larger group to reflect on those experiences. Based on the data, the stakeholders discussed the implementation progress and formulated a plan to improve intervention delivery and coverage. A total of 58 sessions were conducted in all the intervention villages. Integrated MEL Each child received the program for 24 months. To ensure the coverage and fidelity of the intervention, we analyzed intervention data weekly and tracked information on utilization and engagement, as well as the quality of interactions. Data on attendance, meeting schedules, and using the intelligent register application to give personalized feedback to parents were collected on a PC tablet application. Program supervisors used this application to monitor activity, give feedback and initiate discussions with the delivery team to improve the quality and coverage of the intervention (14). The delivery team We created one demonstration center in each PHC area. The caregivers/mothers were invited to this demonstration center monthly for interaction and discussions on creating recipes for pregnant mothers and young children. In addition to these activities, we created a nutrition garden in households, wherever at least 200 square feet of space was available. Eighteen kitchen gardens were set up in the intervention villages. The Project team provided the seeds and saplings, whereas the household members sowed the seeds and maintained the nutrition garden. More preference was given to foods that were locally consumed. A ‘Balsakhi’ community volunteer delivered the intervention. Balsakhi is a local Marathi word, and it means ‘child’s friend.’ Balsakhi were women from the village, preferably married, with some education (able to read and write in the local language), and willing to volunteer for 2–3 h daily. We selected one Balsakhi for each study village by conducting interviews and administering written examinations to evaluate basic skills and trained them.h The initial training spanned 3 days, certifying participants for conducting home visits, group sessions, and community workshops. During the Project’s initial phase, the Project Research Associates accompanied each Balsakhi to provide initial support. The Balsakhi received refresher training on a one-to-one basis whenever required. The Balsakhi were mentored and supported by Anganwadi workers from the same village to deliver the responsive parenting intervention in the respective villages. Competencies of Balsakhis are assessed through regular supervision, observations of their interactions with children and parents, and periodic evaluations to gauge their knowledge and performance in providing childcare and early education services. Each Balsakhi received a monthly honorarium for their services. Community workshops We organize a community workshop to support caregivers in preparing ECD play materials using accessible materials in alternate months. The workshops also demonstrated how to use the toys/play material, what development domain they stimulate, and what to observe while using the play material. The emphasis focused on health and safety issues when using the play materials. We provided the caregivers with a booklet and activity cards to guide them in toy making and play activities. Study design In these meetings, the study team informed the community about the proposed Project and sought their consent for intervention. We engaged the community at all levels including intervention design, implementation, and program monitoring. During these community meetings, through a collaborative approach, community members collectively selected Balsakhi- a community volunteer from the village to implement the intervention. The team also collected data on nutrition practices, including breastfeeding and complementary feeding practices, household decision-making, income levels, and cultural traditions and social norms around childcare. 04 frontiersin.org 10.3389/fpubh.2023.1165728 Gaidhane et al. Gaidhane et al. The study team co-created a curriculum comprising locally/ culturally appropriate play-based activities covering these six groups’ identified parental competencies. Community members, Anganwadi workers and supervisors, schoolteachers/early childhood educators, and other stakeholders participated in the curriculum development process. The enhanced nutrition program included a nutrition demonstration center and locally and culturally relevant recipes for pregnant women and infant and young child complementary feeding. The program linked the study participants with local Anganwadi centers to provide them with supplementary nutrition support. The program also integrated monitoring, evaluation, and learning (MEL) processes to track progress, direct adaptation for improved efficiency, and evaluate impact. Thus, the intervention package comprised age-appropriate content, curriculum, and necessary tools and materials. We designed tools to enable parents to use them with their children to promote their children’s cognitive, language, socioemotional, and physical development at 2 years of age. Balsakhis with guidebooks, manuals, posters, flyers, flip charts, books, and toys to support session delivery. In addition to the material provided to Balsakhi, she was encouraged to use everyday household items in sessions. The trained and motivated Balsakhi to avoid being directive, adapt to the family’s needs, and keep the family actively engaged in developing their knowledge and skills as responsive parents. Implementation The intervention delivery approach was community-driven, as community-based programs are an essential service delivery approach for early childhood intervention in under-resourced and developing contexts. Community-based implementation provides scope for identifying and analyzing specific community issues and prioritizing, designing, and managing activities at the local level (22). Our delivery approach focuses on interactive discussions adapted to the family’s needs and the appropriate use of tools. Delivery was also intended to enable a Cluster Randomized Controlled Trial (cRCT) that assessed effectiveness and feasibility. Frontiers in Public Health frontiersin.org Complementing the government’s ICDS program The focus was on the early initiation of breastfeeding, exclusive breastfeeding, and the appropriate introduction of complementary feeding. The Balsakhi through 44 home visits delivered the intervention over 24 months. We trained the Balsakhi to deliver the session interactively, using activities and reflective practice. We equipped the Every participant in the study was enrolled in the standard services offered by Anganwadi centers, where children under six Frontiers in Public Health 05 frontiersin.org Gaidhane et al. 10.3389/fpubh.2023.1165728 data team developed an XLS file for all devices and then imported those files to Open Data Kit (ODK). Data collection tools were then imported into an Android Tablet-PC. The app had in-built quality checks that monitored score distributions and missing values/data. Using a tablet PC for data collection, we trained evaluators in the ODK process. data team developed an XLS file for all devices and then imported those files to Open Data Kit (ODK). Data collection tools were then imported into an Android Tablet-PC. The app had in-built quality checks that monitored score distributions and missing values/data. Using a tablet PC for data collection, we trained evaluators in the ODK process. receive non-formal education, nutrition supplementation, and growth monitoring. Furthermore, the intervention group benefited from responsive parenting and enhanced nutrition initiatives delivered through home visits, group sessions, and community workshops. This intervention strategy synergizes with the existing components of the ICDS program, introducing an adaptable community-focused, responsive parenting curriculum for children under 2 years of age. The ICDS program’s Anganwadi Workers played a supportive role, guiding the Balsakhi and aiding in addressing challenges related to intervention delivery. At recruitment, we captured the household information and mothers’ maternal characteristics using the Demographic Health Survey tool of the Government of India. At 12 months and 24 months, we assessed all child development outcomes (primary outcomes) – Physical, cognitive, language, and socioemotional development. We also evaluated mother–child interaction and home environment at 12 and 18 months of interventions. Analysis We used STATA version 14 for analysis. We compared household and sociodemographic data from the intervention and the control group to ensure the robustness of the randomization process and to examine the characteristics of participants lost to follow-up. We used the intention-to-treat analysis to compare the child outcomes between the intervention and control groups at 12 months and 24 months by a mixed effect regression model adjusted for cluster and assessors and controlled for potential confounders (mothers’ education, child sex, wealth index, total family members). The mean child development scores for all domains are presented with a 95% confidence interval, considering p < 0.05 for the statistical significance. We also estimated the intervention’s effect size at 12 and 24 months as ‘Cohens d’, as the difference in the adjusted mean between the intervention and the control group divided by the pool SD. The field staff who administered assessment tools received training and certification (17). The outcome assessors were masked to the intervention. Assessors also worked independently with the community volunteer and Anganwadi workers who delivered the intervention. To reduce familiarity with households and caregivers, the research team randomly rotated the assessor team across clusters. The assessors were instructed to refrain from inquiring about the families’ intervention status. Additionally, inter-rater reliability testing was conducted to ensure data quality and consistency. The reliability coefficient for the Development Milestone Checklist (DMC) scale, Observation of Mother–Child Interaction (OMCI) tool, Profile of Socio-emotional Development (PSED), and Home Scale Coding (HSC) was 0.875, 0.691, 0.673 and 0.759, respectively. The Sample size At recruitment, the study team collected household sociodemographic data and antenatal characteristics of pregnant women using the Government of India Demographic Health Survey (DHS) tool (9). Field staff received training and certification in completing DHS forms at recruitment and baseline data collection. We administered a battery of tools to assess child development outcomes at 12 months and 24 months of age. We chose assessment tools previously used in low-middle-income country settings (27). The study team adapted tools to the local context, translated them from English to Marathi (the local language), and back-translated them into English. Language experts validated the translated tools. We aimed to detect differences of 0.3SD between the intervention and the control groups. The preliminary data from the study area calculated the child development score of 67 in the cognitive domain. We assume the Intra-cluster Correlation Coefficient (ICC) of child development as 0.02, with the average number of pregnancies per cluster per year as 30, resulting in the design effect 1.58. Hence, to detect the desired improvement of 0.3SD in development score in the intervention group, with 95% confidence and 80% power, a total sample size of 452 mother–child dyads. Based on the previous experience, we accounted for a 20% loss to follow-up. Thus, the final sample size is 542 from 21 clusters, 271 in each group. However, from an ethical perspective, we enrolled all eligible participants fulfilling the inclusion criteria from the intervention and the control clusters in the study. Developmental Milestones Checklist (DMC) is a reliable and sensitive tool for evaluating motor, language, and personal-social development (23, 24). We considered the gross score of DMC tool for the cognitive development of children under two years. Profile of Socio-Emotional Development (PSED) assesses children’s social and emotional development through observation and parental reports (25). PSED tool was adapted to the local context and incorporated culturally and socially relevant items. The adapted home environment was assessed at baseline and the endpoint using the Infant-Toddler Home Inventory (26–28). The quality of mother–child interaction was evaluated using the Observation of the Mother–Child Interaction (OMCI) (29), and parent behaviors, parental knowledge, and skills for ECD were assessed using the Photostory approach and a parental quiz (17, 21). We used standard established protocols of WHO for anthropometric assessments (30). Frontiers in Public Health Registration We registered the trial with clinical trial registry of India under the CTRI Number: CTRI/2017/05/008553 on 15/05/2017. The Institutional Ethics Committee of Datta Meghe Institute of Medical Sciences (Deemed to be University) approved the trial vide letter with Ref no: DMIMS (DU)/IEC/2017–18/6306 dated 27.03.2017. Frontiers in Public Health 06 frontiersin.org Gaidhane et al. 10.3389/fpubh.2023.1165728 TABLE 1 Baseline characteristics of study participants in the intervention and the control arm. TABLE 1 Baseline characteristics of study participants in the intervention and the control arm. Data are No (%); unless stated otherwise. Project staff monthly meetings We held monthly meetings of Balsakhi (service providers) at each PHC. We conducted 65 Balsakhi meetings to share experiences and reflect on their learnings and challenges. The field supervisors presented the monthly coverage data to the stakeholders and discussed coverage gaps and opportunities to reach out to those who missed out in the previous months. The refresher training sessions were conducted during these meetings, whenever needed, to enhance the skills and competencies of the Balsakhi for conducting home visits. During these meetings, the collaborative re-design process associated with improved delivery indicators was agreed upon. The community and Project staff meetings helped improve the quality and coverage of the intervention. Another paper presents the data on improving the service delivery indicators (16). Table 2 compares the baseline characteristics of study participants lost to follow-up and those who completed the intervention. Sociodemographic characteristics, other than maternal education, were comparable between those who lost to follow-up and those who completed the intervention. Community workshops We assessed 824 participants for eligibility and recruited 656 (79.61%) eligible women in their second trimester of pregnancy and their newborns. The intervention group had 326 (49.69%), and the control group had 330 (50.30%) participants at the enrolment. At 24 months, the study endpoint, 68 (10.36%) participants lost to follow-up. In the intervention group, we conducted 25 toy workshops at the village level to support and train caregivers to prepare low-cost ECD play material from household items. The 295 participants trained to prepare and use a low-cost play material. Four demonstration centers were set up in each PHC area to demonstrate pregnant women and caregivers to prepare locally/socio-culturally acceptable recipes that meet mothers’ and child’s nutrition needs. A recipe book was prepared for complementary feeding and shared with the caregivers. 291 women/caregivers attended sessions in the demonstration center at least once. Table 1 provides characteristics of study participants from the intervention and the control arm at enrolment. Study participants’ sociodemographic characteristics were comparable between the two groups, except for the caste category. Most pregnant women were in the second trimester of pregnancy at enrolment. Maternal education, maternal age, stage of pregnancy, and pregnancy order were similar between the intervention and the control group. Household characteristics, wealth index, and poverty status were comparable in the intervention and control groups. The average wealth index score of participants from the intervention group (0.19, 95%CI 0.04–0.43) and the control group (0.18, 95%CI 0.07– 0.45) was comparable (p = 0.965). At the endpoint, the mean wealth score was comparable to that at recruitment. Thus, the socio-economic status of the families was almost identical throughout the study period. Out of 656 newborns, 49.84% were boys, and 50.16% were girls. Registration Total (n = 656) Intervention (n = 326) Control (n = 330) p value Maternal characteristics Age in years; Mean (SD) 23.94 (3.61) 23.79 (3.57) 24.08 (3.64) 0.288 Education Illiterate 18 (2.74%) 11(3.37%) 7 (2.12%) chi2 = 6.06 p = 0.195 Primary (1–5) 24 (3.66%) 12 (3.68%) 12 (3.64%) Secondary (6–10) 295 (44.97%) 156 (47.85%) 139 (42.12%) Higher Secondary 203 (30.95%) 100 (30.67%) 103 (31.21%) Graduate and more 116 (17.68 5) 47 (14.42%) 69 (20.91%) Pregnancy duration 1st Trimester 132 (20.12%) 68 (20.86%) 64 (19.39%) chi2 = 0.21 p = 0.64 2nd Trimester 524 (79.88%) 258 (79.14%) 266 (80.61%) Gravida First 182 (42.99%) 150 (46.01%) 132 (40%) chi2 = 5.68 p = 0.224 Second 294 (44.82%) 145 (44.48%) 149 (45.15%) Third 64 (9.76%) 26 (7.98%) 38 (11.52%) Fourth 13 (1.98%) 4 (1.23%) 9 (2.73%) Fifth 3 (0.46%) 1 (0.31%) 2 (0.61%) Total of live children; Mean (SD) 0.59 (0.65) 0.61 (0.68) 0.58 (0.65) p = 0.576 Anaemia No Anaemia 173 (30.40%) 82 (29.82%) 91 (30.95%) chi2 = 2.43 p = 0.487 Mild Anaemia 203 (35.68%) 96 (34.91%) 107 (36.39%) Moderate Anaemia 191 (33.57%) 95 (34.55%) 96 (32.65%) Severe Anaemia 2 (0.35%) 2 (0.73%) 0 Father’s characteristics Age in years; Mean (SD) 29.99 (4.13) 29.59 (3.63) 30.4 (4.54) p = 0.012 Education Illiterate 21 (3.20%) 13 (3.99%) 8 (2.42%) chi2 = 4.67 p = 0.322 Primary (1–5) 47 (7.16%) 23 (7.06%) 24 (7.27%) Secondary (6–10) 337 (51.37%) 175 (53.68%) 162 (49.09%) Higher Secondary 170 (25.91%) 82 (25.15%) 88 (26.67%) Graduate 81 (12.35%) 33 (10.12%) 48 (14.55%) Household characteristics Caste category Schedule Caste 52 (8.84%) 24 (8.11%) 28 (9.59%) chi2 = 14.59 p = 0.002 Schedule Tribe 235 (39.97%) 141 (47.64%) 94 (32.19%) Backward classes 279 (47.45%) 123 (41.56%) 156 (53.42%) Open/General 22 (3.74%) 8 (2.70%) 14 (4.79%) Wealth index 1st Quintile 110 (16.77%) 49 (15.03%) 61 (18.48%) chi2 = 7.809 p = 0.099 2nd Quintile 122 (18.60%) 58 (17.79%) 64 (19.39%) 3rd Quintile 143 (21.80%) 83 (25.46%) 60 (18.18%) 4th Quintile 142 (21.65%) 75 (23.01%) 67 (20.30%) 5th Quintile 139 (21.19%) 61 (18.71%) 78 (23.64%) Average family size; mean (SD) 4.66 (1.84) 4.86 (1.91) 4.47 (1.76) p = 0.006 Below poverty line 286 (43.66%) 145 (44.62%) 141 (42.73%) p = 0.626 Data are No (%); unless stated otherwise. 07 Frontiers in Public Health frontiersin.org 10.3389/fpubh.2023.1165728 Gaidhane et al. Frontiers in Public Health Home visits We conducted 6,665 home visits throughout the intervention period. The preliminary review of first-quarter intervention data revealed that home visits were directive, 139 (23%) were family- centered. In 360 (69.62%) home visits, the average duration was less than expected, and 68 (11%) took more than 60 min. Despite rigorous training and certification, monitoring data and interactions in monthly meetings with the Balsakhi revealed low motivation and confidence to deliver interventions. Based on the findings and feedback, we re-designed the intervention delivery approach and introduced the community supervisors to retrain, handhold, and mentor the Balsakhi. We randomly supervised 1,670 (25%) home visits to ensure quality. Community meetings We conducted 450 community meetings in the intervention villages over 24 months. Balsakhi (service provider) and outreach staff coordinated these meetings. Anganwadi workers, community members, mothers, and other care providers attended these meetings. These meetings allowed participants to reflect on their learnings, gaps, or challenges in service delivery and potential solutions. Results (35.37%) households received less than 75% of planned home visits (17). Data errors reduced from 270 to 140 per month on average. Child and mother outcomesh The average weight gain for women from the intervention group was 9.01 (SD 3.74) kilogram weight, which was significantly more than the weight gain in women from the control group (7.67, SD 3.43) during pregnancy (p < 0.001). The intervention arm had a lower proportion of low birth weight newborns 68 (20.86%) than the control arm 88 (26.67%), but this difference was not statistically significant. However, the birth weight was significantly higher in the intervention arm 2.71 (SD 0.44) than in the control arm 2.61 (SD 0.45). Ninety-six percent of women started breastfeeding within 1 h of delivery and the exclusive breastfeeding rate was 89.80%. In the subsequent quarter, the indicator improved. Out of the total of 1,670 home visits supervised, 1,169 (70%) had interactive discussions, 1,458 (87.30%) used tools and other materials effectively during the session delivery, and in 295 (17.66%) home visits, male members from the household participated in the discussion and participation of male members increased from 4.3 to 32.65%. The coverage of the home visits also increased from 31.93% in the first quarter of intervention to 98.6% in the 6th quarter. Out of 294 households from the intervention group, 190 (64.63%) families received more than 75% of home visits, and 104 Table 3 shows the statistically significant effect of the intervention on weight for height (WHZ) in children at 24 months. The effect was comparable for all other anthropometric indicators between the intervention and the control arm. We observed a small effect on cognitive, language, motor, and socio-emotional development at 12 months. A difference in the mean child development outcome scores at 12 months between the intervention and the control group was not statistically significant 08 frontiersin.org Gaidhane et al. 10.3389/fpubh.2023.1165728 TABLE 2 Characteristics of study participants who lost to follow-up during the intervention at 24 months. TABLE 2 Characteristics of study participants who lost to follow-up during the intervention at 24 months. Data are No (%); unless stated otherwise. Frontiers in Public Health Child and mother outcomesh Total (N = 656) Retained (n = 588) Loss to follow-up (n = 68) p value Maternal characteristics Age in years; Mean (SD) 23.94 (3.61) 24.02 (3.64%) 23.20 (3.23%) 0.076 Education Illiterate 18 (2.74%) 13 (2.21%) 5 (7.35%) chi2 = 9.694 p = 0.046 Primary (1–5) 24 (3.66%) 20 (3.40%) 4 (5.88%) Secondary (6–10) 295 (44.97%) 261 (44.39%) 34 (50.00%) Higher Secondary 203 (30.95%) 186 (31.63%) 17 (25.00%) Graduate and more 116 (17.68%) 108 (18.37%) 8 (11.76%) Pregnancy duration 1st Trimester 132 (20.12%) 111 (18.88%) 21 (30.88%) chi2 = 5.465 p = 0.019 2nd Trimester 524 (79.88%) 477 (81.12%) 47 (69.12%) Gravida First 132 (40%) 112 (38.36%) 20 (52.63%) chi2 = 6.937 p = 0.139 Second 149 (45.15%) 135 (46.23%) 14 (36.84%) Third 38 (11.52%) 35 (11.99%) 3 (7.89%) Fourth 9 (2.73%) 9 (3.08%) 0 (0.00%) Fifth 2 (0.61%) 1 (0.34%) 1(2.63%) Anaemia No Anaemia 173 (30.40%) 159 (30.93%) 14 (25.45%) chi2 = 6.858 p = 0.077 Mild Anaemia 203 (35.68%) 189 (36.77%) 14 (25.45%) Moderate Anaemia 191 (33.57%) 164 (31.91%) 27 (49.09%) Severe Anaemia 2 (0.35%) 2 (0.39%) 0 (0%) Father’s characteristics Age in years; Mean (SD) 29.99 (4.13) 30.06 (3.96) 29.44 (5.36) p = 0.241 Education Illiterate 21 (3.20%) 17 (2.89%) 4 (5.88%) chi2 = 2.382 p = 0.666 Primary (1–5) 47 (7.16%) 41 (6.97%) 6 (8.82%) Secondary (6–10) 337 (51.37%) 304 (51.70%) 33 (48.53%) Higher secondary 170 (25.91%) 152 (25.85%) 18 (26.47%) Graduate 81 (12.35%) 74 (12.59%) 7 (10.29%) Household characteristics Caste category Schedule caste 61 (9.30%) 52 (8.84%) 9 (13.24%) chi2 = 4.806 p = 0.187 Schedule Tribe 264 (40.24%) 235 (39.97%) 29 (42.65%) Backward classes 304 (46.34%) 279 (47.45%) 25 (36.76%) Open/General 27 (4.12%) 22 (3.74%) 5 (7.35%) Wealth index 1st Quintile 110 (16.77%) 94 (15.99%) 16 (23.53%) chi2 = 6.257 p = 0.181 2nd Quintile 122 (18.60%) 113 (19.22%) 9 (13.24%) 3rd Quintile 143 (21.80%) 126 (21.43%) 17 (25%) 4th Quintile 142 (21.65%) 125 (21.26%) 17 (25%) 5th Quintile 139 (21.19%) 130 (22.11%) 9 (13.24%) Average family size; mean (SD) 4.66 (1.84) 4.47 (1.87) 4.68 (1.84) p = 0.360 Below poverty line 286 (43.66%) 255 (43.44%) 31 (45.59%) chi2 = 0.114 p = 0.735 Data are No (%); unless stated otherwise. 09 Frontiers in Public Health frontiersin.org 10.3389/fpubh.2023.1165728 Gaidhane et al. TABLE 3 Comparison of anthropometric indicators in the intervention and the control group. frontiersin.org Child and mother outcomesh TABLE 3 Comparison of anthropometric indicators in the intervention and the control group. Developmental domain Intervention arm Control arm p value n = 303 at 12 months n = 296 at 24 months n = 293 at 12 months n = 292 at 24 months WAZ 12 months −1.51 (1.12; −1.63. −1.38) −1.57 (1.03; −1.69 −1.45) 0.493 24 months −1.73 (1.01; −1.85 −1.62) −1.78 (0.92; −1.89 −1.67) 0.515 HAZ 12 months −0.94 (1.28; −1.08. −0.79) −0.97 (1.33; −1.12 −0.82) 0.778 24 months −1.37 (1.09; −1.49 −1.24) −1.21 (1.07; −1.34 −1.09) 0.085 WHZ 12 months −1.36 (1.28; −1.51. −1.21) −1.47 (1.2–1.61 −1.33) 0.286 24 months −1.47 (1.13; −1.60 −1.34) −1.66 (1.07; −1.78 −1.54) 0.033 Data are mean (SD; 95%CI). The analysis is adjusted for the cluster & assessors and controlled for covariates (maternal age, maternal education, wealth index, poverty status, caste category, f son of child development outcome, mother–child interaction and home environment scores in the intervention and the control TABLE 4 Comparison of child development outcome, mother–child interaction and home environment scores in the intervention and the control group. Developmental domain Intervention arm Control arm Value of p n = 303 at 12 months n = 296 at 24 months n = 293 at 12 months n = 292 at 24 months Cognitive 12 months 56.91 (8.84; 55.91–57.91) 56.29 (10.1; 55.13–57.44) 0.239 24 months 70.18 (8.71; 69.18–71.18) 67.65 (9.70; 66.53–68.77) 0.001 Language 12 months 9.03 (3.10; 8.68–9.38) 8.84 (3.38; 8.45–9.23) 0.117 24 months 18.25 (4.86; 17.70–18.81) 17.41 (5.35; 16.79–18.03) 0.002 Motor 12 months 38.36 (6.63; 37.61–39.11) 38.34 (7.32;37.49–39.18) 0.042 24 months 53.36 (5.21; 52.76–53.96) 51.97 (6.01; 51.28–52.67) 0.046 Socioemotional 12 months 20.29 (6.54; 19.55–21.03) 20.37 (6.98; 19.56–21.17) 0.443 24 months 21.47 (4.86; 20.88–22.07) 20.51 (5.62; 19.86–21.16) 0.031 Home inventory 12 months 37.06 (4.61; 36.54–37.58) 35.65 (5.05; 35.06–36.23) 0.035 24 months 36.58 (5.21; 36.08–37.06) 35.61 (4.65; 35.07–36.14) 0.008 Mother–child interaction 12 months 36.01 (7.34; 35.17–36.83) 34.54 (7.63; 33.66–35.42) 0.014 24 months 40.37 (5.42; 39.75–40.99) 38.2 (6.01; 37.50–38.89) <0.001 Data are mean (SD; 95%CI). The analysis is adjusted for the cluster & accessors and controlled for covariates (maternal age, maternal education, wealth index, poverty status, caste category, gravida and maternal anaemia) by a mixed effect model. Data are mean (SD; 95%CI). Child and mother outcomesh The analysis is adjusted for the cluster & accessors and controlled for covariates (maternal age, maternal education, wealth index, poverty status, caste category, gravida and maternal anaemia) by a mixed effect model. (p > 0.05). The effect sizes increased for cognitive (Cohens d = 0.31; 95% CI: 0.13–0.48), language (Cohens d = 0.2; 95% CI: 0.01 0.39), motor (Cohens d = 0.27; 95% CI: 0.11–0.43); socioemotional development (Cohens d = 0.19; 95% CI: 0.01 0.37) at 24 months compared to 12 months of intervention. The intervention had a statistically significant effect on child development outcomes between the intervention and the control arm at 24 months (p < 0.05; Table 4). The intervention statistically impacted the home environment and mother–child interaction at 12 months (Figure 2). We observed moderate to mild but statistically significant effects on the home environment at 24 months (0.3, 95% CI: 0.05–0.43). The effect of the intervention was maximum for mother–child interaction at 24 months (0.4, 95% CI, 0.22–0.58; Figure 2). The number of children with improved scores in the intervention group at 24 months was more compared to 12 months for the cognitive Table 4). 10 frontiersin.org frontiersin.org Gaidhane et al. 10.3389/fpubh.2023.1165728 FIGURE 2 Effect size (Cohens d) with 95%CI of intervention on child development domains at 12 and 24 months. FIGURE 2 Effect size (Cohens d) with 95%CI of intervention on child development domains at 12 and 24 months. We co-created and implemented a responsive parenting and nutrition program delivered through community networks that included Government Anganwadi Centers for 24 months. The findings emphasized that community engagement, a theory-driven conceptual framework, and formative research are needed to design and implement complex interventions effectively. A paper by Bentley and colleagues also stressed a need to contextualize a program through an inclusive process and sustained stakeholder engagement to improve the quality of delivery (32). domain (181, 61.15% versus 157, 51.82%, p = 0.021) and motor domain (224, 75.68% versus 139, 45.87%, p < 0.001). However, the difference was not statistically significant for language and socioemotional domains. The lowest wealth quantile shows the maximum and statistically significant impact of the intervention on cognitive development (Cohens d = 0.92; 95% CI = 0.53–1.30), motor development (Cohens d = 0.72; 95% CI = 0.29–1.14); language development (Cohens d = 0.79; 95% CI = 0.43–1.16). The intervention had a lower effect on other wealth quantiles. Child and mother outcomesh Study participants below the poverty line as per the Indian Government’s categorization had a statistically significant minimal to moderate effect size on cognitive (Cohens d = 0.43; 95%CI = 0.19–0.67), motor (Cohens d = 0.19; 95% CI = 0.03–0.42), language (Cohens d = 0.25; 95% CI = 0.02–0.49) and socio-emotional development (Cohens d = 0.36; 95% CI = 0.10–0.62) (Supplementary Table S1). Our program’s key highlight was sustained community engagement. A ‘Balsakhi,’ meaning the friend of a child in the local language, delivered the entire intervention. Despite the well- conceptualized intervention, which draws upon the community’s strengths and is contextually appropriate, we faced challenges in intervention delivery by the community volunteers in the initial stages. Quality of service delivery was a primary concern, and data revealed coverage gaps, including community volunteers’ low motivation and engagement. Thus, we redesigned the implementation approach to motivate and engage service providers to deliver the intervention with fidelity. We appointed a community supervisor for handholding and mentoring community volunteers. Supervisors accompanied the community volunteers in at least 25% of the home visits. The implementation data revealed that the coverage improved over time and significantly improved the quality of intervention delivery. We presented the data in a separate paper, (16). We adopted a data-driven approach to improve coverage and quality of intervention delivery, and changes in practice developed out of the evidence shared across the network of stakeholders. Thus, Frontiers in Public Health Discussion Despite the strong evidence from neurosciences and economics regarding the benefit of the intervention in the early years, most parenting interventions for cognitive and behavioral development are targeted at older children, at preschoolers (42–46). Our study recruited pregnant women, and the intervention continued till 24 months of the child’s age, providing evidence of impact and engagement at the foundational stages of growth. Another strength of the study is an integrated intervention delivery through community-led channels. In resource-constrained settings, such an approach may be cost- effective. In addition to program-level advantage, available evidence suggests no significant loss in effect size when intervention is delivered in community settings through community volunteers (12). Our data showed a positive and statistically significant impact on the home environment and mother–child interaction. Mothers from the intervention group showed improved knowledge and skills for responsive parenting. Our study substantiates the findings of a systematic review that parenting interventions improve parenting knowledge, skills, parent–child interactions, and home environment are the critical pathways to bringing positive change in child development (42).hf In addition to the evidence of the combined effect of nutrition and responsive parenting programs on child development, our study has provided program and delivery level guidance to positively influence interventions’ quality, engagement, and sustainability. The needs- based approach we employed guides the rapid re-design of delivery mechanisms, which was associated with acceptance by the community and led to a shift in responsibility and accountability at a local level (22). To further understand effective delivery mechanisms, rather than purely focusing on assessing responsive parenting in the mother, we recommend future studies that consider others in the support network for ECD (47), such as older siblings, grandparents, and other relatives, who can play a more prominent role on ECD in extended family or joint families structures. The intervention had a maximum effect on Cognitive development, followed by language, motor, and socio-emotional development. Our results were comparable to the systematic review by Jeong and colleagues, which included 102 studies from 33 countries, concluding that parenting interventions in the first 3 years of life improve a child’s cognitive, language, motor, and socioemotional development and reduce behavioral problems (42). We observed that these effects on child developmental outcomes increased over time. Discussion Our study also demonstrated that the effectiveness of a responsive parenting program integrated with nutrition intervention significantly affected the development of children under 2 years of age and promoted a conducive home environment and mother-to-child interactions. The optimum growth and development of children under three may break the cycle of inequality and vulnerability and lay the foundation for achieving sustainable development goals (22, 31). 11 frontiersin.org 10.3389/fpubh.2023.1165728 Gaidhane et al. significant benefits for 24 months of child age (34, 43). The integrated nutrition components in our study focused on information and practical ideas. The effective delivery of nutrition messages is essential, but more is needed. Nutrition-specific and sensitive interventions are needed, which include food security issues to improve feeding behaviors, the sufficiency and quality of complementary foods, maternal nutrition (preconception and during pregnancy), and birth spacing (45, 46). These issues were beyond the scope of this study. Another major challenge was that the families found difficulty maintaining vegetable gardens in April and May due to intense heat in the region and water scarcity. Our study suggests these actionable components require further contextualization to embed more firmly into local practices for sustainability and scalability. our study emphasized that engaging the remotest field staff, community members, and other stakeholders in the data review and decision-making process motivates and improves their engagement, creates an ecosystem that improves accountability and efficiency, and empowers everyone involved. If a community volunteer is mentored, supported, and monitored, they can deliver the complex integrated intervention in early childhood with the desired fidelity. i Our program included fortnightly home visits along with monthly group sessions. Our trial’s frequency of contact with caregivers was similar to earlier studies from India (33) and Bangladesh (34, 35). However, it was less than earlier Jamaican trials, which reported weekly play sessions through home visits (36–39). Even though the group-based parenting education programs are practical and potentially cost-effective options (40), we decided to adopt a combination of methods, both the home visits and group sessions, based on the local context and caregivers’ needs. Our approach of home visits to tailor the intervention to caregivers’ specific needs, while group sessions facilitated peer learning through experience sharing, is supported by the evidence. Group sessions enable ECD culture across the community, and the home visit strengthens family processes (40, 41).i Our program has several strengths. Frontiers in Public Health Discussion Assessing the impact of the duration of the intervention on child development outcomes was not the primary objective of this trial; however, our data highlighted that the intervention given for a longer duration, that is 24 months, shows more benefits than an intervention delivered over 12 months. A systematic review published in 2021 reported a lack of evidence on the effect of variable program duration on child development outcomes (38). Further study is needed to separate the influence of age at assessment from that of the duration of the intervention. It would be pertinent to enroll families for variable periods to explore the benefits over time and follow up a long-term cohort to understand how intervention benefits can be sustained beyond 24 months, even up until adolescence. The strength of our innovation is that it aligns with the recommendation of 2013 National Early Childhood Education and Care Policy of the Government of India, enhancing the potential for sustainable scaling. Our innovation was designed to address scalability and replicability, to establish self-sustaining village-level units that serve as models for neighboring communities, and to foster expansion. To ensure the sustainability and expansion of our program, we prioritized community engagement and ownership, engaging local stakeholders, parents, and community leaders in the design and implementation of our intervention. We optimized resource utilization by leveraging locally local assets and investing in capacity building, thereby reducing reliance on external funding. A robust monitoring and evaluation system built around shared accountability, ensures continuous assessment. Regular review of effectiveness and impact of the program enables data-driven adjustments to meet evolving needs. This comprehensive approach ensures our program’s lasting impact and continued expansion of the One of the limitations of our study is that we should have included nutrition supplementation. However, to avoid duplication of services offered at the Anganwadi Centers under the current ICDS program of India, we linked the beneficiaries to the Anganwadi Centers for nutritional supplementation. In previous studies, direct nutrition supplementation within a parenting intervention has shown 12 frontiersin.org Gaidhane et al. 10.3389/fpubh.2023.1165728 with additional support from the Indian Council of Medical Research (File No: 5/7/1693/CH/Adhoc/RBMCH-2020). with additional support from the Indian Council of Medical Research (File No: 5/7/1693/CH/Adhoc/RBMCH-2020). innovation, benefiting a wider population of children, their families and communities. 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. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ethics statement The studies involving humans were approved by Institutional Ethics Committee of Datta Meghe Institute of Medical Sciences (Deemed to be University) approved the trial vide letter with Ref no: DMIMS (DU)/IEC/2017-18/6306 dated 27.03.2017. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Discussion To conclude, our study emphasized the importance of developing a conceptual framework integrating a theoretical model with formative research for designing and redesigning a complex intervention. The study provides an evidence-based, responsive curriculum, with implementation strategies grounded in social learning theory that enhance caregivers’ knowledge and skills for promoting early child development. Due to the pragmatic nature of the study, our intervention also has the potential to integrate within the existing Integrated Child Development Program in India. 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. Author contributions ZQS and AG conceptualized the study and led the design as the primary author, analyzed data, and led the write-up. ST developed the data collection materials with inputs from ZQS, AG, and PH. PH and MNK oversaw the study, data analysis, and interpretation, and drafted the manuscript. PK and MP trained and supervised the data collection team. ST and AG oversaw the quality assurance. MP administrated the project work. SG, PK, PH, DS, and SC assisted with the write-up and participated in the study design, data analysis, and interpretation. All authors contributed to the article and approved the submitted version. All authors critically reviewed drafts of the manuscript. Acknowledgments We thank the Grand Challenges Canada team for generous funding and time-to-time support through online sessions and orientation programs. We are grateful to all the mothers and their families from the 110 villages of Wardha and Nagpur districts of rural India who consented to be interviewed and gave their valuable time. It would not have been possible to conduct this study without them. We thank the Village Authorities and Gram Panchayat members for cooperating in conducting baseline surveys, intervention delivery, and assessments. We also acknowledge the cooperation and support of the Child Development Programme Officers of Wardha and Nagpur districts and the Anganwadi workers. We acknowledge the efforts of Dr. Sylvia Fernandez, Scientist, National Institute of Nutrition, Hyderabad (India), for participation in the Induction Training of the Baseline Survey team and adaptation of different developmental tools to the local context. We are thankful to Frances Glascoe, Professor of Pediatrics (Adjunct), Vanderbilt University, for orientation about and for permitting us to use the PEDS-DM Tool in our context. We thank Muneera A. Rasheed and Aisha K. Yousafzai from Pakistan for sharing and helping us with the Observation of Mother–Child Interactions (OMCI) tool. We also thank all the study staff, the data collection research team, ECD facilitators, and community volunteers. Funding The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpubh.2023.1165728/ full#supplementary-material The trial received funding from the Saving Brains Round 5 Initiative of Grand Challenges Canada (Grant no. SB- 1707-05084), 4. Walker SP, Wachs TD, Gardner JM, Lozoff B, Wasserman GA, Pollitt E, et al. Child development: risk factors for adverse outcomes in developing countries. Lancet. (2007) 369:145–57. doi: 10.1016/S0140-6736(07)60076-2 3. Walker SP, Wachs TD, Grantham-McGregor S, Black MM, Nelson CA, Huffman SL, et al. Inequality in early childhood: risk and protective factors for early child development. Lancet. (2011) 378:1325–38. doi: 10.1016/S0140-6736(11)60555-2 2. Black MM, Walker SP, Fernald LCH, Andersen CT, DiGirolamo AM, Lu C, et al. 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https://openalex.org/W3094385235	https://revistas.pj.gob.pe/revista/index.php/ropj/article/download/109/174	es		La enseñanza por competencias jurídicas	Revista oficial del poder judicial	2,007	cc-by	4,661	LA ENSEÑANZA POR COMPETENCIAS JURÍDICAS H ERNÁN A LEJANDRO OLANO G ARCÍA* Resumen: El autor desea presentar un propuesta de enseñanza por competencias específicas en Derecho dentro del proyecto Tuning América Latina, para lo cual, con las restringidas fuentes de información existentes, desarrolla la historia del proceso y nos presenta además algunas consideraciones sobre la formación por competencias y el proyecto formativo, o programa de Derecho Constitucional colombiano, en el que incluye cuatro de las competencias Tuning europeas para el desarrollo de la ruta formativa de cualquier asignatura jurídica. Palabras clave: Tuning - Competencias - Programa - Derecho - Educación por Competencias Abstract: The author presents a proposal of education by specific competitions in Law within the project Tuning Latin America, to do so, with the restricted existing sources of intelligence, develops the history of the process and presents in addition some considerations to us on the formation by competitions and the formative project, or program of Colombian Constitutional Law, in which includes four of the European Tuning competitions for the development of the formative route of any legal subject. Key words: Tuning - Competence - Program - Law - Education by Competitions. Sumario: 1. La educación por competencias. 2. Objetivos del Proyecto Tuning. 3. Las competencias. 4. Competencias genéricas para América Latina. 5. Competencias específicas latinoamericanas para Derecho. 6. Un ejemplo: las competencias ecuatorianas. 7. Una propuesta específica para Colombia. 8. Bibliografía. * Profesor de la Universidad de La Sabana de Chía Colombia. Revista Oficial del Poder Judicial 1/1 2007 383 Hernán Olano García La enseñanza por competencias jurídicas El tema de competencias, especialmente en procesos de diseño curricular, es una nueva exigencia para que como profesores podamos aplicar en nuestras asignaturas las competencias Tuning para América Latina1 . En los dos años anteriores, profesores de un total de 18 universidades participaron en el desarrollo del proyecto así: Dos de Argentina (Universidad del Museo Social Argentino y Universidad Nacional del Litoral; una de Bolivia la Universidad Autónoma Juan Misael Caracho; de Brasil dos, la Universidad Presbiteriana Mackenzie Sao Paulo y la Universidad de Brasilia; de Chile la Universidad Católica de Temuco; de Colombia, el Externado; del Ecuador la Universidad del Azuay; de El Salvador la Universidad Salvadoreña Alberto Masferrer; en México dos, la Universidad de Colina y la Universidad de Guadalajara; en Nicaragua la Universidad Centroamericana; en Paraguay dos, la Universidad Autónoma de Asunción y la Universidad Católica Nuestra Señora de La Asunción; en Perú la Universidad San Martín de Porres, en Uruguay la Universidad Católica del Uruguay y, en Venezuela la Universidad Católica del Táchira. La Coordinadora General en Derecho es Loussia Penha MUSSE FELIX de la Universidad de Brasilia. A solicitud de un grupo de universidades de América Latina, el Proyecto Tuning tiene aplicación en América Latina y el doctor Juan Morales Ordóñez, de la Universidad Azuay del Ecuador es su representante. Los instrumentos de la metodología de las competencias Tuning y los resultados más específicos a través de la consulta de profesores, estudiantes, egresados y empleadores, servirá para contar con elementos interesantes para la evaluación del currículo actual, sobre lo cual se centrará nuestra propuesta. Alfa Tuning América Latina es un proyecto independiente, impulsado y coordinado por Universidades de distintos países, tanto latinoamericanos como europeos que busca identificar e intercambiar información y de acuerdo con lo propuesto desde Europa, busca mejorar la colaboración entre las instituciones de educación superior para el desarrollo de la calidad, efectividad y la transparencia en la misión de educar, particularmente ahora por competencias. Según la página oficial en castellano2 , el proyecto ALFA Tuning América Latina surge en un contexto de intensa reflexión sobre educación superior tanto a nivel regional como internacional. Hasta el momento Tuning había sido una experiencia y un logro de 384 Revista Oficial del Poder Judicial 1/1 2007 Hernán Olano García La enseñanza por competencias jurídicas más de 135 universidades europeas que desde el año 2001 llevan adelante un intenso trabajo en pos de la creación del Espacio Europeo de Educación Superior. Y continúa así la descripción: Durante la IV Reunión de Seguimiento del Espacio Común de Enseñanza Superior de la Unión europea, América Latina y el Caribe (UEALC) en la ciudad de Córdoba (España) en Octubre de 2002, los representantes de América Latina que participaban del encuentro, luego de escuchar la presentación de los resultados de la primera fase del Tuning, acercaron la inquietud de pensar un proyecto similar con América Latina. Desde este momento se comenzó a preparar el proyecto que fue presentado por un grupo de universidades europeas y latinoamericanas a la Comisión Europea a finales de Octubre de 2003. La propuesta Tuning para América Latina es una idea intercontinental, un proyecto que se ha nutrido de los aportes de académicos tanto europeos como latinoamericanos. La idea de búsqueda de consensos es la misma, es única e universal, lo que cambian son los actores y la impronta que brinda cada realidad. Con fecha de 15 de Julio 2005, la Comisión Europea ha informado sobre la aprobación en la convocatoria de la Décima Ronda del Programa ALFA de una ampliación del Proyecto Tuning América Latina, a ocho nuevas áreas del conocimiento: Arquitectura, Derecho, Enfermería, Física, Geología, Ingeniería Civil, Medicina y Química, incorporando 120 nuevas Universidades Latinoamericanas. 1. LA EDUCACIÓN POR COMPETENCIAS Según el tratadista de las competencias, Sergio Tobón3, la formación basada en competencias es el nuevo enfoque para la educación en sus diversos niveles (primaria, secundaria, técnica, superior), debido a que posibilita una serie de cambios y transformaciones que vienen siendo demandadas por la sociedad, los estudiantes y los mismos docentes. Es así como el enfoque de competencias viene construyendo una serie de principios conceptuales y herramientas para pasar del énfasis en la transmisión de la información al aseguramiento de saberes esenciales, no solo en lo cognoscitivo, sino también en el ser y el hacer, que les permita a los estudiantes desempeñarse con pertinencia y pertenencia ante las actividades y problemas propios de los diferentes contextos (sociales, disciplinares, investigativos, profesionales, ambientales, políticos, económicos y laborales). Revista Oficial del Poder Judicial 1/1 2007 385 Hernán Olano García La enseñanza por competencias jurídicas Y es que las competencias, según Levy-Leboyer 4 , son repertorios de comportamientos que algunas personas dominan mejor que otras, lo que las hace eficaces en una situación determinada. Eso quiere decir, que con las competencias se busca basar la docencia en el aprendizaje, no en la enseñanza, por tanto, el concepto de educación por competencias relaciona toda actividad académica con el desarrollo de ciertos perfiles que se consideran necesarios para cada una de las diferentes profesiones o carreras que se estudian en las universidades del mundo. Los programas de estudio y la metodología pedagógica, deben estar dirigidas a la formación del nuevo profesional de acuerdo a las competencias requeridas por su carrera. Lo anterior implica, de acuerdo con el mismo Tobón, que han de tenerse en cuenta al menos cuatro aspectos que transforman el concepto de educación en un proceso amplio e integral asumido por cada persona para lograr su autorrealización. Dichos aspectos son: a) tener como base los aprendizajes previos de los estudiantes para planear los propósitos a partir de ellos b) tener un conocimiento profundo de los estilo y ritmos de aprendizaje, para orientar y mediar las estrategias didácticas acordes con ellos; c) hacer partícipe al estudiante de su aprendizaje, guiándole en la forma en que puede planificar, investigar y regular el estudio; d) tener presente en la docencia la formación y afianzamiento de estrategias cognoscitivas y metacognoscitivas que ayuden a los estudiantes a buscar, organizar, asimilar, comprender y aplicar el conocimiento con pertinencia. De esta manera se favorece la autonomía en los estudiantes, principio esencial de la pedagogía actual. A raíz de varias inquietudes detectadas por los expertos en cuanto a la claridad de la aplicación de lo que en realidad son las competencias y, para poder dejar de abordarlas como un mero hacer procedimiental enfocado a la realización de actividades, enfatizando en la aplicabilidad del conocimiento, o como atributos separados entre sí5 , como diría Tobón, surgió el proyecto Tuning, pionero en la definición de las competencias como una dinámica compleja formada por una suma integrada de conocimientos y aplicación práctica de los mismos que deberían caracterizar todo el proceso de convergencia europea de la educación superior.6 El proyecto Tuning, es la base en torno a la cual se construyó el denominado Proceso de Bolonia, que generó el EEES (Espacio Europeo de Educación Superior), uno de los programas de mayor impacto en el ámbito de la educación 386 Revista Oficial del Poder Judicial 1/1 2007 Hernán Olano García La enseñanza por competencias jurídicas superior europea y busca afinar las estructuras de sus universidades con el fin de mejorar la calidad académica de sus centros de educación superior, priorizando los procesos de aprendizaje a través de la investigación, con el objetivo fundamental de formar de acuerdo a las competencias profesionales previamente definidas, que más adelante apreciaremos. Según Juan Morales Ordóñez7 , la determinación de las competencias para cada una de las carreras universitarias, se realiza a través de consultas sistemáticas a los diferentes actores sociales relacionados con el proceso educativo. De esta forma, grupos de académicos, graduados, estudiantes, empleadores y sociedad civil en general, aportan con sus criterios para la definición de las competencias que deben tener los profesionales que se titulan en las universidades. Las competencias de quienes se gradúan en las facultades de Derecho, deben determinarse a través del proceso mencionado. Es probable que el perfil profesional de los abogados exija un claro conocimiento y sensibilidad frente a los verdaderos objetivos del Derecho, que tienen que ver con la búsqueda permanente de la justicia y el bien común; así como adecuados conocimientos sobre las estructuras jurídicas que organizan y determinan las formas de vida del País y del mundo. Además, es probable, que abogados y juristas deban ser profesionales con una alta formación social y humanista, considerando que su labor afecta los destinos de personas individuales y por ende el destino de las colectividades. La Ética que se constituye en una competencia básica en todas las profesiones, adquiere características de exigencia ineludible en el jurista, pues los valores y el deber ser moral son los fundamentos esenciales de todo ordenamiento jurídico. Las mallas curriculares y la estructura educativa de las facultades de Derecho, deberán adaptarse a las competencias para América Latina para lograr una adecuada formación en sus propias competencias. Y es que las competencias, como procesos complejos de desempeño, buscan fortalecer y desarrollar no sólo la formación de los estudiantes, mediante el diseño de programas de formación pertinentes a las competencias, sino también, de acuerdo con las recomendaciones de Tobón8 , con ellas se busca que los estudiantes sean protagonistas tanto de su vida, como de su proceso de aprendizaje y, particularmente, cumpliendo con cinco principios que se han de tener en cuenta en el proceso de aplicación de Tuning: a. La formación de competencias para hacer estudiantes protagonistas, implica tener como base el proyecto ético de vida, el cual se refiere a planear la vida Revista Oficial del Poder Judicial 1/1 2007 387 Hernán Olano García La enseñanza por competencias jurídicas con base en valores personales y sociales. Esto significa que las competencias no se forman en abstracto, sino en el marco de unas expectativas y metas, y es necesario abordar este ámbito como algo transversal a todo el plan de estudios. b. La enseñanza se orienta tomando como base los módulos, los cuales constituyen programas completos de formación de una o varias competencias mediante estrategias didácticas enfocadas a las tres dimensiones competenciales: afectivo-motivacional, cognoscitiva y actuacional, a través del trabajo centrado en problemas, mapas cognitivos y conceptuales, experimentos, simulaciones, prácticas laborales y proyectos. c. La instrucción se dirige a cada una de las competencias identificadas en el estudio del contexto y sistematizadas en el perfil de los diplomados y licenciados. Esto significa que las actividades didácticas en los módulos se planean por cada una de las competencias tomadas en forma individual. d. Las actividades de aprendizaje tienen como base la continua retroalimentación, con el fin de posibilitarles a los estudiantes el reconocimiento de sus logros y aspectos a mejorar, como también para ajustar mejor tales actividades a los propósitos formativos establecidos en un determinado módulo. e. Con el enfoque de las competencias se trabaja tomando como referencia los resultados verificables. Se comprende que diversos aspectos del aprendizaje no es posible medirlos de forma exacta, para así determinar el grado de impacto de la educación; sin embargo, sí se pueden buscar algunos aspectos que puedan ser plenamente contrastables, con el fin de tener algunos criterios para evaluar la calidad de la docencia. 2. OBJETIVOS DEL PROYECTO TUNING El proyecto Tuning posee cuatro líneas de trabajo: a. b. c. d. Competencias (genéricas y específicas) Enfoques de enseñanza, aprendizaje y evaluación Créditos académicos y, Calidad de los programas a. Competencias (genéricas y específicas): En cuanto a las competencias genéricas, que más adelante presentaremos, el 388 Revista Oficial del Poder Judicial 1/1 2007 Hernán Olano García La enseñanza por competencias jurídicas proyecto trata de identificar atributos compartidos que pudieran generarse en cualquier carrera y que son considerados importantes por la sociedad. Según los expertos, hay ciertos atributos como la capacidad de aprender, la capacidad de análisis y síntesis, etc., que son comunes a todas o casi todas los programas de formación, pero también puede haber unas competencias en cada área temática, a que se consideran como cruciales para cualquier titulación y sobre las cuales haremos una propuesta para Colombia, precisamente porque están específicamente relacionadas con el conocimiento concreto de un área temática, en nuestro caso: el Derecho. Se conocen también como destrezas y competencias relacionadas con las disciplinas académicas y son las que confieren identidad y consistencia a cualquier programa 9 . b. Enfoques de enseñanza, aprendizaje y evaluación Se trabaja en profundidad la traducción de las competencias tanto genéricas como específicas en actividades dentro del proceso de enseñanza, aprendizaje y evaluación. Para ello se propone preparar una serie de materiales que permitan visualizar cuales serán los métodos de enseñanza, aprendizaje y evaluación más eficaces para el logro de los resultados del aprendizaje y las competencias identificadas. Cada estudiante debe experimentar una variedad de enfoques y tener acceso a diferentes contextos de aprendizaje, cualquiera que sea su área de estudio. 10 c. Créditos académicos: En esta línea se llevará adelante una intensa reflexión sobre la vinculación de las competencias con el trabajo del estudiante, su medida y conexión con el tiempo calculado en créditos académicos. 11 d. Calidad de los programas: Está línea asume que la calidad es una parte integrante del diseño del currículo basado en competencias, lo que resulta fundamental para articular con las otras líneas expuestas. Si un grupo de académicos desean elaborar un programa de estudios o redefinirlo necesita un conjunto de elementos para brindar calidad a esos programas y titulaciones.12 Basados en el modelo de competencias, Tuning busca lograr unos objetivos, que en Derecho se centran en crear puentes entre las universidades y otras entidades apropiadas y calificadas para producir convergencia en las áreas Revista Oficial del Poder Judicial 1/1 2007 389 Hernán Olano García La enseñanza por competencias jurídicas de las disciplinas seleccionadas, así como crear redes capaces de presentar ejemplos de prácticas eficaces, estimular la innovación y la calidad mediante la reflexión y el intercambio mutuo y desarrollar e intercambiar información relativa al desarrollo de los currículos en las áreas seleccionadas y crear una estructura curricular modelo expresada por puntos de referencia para cada área, promoviendo el reconocimiento y la integración latinoamericana de titulaciones. 3. LAS COMPETENCIAS Para poderlas presentar, debemos remontarnos históricamente tan sólo hasta marzo de 2005, cuando se llevó a cabo la Primera Reunión General en Buenos Aires, donde los grupos de trabajo en consenso elaboraron la lista de competencias genéricas que se consultarían a académicos, estudiantes, graduados y empleadores de América Latina, los que se logró de Abril a Julio de 2005. La Segunda Reunión General del Proyecto, realizada en Belo Horizonte, en agosto del mismo año, se presentó el informe del análisis de los resultados de la consulta de competencias genéricas. En esa misma reunión los grupos de trabajo discutieron acerca de las competencias específicas y lograron definir la lista de competencias específicas para las áreas temáticas de Administración de Empresas, Educación, Historia y Matemáticas y fueron consultados académicos, estudiantes, graduados y empleadores de cada área temática en los meses de Octubre a Diciembre de 2005. En la Tercera Reunión General Tuning, que se realizó en San José durante el mes de febrero de 2006, se incorporaron nuevos grupos de trabajo: arquitectura, derecho, enfermería, física, geología, ingeniería civil, medicina y química, los cuales definieron las listas de competencias específicas para cada área. Los grupos que venían trabajando con anterioridad analizaron los resultados de las consultas llevadas a cabo. En Bruselas, Junio de 2006, se realizó la primera reunión conjunta de Tuning América Latina con Tuning Europa, donde se compararon las listas de competencias alcanzadas por los distintos grupos de trabajo, identificando similitudes y diferencias entre ambas reflexiones. La reunión de cierre del Proyecto se adelantó en Ciudad de México en el mes de Febrero 2007, programada con el objeto de hacer un balance sobre los resultados del proyecto, así como su impacto en las instituciones participantes. Además los grupos 390 Revista Oficial del Poder Judicial 1/1 2007 Hernán Olano García La enseñanza por competencias jurídicas de trabajos terminaron de revisar los documentos que se incluirán en el informe final del Proyecto. 13 4. COMPETENCIAS GENÉRICAS PARA AMÉRICA LATINA Estas competencias genéricas Tuning para América Latina, comprenden todas las áreas enunciadas, con base en ellas se realizará la confección de unas competencias específicas nacionales en cada área, lo mismo que siguiendo las competencias específicas del área para América Latina: 1. 2. 3. 4. 5. 6. 7. 8. Capacidad de abstracción, análisis y síntesis, Capacidad de aplicar los conocimientos en la práctica, Capacidad para organizar y planificar el tiempo, Conocimientos sobre el área de estudio y la profesión, Responsabilidad social y compromiso ciudadano, Capacidad de comunicación oral y escrita, Capacidad de comunicación en un segundo idioma, Habilidades en el uso de las tecnologías de la información y de la comunicación, 9. Capacidad de investigación, 10. Capacidad de aprender y actualizarse permanentemente, 11. Habilidades para buscar, procesar y analizar información procedente de fuentes diversas, 12. Capacidad crítica y autocrítica, 13. Capacidad para actuar en nuevas situaciones, 14. Capacidad creativa, 15. Capacidad para identificar, plantear y resolver problemas, 16. Capacidad para tomar decisiones, 17. Capacidad de trabajo en equipo, 18. Habilidades interpersonales, 19. Capacidad de motivar y conducir hacia metas comunes, 20. Compromiso con la preservación del medio ambiente, 21. Compromiso con su medio socio-cultural, 22. Valoración y respeto por la diversidad y multiculturalidad, 23. Habilidad para trabajar en contextos internacionales, 24. Habilidad para trabajar en forma autónoma, 25. Capacidad para formular y gestionar proyectos, 26. Compromiso ético, 27. Compromiso con la calidad Revista Oficial del Poder Judicial 1/1 2007 391 Hernán Olano García La enseñanza por competencias jurídicas 5. COMPETENCIAS ESPECÍFICAS LATINOAMERICANAS PARA DERECHO Éstas fueron elaboradas por las universidades participantes, con base en los borradores nacionales que cada universidad aportó, y a través del debate alcanzaron consenso sobre una lista de competencias específicas para cada el temática. Derecho definió consultar las competencias específicas a académicos, graduados, estudiantes y empleadores. Después se puso a disposición de los grupos un formato en línea para llevar adelante la consulta, además de las alternativas presenciales y de correo postal propuestas en la consulta de competencias genéricas. La consulta en línea se abrió del 3 de Abril de 2006 al 5 de Mayo de 2006 y, el análisis de los resultados de la consulta de competencias específicas se realizó en la reunión de Bruselas en Junio de 2006. Además, en todos los grupos de trabajo del proyecto se reflexionó en un ejemplo de como enseñar y evaluar una competencia de su área temática. Las competencias, a mi juicio, son una traducción, razón por la cual, poseen algunos errores gramaticales: 1. Conocer, interpretar y aplicar los principios generales del Derecho y del ordenamiento jurídico. 2. Conocer, interpretar y aplicar las normas y principios del sistema jurídico nacional e internacional en casos concretos. 3. Buscar la justicia y equidad en todas las situaciones en las que interviene. 4. Estar comprometido con los Derechos Humanos y con el Estado social y democrático de Derecho. 5. Capacidad de ejercer su profesión trabajando en equipo con colegas. 6. Capacidad de trabajar en equipos interdisciplinarios como experto en Derecho contribuyendo de manera efectiva a sus tareas. 7. Comprender adecuadamente los fenómenos políticos, sociales, económicos, personales y psicológicos -entre otros- , considerándolos en la interpretación y aplicación del Derecho. 8. Ser conciente de la dimensión ética de las profesiones jurídicas y de la responsabilidad social del graduado en Derecho, y actuar en consecuencia. 9. Capacidad de razonar y argumentar jurídicamente. 10. Capacidad de dialogar y debatir desde una perspectiva jurídica, 392 Revista Oficial del Poder Judicial 1/1 2007 Hernán Olano García La enseñanza por competencias jurídicas comprendiendo los distintos puntos de vista y articulándolos a efecto de proponer una solución razonable. 11. Considerar la pertinencia del uso de medios alternativos en la solución de conflictos. 12. Conocer una lengua extranjera que permita el desempeño eficiente en el ámbito jurídico (inglés, portugués y español). 13. Capacidad para usar la tecnología necesaria en la búsqueda de la información relevante para el desempeño y actualización profesional. 14. Capacidad para aplicar criterios de investigación científica en su actividad profesional. 15. Capacidad para aplicar sus conocimientos de manera especialmente eficaz en un área determinada de su profesión. 16. Capacidad de enfrentar nuevas situaciones y contribuir a la creación de instituciones y soluciones jurídicas en casos generales y particulares. 17. Capacidad para redactar textos y expresarse oralmente en un lenguaje fluido y técnico, usando términos jurídicos precisos y claros. 18. Capacidad para analizar una amplia diversidad de trabajos complejos en relación con el Derecho y sintetizar sus argumentos de forma precisa. 19. Capacidad para tomar decisiones jurídicas razonadas. 20. Comprender y relacionar los fundamentos filosóficos y teóricos del Derecho con su aplicación práctica. 21. Demostrar conciencia crítica en el análisis del ordenamiento jurídico. 22. Capacidad de actuar jurídica y técnicamente en diferentes instancias administrativas o judiciales con la debida utilización de procesos, actos y procedimientos. 23. Capacidad para decidir si las circunstancias de hecho están suficientemente claras para poder adoptar una decisión fundada en Derecho. 24. Actuar de manera leal, diligente y transparente en la defensa de intereses de las personas a las que representa. Hasta ahora, sólo hemos podido encontrar que Ecuador es el único país del grupo que ha propuesto sus competencias específicas para Derecho. 6. UN EJEMPLO, LAS COMPETENCIAS ESPECÍFICAS ECUATORIANAS 1. Buscar la verdad jurídica en todas las situaciones en las que intervenga. 2. Comprensión de problemas individuales y sociales en su relación con la vida colectiva y con el medio ambiente. Revista Oficial del Poder Judicial 1/1 2007 393 Hernán Olano García La enseñanza por competencias jurídicas 3. Comprensión de la pluralidad de criterios sociales y adaptación en los diferentes medios culturales. 4. Orientación a la búsqueda de soluciones alternativas de los conflictos. 5. Orientación a la mediación y al arreglo amistoso de las divergencias. 6. Compromiso con el desarrollo del Ecuador desde los roles de jurista y abogado. 7. Motivación para el servicio privado y público. 8. Destrezas en los campos de la argumentación y oratoria. 9. Destrezas en el campo de la presentación escrita de argumentos. 10. Disposición al trabajo en equipo. 11. Manejo de un segundo idioma. 12. Manejo de las nuevas tecnologías de información y comunicación. 13. Destrezas administrativas y gerenciales. 14. Conocimientos contables. 15. Manejo de criterios de planificación, elaboración y ejecución de proyectos en diferentes áreas sociales. 16. Conocimientos relacionados con técnicas de investigación académica. 17. Profundo conocimiento de las leyes y del sistema jurídico nacional y de sus relaciones con el mundo internacional. Sin embargo, yo propongo dar una ponderación de importancia a las competencias genéricas de Derecho, al parecer inmodificables y obligatorias punto a punto según los europeos, así como incluir otras o también mejorar la redacción a las existentes y comprometen al profesional del Derecho en principio, a que se le de una formación en valores, en estructura de su ciencia y por último, en un nivel instrumental complementario, como el que surge de la necesidad de una segunda lengua: 7. UNA PROPUESTA ESPECÍFICA PARA COLOMBIA Mi propuesta, para la enseñanza del Derecho por competencias, se basa en dieciséis puntos, de los cuales, cada profesor escogerá uno o varios para poder elaborar el programa de cada una de sus asignaturas: 1. Actúa en forma ética y transparente con responsabilidad social, buscando de manera leal y eficiente la justicia y la equidad en sus actuaciones, para defender adecuadamente los intereses de quienes representa. 2. Actúan con capacidad jurídica, con el apoyo técnico necesario para ejercer el debido proceso, razonado y con argumentos, ante autoridades judiciales o administrativas. 394 Revista Oficial del Poder Judicial 1/1 2007 Hernán Olano García La enseñanza por competencias jurídicas 3. Conoce, interpreta y aplica los principios generales del Derecho y del ordenamiento propio de su país, así como las diferentes normas del sistema jurídico nacional e internacional en casos concretos. 4. Posee capacidad para dialogar y debatir desde una perspectiva jurídica y con conciencia crítica, comprendiendo las distintas teorías y conceptos, jurídicos y filosóficos del Derecho, con el efecto de articularlos y proponer y tomar una solución jurídica razonada. 5. Posee capacidad para decidir si las circunstancias de hecho son suficientemente claras para poder adoptar en Derecho una decisión bien fundada. 6. Considera la importancia y la pertinencia del uso de los Medios Alternativos de Solución de Conflictos MASC. 7. Posee capacidad para redactar textos y expresarse de manera adecuada en forma verbal y escrita con un lenguaje fluido y técnico-jurídico, así como con una adecuada gramática acorde con las actualizaciones idiomáticas recientes. 8. Es capaz de enfrentar nuevas situaciones y de contribuir a formular soluciones jurídicas en casos generales y particulares. 9. Posee capacidad para aplicar sus conocimientos de manera eficaz en un área determinada de su profesión. 10. Posee capacidad para analizar una diversidad muy amplia de trabajos complejos en relación con el Derecho y de sintetizar sus argumentos en forma precisa. 11. Comprende adecuadamente los fenómenos políticos, sociales, económicos, personales y psicológicos entre otros-, considerándolos en la interpretación y aplicación del Derecho. 12. Posee capacidad de ejercer su profesión trabajando en equipo, bien sea con colegas o con expertos de otras carreras, contribuyendo de manera efectiva en la solución de casos. 13. Posee capacidad para ejercer la investigación científica en su actividad profesional. 14. Posee capacidad para utilizar la tecnología, así como los avances de ésta, en la búsqueda de la información relevante para ejercer su carrera, así como para actualizarse profesionalmente. 15. Conocer al menos una lengua extranjera distinta a la materna, que le permita actuar eficientemente en el ámbito jurídico. 16. Está comprometido con los Derechos Humanos y con el Estado Social y Democrático de Derecho. Revista Oficial del Poder Judicial 1/1 2007 395 Hernán Olano García La enseñanza por competencias jurídicas Esperamos que en Perú, también la enseñanza por competencias sea una realidad. Disponible en: http://www.unideusto.org/tuning/tuningal/ Disponible en: http://www.unideusto.org/tuning/tuningal/ 3 Tobón, S. El Enfoque de las Competencias en el Marco de la Educación Superior. Módulo Uno. CIFE. Madrid, 2006, página 1. 4 Citado por Tobón, página 3. 5 Tobón, S. Op. Cit., página 3. 6 Ver cita # 2 en Tobón, S. Op. Cit., página 3. 7 Morales, J. La Educación por Competetencias. Disponible en: http://www.uazuay.edu.ec/tuningderecho/ articulos.htm, acceso en abril de 2007. 8 Tobón, S. Op. Cit., página 9. 9 Disponible en: http://www.unideusto.org/tuning/tuningal/ 10 Disponible en: http://www.unideusto.org/tuning/tuningal/ 11 Disponible en: http://www.unideusto.org/tuning/tuningal/ 12 Disponible en: http://www.unideusto.org/tuning/tuningal/ 13 Disponible en: http://www.unideusto.org/tuning/tuningal/ 1 2 396 Revista Oficial del Poder Judicial 1/1 2007
W3153838355.txt	https://revistas2.uepg.br/index.php/pauta/article/download/15837/209209214158	en		Crying girl on the border: a colonialidade de gênero na fronteira das imagens	Pauta Geral	2,020	cc-by-sa	7,280	REVISTA PAUTA GERAL ESTUDOS EM JORNALISMO 10.5212/RevistaPautaGeral.v.7.15837 Crying girl on the border: a colonialidade de gênero na fronteira das imagens Angie Biondi1 Ângela Cristina Salgueiro Marques2 Resumo As discussões empreendidas pelo pensamento decolonial têm levado pesquisadoras e pesquisadores à revisão crítica das práticas mediáticas no âmbito da produção e circulação de imagens. As maneiras de representar sujeitos sem considerar os marcadores de gênero, raça, classe, etnia, em interseccionalidade, são questionadas como sínteses visuais redutoras de complexos processos engendrados por formas subjacentes do capitalismo. Neste texto, propomos uma reflexão crítica acerca dos modos como a imagem jornalística ainda circunscreve um lugar específico ao sujeito vulnerável, tomando como uma base exemplar a fotografia vencedora do prêmio internacional World Press Photo, em 2019. Palavras-chave: Fotojornalismo. Gênero 2. Migrantes 3. Crying girl on the border: gender coloniality on the border of images Abstract The discussions undertaken by decolonial thought have led researchers to a critical review of media practices in the scope of image production and circulation. The different ways to represent subjects without considering the markers of gender, race, class, ethnicity, in intersectionality, are questioned as visual syntheses that reduces complex processes engendered by the underlying forms of capitalism. In this text, we propose a critical reflection on the ways in which journalistic image circumscribes a specific role for vulnerable people, taking as an exemplary base the photograph that won the World Press Photo international award in 2019. 1 Professora do Programa de Pós-graduação em Comunicação e Linguagens da Universidade Tuiuti do Paraná. Doutora em Comunicação Social pela UFMG. E-mail: angiebiondina@gmail.com 2 Professora do Programa de Pós-graduação em Comunicação Social da UFMG. Doutora em Comunicação Social pela UFMG. E-mail: angelasalgueiro@gmail.com Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa.v.7.e2015837, p.1-20, 2020. 1 REVISTA PAUTA GERAL ESTUDOS EM JORNALISMO 10.5212/RevistaPautaGeral.v.7.15837 Keywords: Photojournalism1. Gender 2. Migrants 3. Introdução Há algum tempo, as pesquisas em comunicação têm empreendido esforços para compreender como imagens concernentes ao jornalismo se referem, demonstram, indicam, retratam, enfim, representam os sujeitos comuns, anônimos, imersos em suas vidas e acontecimentos cotidianos. A observação e análise da circulação destas diversas imagens, bem como a produção de discursos e significados que lhe são extensivos são recorrentes objetos de estudo na área. Interessados não tanto nas imagens em si, mas nas relações que podem ser traçadas entre elas a partir de sua presença em contextos diversos, esses estudos se concentram em observar as potências da imagem no tempo – sua aparição, suas reverberações, a dissolução e desaparecimento, bem como seu retorno a partir da persistência como parte de um imaginário (MARTINO; MARQUES, 2020, p. 85) Embora figurem frequentemente nos espaços visuais e narrativos das diferentes mídias, observa-se, no entanto, que a condição de homens, mulheres e crianças, muitas vezes, são mantidas invisíveis mesmo quando alcançadas pelas câmeras fotográficas e profissionais da imprensa, uma vez que lhe é conferido apenas um caráter ilustrativo ou exemplar junto aos diversos temas que constituem acontecimentos e perfazem o contexto jornalístico. As narrativas jornalísticas, muitas vezes, buscam representar as camadas populares e vulneráveis localizando-os a partir de matrizes socioculturais pré-definidas. E, deste modo, demarcam tanto os notórios “condicionantes estruturais quanto os processos de subjetivação em que se encontram inseridos empiricamente os diferentes atores” (LOPES, 2018, p.41). Segundo Persichetti (2006, p. 184), a revisão dos critérios que animam o fotojornalismo, em especial, não é uma tarefa nova e apresenta uma literatura crítica que destaca pelo menos dois grandes momentos de sua prática, a saber, uma ligada ao aspecto idealizado (ainda moderno) de retratar a realidade mesma, na qual a imagem seria dada como a própria informação, e outra ligada ao aspecto expressivo (advindo a partir dos anos de 1990), na qual a prevalência da imagem passa a compor ou criar o próprio fato ou o acontecimento. A discussão sobre os meios de comunicação nos leva ao uso político da imagem. As fotografias servem para construir fatos. Muitas imagens foram feitas por fotojornalistas, mas por motivos muitos e diversos, só Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa.v.7.e2015837, p.1-20, 2020. 2 REVISTA PAUTA GERAL ESTUDOS EM JORNALISMO 10.5212/RevistaPautaGeral.v.7.15837 algumas destas imagens se tornaram públicas, a imagem de um evento. Estas fotografias se transformaram no próprio evento, são monumentos. (D’AUTILIA apud PERSICHETTI, 2006, p. 185). Vale observar, portanto, se – e como - sujeitos comuns ainda figurariam como exemplares notórios dos acontecimentos através das imagens veiculadas em páginas de jornais, revistas ou portais de notícias. E, extensivamente, como tal representação estaria engendrada aos aspectos expressivos elaborados em imagens fotojornalísticas ainda hoje. Em trabalhos anteriores (MARQUES; BIONDI, 2017, 2018, 2019) exploramos o tensionamento entre duas formas de representar sujeitos em situação de vulnerabilidade no fotojornalismo. Salientamos que, apesar da constante utilização de um léxico que geralmente pretende conferir-lhes visibilidade, as imagens ainda tendem a invisibilizá-los a partir da reiteração de uma lógica de registro que considera discursos já enraizados sobre pobreza, dependência, vulnerabilidade e estigmas de gênero. Há racionalidades que reforçam modos de legibilidade e inteligibilidade das imagens a partir do acionamento de premissas, julgamentos, valores, predisposições afetivas, que permitem aos atores sociais reconhecerem e compreenderem os fatos a partir do que chamamos de enquadramentos consensuais. Assim, mesmo ganhando “visibilidade” nas páginas de jornais, sujeitos e grupos mais vulneráveis não se tornam socialmente inteligíveis e visualmente reconhecíveis. Como se esses sujeitos e grupos fossem menos dignos de valor diante do olhar de um espectador que, presumivelmente, as interroga e avalia seus modos de vida e condutas. Todavia, acreditamos que as imagens fotojornalísticas são operações dialéticas que trabalham tanto para “representar” de maneira documental as vidas precárias, quanto no sentido de não apagar todos os vestígios e brechas que permitem o “aparecimento” e a figuração dos povos vulneráveis. Dito de outro modo, há uma possibilidade de, a partir de um deslocamento do olhar, revelar os desencaixes que escapam aos modos de captura e controle pelos aparatos governamentais e midiáticos. Assim, permitir a figuração e o aparecimento de corpos produzidos visualmente como precários e expostos na fotografia jornalística implica tanto explorar as formas discursivas de enquadramento consensual que acentuam sua desaparição, quanto em buscar indícios que possam evidenciar como tal enquadramento muitas vezes não resiste aos deslocamentos do olhar que, ao percorrer a imagem, duvida, oscila, encontra uma forma de conferir dignidade e reconhecimento aos sujeitos que ali figuram (MARQUES; BIONDI, 2017, 2018). Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa.v.7.e2015837, p.1-20, 2020. 3 REVISTA PAUTA GERAL ESTUDOS EM JORNALISMO 10.5212/RevistaPautaGeral.v.7.15837 Ao mesmo tempo, nos últimos anos, o acirramento das discussões acerca do decolonialismo, sobretudo, a partir de pesquisas e publicações de obras centrais advindas de fora do eixo acadêmico europeu e norte-americano, têm levado pesquisadoras e pesquisadores à revisão crítica de certas práticas mediáticas, sobretudo, aquelas que se referem ao âmbito da produção e circulação de imagens como conhecidos espaços produtores de enquadramentos e sentidos que conduzem à (in)visibilidade dos sujeitos comuns quando representados no âmbito jornalístico. Pesquisadores como CASTROGÓMEZ; GROSFOGUEL, 2007; CALLEGARO; LAGO, 2012; URANGA, 2005; CRUZ, 2017; 2019, têm destacado como certas produções mediáticas contemporâneas têm sido analisadas e refletidas a partir de uma perspectiva que lhes exige a desconstrução de paradigmas hegemônicos que ainda balizam as formas narrativas quando conferem “um rosto humano à notícia” (CRUZ, 2017, p.2). No jornalismo, atualmente, as maneiras de representar sujeitos sem considerar os marcadores de gênero, raça, classe, sexualidade ou ainda etnia, em interseccionalidade 3, são questionadas como sínteses visuais redutoras de complexos processos engendrados por formas subjacentes do capitalismo – entendido aqui tanto um modo de produção, quanto uma lógica ocidental moderna -, que ainda reverbera modelos de opressão e marcadores culturais estigmatizantes. Assim, a expansão e aprofundamento de crises e sofrimentos de diversas populações e certos grupos sociais têm sido discutidas como tributárias dos modelos de opressão vividos, e ainda em vivências. A partir destas premissas iniciais, buscamos, neste texto, dar passagem a uma reflexão que contemple a crítica advinda do pensamento decolonial enfatizando um grave problema elaborado como um tema visual dos mais recorrentes no jornalismo: a migração. Para isso, tomamos como exemplo da discussão reflexiva aqui proposta, a imagem classificada como “a fotografia do ano”, vencedora da maior láurea de fotojornalismo internacional, o World Press Photo, por entender que este tipo de premiação tanto postula as bases referencias da prática fotojornalística contemporânea quanto atribui legitimidade à produção de conteúdo veiculado de modo adjunto. Intitulada Crying girl on the border, a fotografia de John Para fins de aprofundamento de pesquisa indicamos que a interseccionalidade, enquanto um termo conceitual, aparece cunhada no âmbito dos direitos humanos pela pesquisadora afro-estadunidense Kimberlé Crenshaw, em 2001. Seu potencial heurístico, contudo, já comparecia nas discussões empreendidas por pensadoras como Angela Davis, na obra Mulheres, raça e classe, de 1981. 3 Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa.v.7.e2015837, p.1-20, 2020. 4 REVISTA PAUTA GERAL ESTUDOS EM JORNALISMO 10.5212/RevistaPautaGeral.v.7.15837 Moore, vencedora da edição de 2019, retrata a abordagem de agentes policiais norteamericanos a um grupo latino-americano composto, em sua maioria, por mulheres e crianças. Nas fronteiras, a colonialidade de gênero O pensamento decolonial tem buscado promover uma ruptura no grande modelo teórico e epistemológico moderno que até hoje ampara conceitos hegemônicos e universalismos. Segundo Castro-Gómez; Grosfoguel (2007, p.14), mesmo as produções e análises acadêmicas atuais ignoram epistemologias que são produzidas desde as margens, reproduzindo um eixo centralizador que uniformiza os saberes e mantém discursos e sujeitos aderidos às posições e categorias estigmatizantes. Contemporaneamente, o que se apresenta é ainda uma espécie de modulação da agência de poder que constitui diferentes formas de assujeitamentos. Segundo Achille Mbembe (2018), esta “ocupação colonial tardia” apresenta facetas diferenciadas da primeira ocupação moderna, especificamente em sua combinação entre o disciplinar, a biopolítica e a necropolítica; arranjo denominado pelo autor de “necropoder”. Se anteriormente, conforme indica Mbembe (2018), a colonialidade moderna ocorria em termos de ocupação forçada, com objetivos de conquista, aquisição e gerência de territórios, nos tempos atuais, as “máquinas de guerra” estão encarnadas em práticas difusas e polimorfas, cujo exercício maior é o de decisão sobre vida e morte, no sentido diluído de suas percepções de valor, mas também de materializações acerca das diferentes formas de inclusão e exclusão de sujeitos. Trata-se, portanto, de um outro modo de governamentalidade, segundo o autor, que não se importaria com dimensões internas ou externas, em termos geográficos e físicos, mas de corpos feitos territórios que deveriam seguir o fluxo controlado e a demarcação dos movimentos de interesses de um capital imaterial, simbólico, indicado como “esta nova era da mobilidade global” (MBEMBE, 2018, p. 52). A afirmação de uma autoridade suprema em um determinado espaço político não se dá facilmente. Em vez disso, emerge um mosaico de direitos de governar incompletos e sobrepostos, disfarçados e emaranhados, nos quais sobejam diferentes instâncias jurídicas de facto geograficamente entrelaçadas, e nas quais abundam fidelidades plurais, suseranias assimétricas e enclaves. Nessa organização heterônima de direitos territoriais e reivindicações, faz pouco sentido insistir na distinção entre campos políticos ‘interno’ e ‘externo’, separados por limites claramente demarcados. (MBEMBE, 2018, p.51) Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa.v.7.e2015837, p.1-20, 2020. 5 REVISTA PAUTA GERAL ESTUDOS EM JORNALISMO 10.5212/RevistaPautaGeral.v.7.15837 Deste modo, nos parece importante sublinhar o modo através do qual o pensamento decolonial tem observado discursos e práticas que instituem modelos de exclusões, invisibilidades e assujeitamentos, sobretudo quando inscritos em práticas mediáticas, cujos discursos constituem ainda importantes elementos socializantes de linguagem e significação acerca dos sujeitos figurados. Em diálogo com o pensamento da pesquisadora María Lugones (2014), a lógica categorial, atomizada e hierárquica sempre foi necessária à manutenção de um sistema dicotômico que ainda separa sujeitos e posições econômicas, mas também ecológicas, cosmológicas e espirituais, como aspecto central da roupagem de um capitalismo colonialista. Neste processo, grandes contingentes populacionais padeceram dos modelos de opressão colonial, tradicionalmente baseados no escravismo, mas que reverbera, ainda hoje, nas vidas, corpos e subjetividades de sujeitos vilipendiados de direitos e cidadania, quando não da própria condição de humanos. Segundo ela, o exercício de revisar os modelos hegemônicos que impuseram categorias como universais perpassa não apenas uma revisão teórica e conceitual das mais urgentes, mas procura fazer jus a um modo de pensamento que já demarcava a distinção às maneiras de compreender e dar visibilidade aos aspectos constituidores de assujeitamentos. Neste contexto é que comparecem, nas discussões atuais, os marcadores de gênero, raça, classe, etnia, sexualidade que, quando sobrepostos uns aos outros, correspondem a uma classificação identitária estanque e fragmentada que aparta ainda mais os sujeitos em uma dinâmica social, cultural e política, quando, na realidade, precisam ser pensados em seus contextos materiais e nas situações concretas de atravessamentos, ou seja, em intersecção. A interseccionalidade apresenta um potencial heurístico que tem sido cada vez mais debatido como uma forma de necessária resistência também metodológica, opositiva – e criativa - aos cânones interpretativos ainda baseados em uma visão de humanidade advinda, quase que exclusivamente, do norte global e reproduzindo modelos de opressão que reverberam em práticas e atividades que movimentam o conhecimento, a cultura e a sociedade (AKOTIRENE, 2019, p.40). É preciso frisar que a discussão e o recurso à interseccionalidade aqui comunga da crítica no sentido de que não se pode propor hierarquias de opressão e nem mesmo subsumir lutas e reivindicações justificadamente colocadas pelo pensamento decolonial, Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa.v.7.e2015837, p.1-20, 2020. 6 REVISTA PAUTA GERAL ESTUDOS EM JORNALISMO 10.5212/RevistaPautaGeral.v.7.15837 mas procurar dar centralidade aos modos que repercutem e/ou reproduzem opressões contra grupos e contingentes populacionais historicamente marginalizados, a fim de desconstruí-los. Recomenda-se, pela interseccionalidade, a articulação das clivagens identitárias, repetidas vezes reposicionadas pelos negros, mulheres, deficientes, entre outros grupos considerados minoritários, para finalmente defender a identidade política contra a matriz de opressão colonialista, que sobrevive graças às engrenagens de um racismo cisheteropatriarcal capitalista (AKOTIRENE, 2019, p. 45). Portanto, a partir desta matriz teórica trazida pelo pensamento decolonial, uma observação das práticas mediáticas, do campo jornalístico, e das imagens fotojornalísticas, em particular, não pode prescindir de um olhar crítico acerca dos atravessamentos destes marcadores, onde os acontecimentos e sujeitos aos quais se reportam, cotidianamente, permaneçam isentos de uma discussão aprofundada sobre seu posicionamento neste contexto, pois mesmo quando em tom de denúncia, são, muitas vezes, submetidos aos quadros visuais e informativos pré-fixados por critérios limitadores, tais como noticiabilidade, agendamento ou protocolos de cobertura, já que são constituintes e constituidores de uma prática jornalística, e comunicacional, linear (LEAL; ANTUNES, 2019). Trata-se do desafio de debater em que medida as imagens, no âmbito jornalístico, tem alcançado os sujeitos que retratam, ou ainda, se somos nós, espectadores moralmente enquadrados, que atribuímos a estas imagens jornalísticas o empenho de realidade esperado. Espera-se que exerçam uma função emancipadora ou prescritiva, afinal? Há várias dificuldades para que o “aparecimento” de sujeitos vulneráveis nas imagens não seja reduzido à desumanização, mas permita entrever seres dotados de dignidade, de humanidade e de agência. Enquadramentos vitimizantes impedem que os sujeitos consigam libertar sua capacidade de aparecer na cena pública, reforçando o regime representativo das imagens (RANCIÈRE, 2010, 2012). “Aparecer”, como mencionamos, não é só adquirir visibilidade, mas envolve alterar o modo como sujeitos são percebidos e reconhecidos diante dos outros, o que demanda um deslocamento do olhar, uma outra forma de imaginar as relações com a alteridade e de considerar as formas de vida daqueles que se apresentam diante de nós (MARQUES; BIONDI, 2019). Sob esse aspecto, a imagem funciona como operação sensível que expressa uma batalha constante entre regimes figurativos e representativos de visibilidade, que Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa.v.7.e2015837, p.1-20, 2020. 7 REVISTA PAUTA GERAL ESTUDOS EM JORNALISMO 10.5212/RevistaPautaGeral.v.7.15837 persistem em reduzir a multipliciade de formas de vida, de temporalidades e de modos de expressão que tecem a rede de manutenção cotidiana da vida dos sujeitos. Segundo Rancière (2010), a capacidade de aparecer manifesta-se em uma espécie de perturbação da ordem sensível que pode revelar a fratura causada por representações preconceituosas que aprofundam desigualdades. Nem sempre as imagens que encontramos promovem uma abertura para que ocorra o deslocamento do olhar do espectador. Contudo, quando algumas delas escapam à rotulação imediata, alterando, na circulação ampliada, sentidos já fixados por valores e quadros de julgamento, há uma ruptura, um intervalo. Esse intervalo requer do espectador uma contemplação mais detida, tornando possível refletir acerca de uma dimensão política das imagens, entendendo a política em um sentido amplo, como os jogos discursivos que influenciam na percepção do que é dado a ver, sentir, ouvir e falar (RANCIÈRE, 2012). Partimos do pressuposto de que imagens destinadas a produzir um certo atestado de realidade, como aquelas feitas sob o regime jornalístico, podem oscilar, em uma tensão dialética, entre uma representação que afirma expectativas e uma ruptura capaz de instaurar uma prática de dissenso fundada em um intervalo, uma fratura que permite outras aberturas de sentido. Deste modo, o esforço desta discussão passa por observar e analisar os modos através dos quais o jornalismo, enquanto um dos mediadores centrais em nossa sociedade, promove ou referencia lugares e posições aos sujeitos, a partir de suas próprias imagens produzidas, elaboradas e difundidas. Neste intento, tomamos a fotografia vencedora do maior prêmio de fotojornalismo internacional, o World Press Photo, laureada na edição de 2019. Intitulada Crying girl on the border, a foto foi registrada por John Moore, profissional norte-americano, que cobria a chegada de um grupo latino-americano, composto por mulheres e crianças, à fronteira dos EUA com o México, situado em McAllen, Texas, em junho de 2018. Na imagem (Figura 1), o flagrante de uma revista a uma mulher abordada por uma/um agente de fiscalização da fronteira. Identificada como mulher apenas pelo reconhecimento da compleição física entrevista no enquadramento dado ao seu corpo, a personagem aparece de perfil apoiando os braços em um automóvel e, enquanto as mãos da/do agente (também não identificada/o para além do uniforme) lhe fixam a cintura, uma criança, em pé, ao seu lado, é a única a ter parte do rosto revelado e visto em choro. De Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa.v.7.e2015837, p.1-20, 2020. 8 REVISTA PAUTA GERAL ESTUDOS EM JORNALISMO 10.5212/RevistaPautaGeral.v.7.15837 frente para a situação de revista, a criança é a única personagem com uma reação e expressão retratadas na imagem. Por inferência, atribuímos uma identidade feminina à personagem abordada, por inferência, também atribuímos a condição de maternidade desta mulher. Abordadas, fiscalizadas, mulher e criança são as personagens femininas que, frequentemente, figuram a travessia de grupos latino-americanas que arriscam suas vidas deixando para trás, não raro, histórias de violência, precariedade, insegurança, perseguição, entre outras circunstâncias, em tentativas, em geral, precárias, de entrar nos EUA em busca de melhores condições de vida. Porém, interditadas, tais personagens femininas aparecem tanto no limite da fronteira quanto da imagem em questão. O médio plano, a tomada da distância, a iluminação ambiente entre as sombras projetadas e o veículo, a ênfase no perfil e alinhamento dos corpos no momento flagrante da revista constituem elementos visuais que, em conjunto, tem a finalidade de garantir o caráter ilustrativo da ação corriqueira de abordagem policial a estas pessoas. Figura 1: John Moore, 12 junho 2018. Fonte: Disponível em https://www.worldpressphoto.org/collection/photo/2019/37620/1/John-Moore Acesso em junho 2019. É importante notar que a mulher, mestiça, terceiro-mundista, latina, carrega a tradição dos rótulos e do interdito. O tema da fotografia, que ilustra as ações de abordagem e captura de mulheres e crianças na divisa estadunidense com o México, tem sido, inclusive, alvo de inúmeras críticas na imprensa mundial, além das declarações e relatórios publicados por diversas instituições de Direitos Humanos, justamente por Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa.v.7.e2015837, p.1-20, 2020. 9 REVISTA PAUTA GERAL ESTUDOS EM JORNALISMO 10.5212/RevistaPautaGeral.v.7.15837 terminar na separação entre mães e suas crianças. Entre outras situações registradas de mulheres grávidas sem atendimento, mães com filhos portadores de síndrome de Down, entre outras deficiências, mulheres e crianças enfrentam, indistintamente, situações de vigilância, detenção, atendimento médico precário ou insuficiente, ofensas pessoais, além de deportações solitárias. Deste modo, diferentes interdições se sobrepõem a estas mulheres, uma vez que, impedidas de entrar no país são quase sempre deportadas desacompanhadas de seus filhos e filhas, que permanecem cativos por semanas ou meses em campos de detenção4 para imigrantes, muitos deles, sem qualquer documento. Neste contexto, discutir a colonialidade do gênero, como explica Lugones, possibilita a entender a opressão como uma interação complexa de um sistema, simultaneamente econômico, racializante e engendrado culturalmente. No exemplar aqui discutido, a intrínseca relação entre as políticas racializadas de migração e policiamento não passam despercebidas de outros veículos de comunicação, que a repercutem. Começo aqui a fornecer uma forma de compreender a opressão de mulheres subalternizadas através de processos combinados de racialização, colonização, exploração capitalista, e heterossexualismo. [...] Chamo a análise da opressão de gênero racializada capitalista de ‘colonialidade do gênero’. Chamo a possibilidade de superar a colonialidade do gênero de ‘feminismo descolonial’ (LUGONES, 2014, p. 941). Figura 2: Time. Edição de 02 julho 2018. Fonte: Disponível em https://www.tellerreport.com/life/--john-moore-wins-world-press-photo-with-the-iconicimage-of-the-honduran-girl-separated-from-her-mother-on-the-us-border-.HJ41h2TYV.html Acesso em junho 2019. Informações disponibilizadas pelo Relatório Mundial 2019, Human Rights Watch. Disponível em https://www.hrw.org/pt/world-report/2019 Acesso em janeiro 2020. 4 Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa.v.7.e2015837, p.1-20, 2020. 10 REVISTA PAUTA GERAL ESTUDOS EM JORNALISMO 10.5212/RevistaPautaGeral.v.7.15837 Na edição de 02 de julho de 2018 (Figura 2), a capa da revista Time utiliza da mesma fotografia para compor outra ilustração acerca das interdições na fronteira e mistura o registro fotográfico do flagrante da abordagem policial registrada por Moore com a fotografia de Donald Trump, retirada em outra ocasião. Os personagens são colocados frente a frente, como em um encontro/confronto de olhares. Suas diferenças de tamanho podem ser compreendidas como a reiteração irônica das diferenças de escalas entre dois povos, dois mundos, duas naturezas, dois tipos de sujeitos. Assimétrica, a distinção marcada e encarnada no olhar, na postura e na desproporção entre Trump e a criança migrante, ilustra o próprio sistema colonial ainda postulado no cruzamento das normatizações opressoras de uma forma de racismo cisheteropatriarcal atribuindo à garota migrante, a figuração das desigualdades estruturantes da posição de gênero, raça, etnia, classe e discriminações. A revista Time nos propõe, em suma, uma legenda (implícita) que vai explorar ainda mais o sentido destas fraturas promovidas pela dualidade e polarização existente nas figuras do par colonizador/colonizado estabelecidas, na imagem, pela hierarquia entre os sujeitos feita, também, como uma hierarquização de olhares. Daí, não poder escapar a ironia colocada pelo título que “mancheta” a imagem: Welcome to America aparece em um fundo uniforme vermelho que poderia facilmente remeter à histórica, mas sempre atual, violência dos EUA para com os povos latinos, fronteiriços, com todas as implicações problemáticas de assujeitamento que esta relação de poder ainda coloca. “O processo de colonização inventou os/as colonizados/as e investiu em sua plena redução a seres primitivos, menos que humanos, possuídos satanicamente, infantis, agressivamente sexuais, e que precisavam ser transformados” (LUGONES, 2014, p. 941). Na imagem, ao se descaracterizar, ao desinvestir as migrantes pela interdição, elas não poderiam estar em nenhuma outra posição senão o lugar do outro, diminuído, assujeitado, infans sem fala. E aqui, é preciso sublinhar que a elaboração da imagem jornalística, embora em viés irônico, acaba mantendo as posições dicotômicas e hierárquicas presentes na relação dual entre as personagens. Ainda não se pode ouvir o choro e nem a voz da hondurenha Sandra Sanchez, nem da criança, Yanela Sanchez. Deste modo, também pela imagem se exerce a colonialidade de gênero dada pela linguagem, pois uma tradução vigora como uma prática colonial quando apaga a possibilidade de resistência a ela oferecendo, em seu lugar, uma legibilidade específica. Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa.v.7.e2015837, p.1-20, 2020. 11 REVISTA PAUTA GERAL ESTUDOS EM JORNALISMO 10.5212/RevistaPautaGeral.v.7.15837 “Assim, ver a colonialidade é revelar a degradação mesma que nos dá duas interpretações da vida e um ser interpretado por elas” (LUGONES, 2014, p.946). A consideração das formas de vida na figuração das migrantes pela imagem Um dos aspectos desfigurantes da identidade dessas duas mulheres migrantes é justamente essa construção colonial que quer acentuar, de um lado, a posição de vítima dos sujeitos retratados e, de outro, o lugar virtuoso ocupado pelo espectador que pode se dizer indignado e compadecido pela situação enquadrada na narrativa jornalística. A imagem em questão tende a reforçar o fato de que quando as fotografias jornalísticas mostram mulheres em situações de sujeição e opressão, parece que elas acionam um enquadramento que predispõe o público a assumir um ponto de vista moral que oscila entre a condenação e a indignação, a sideração e a consideração, num movimento que não contraria o direcionamento dos espectadores a um lugar de avaliadores virtuosos diante da precariedade e vulnerabilidade daqueles que são mostrados como “incapazes” de sobreviver e de agir. Como afirma a pesquisadora Tania Perlini (2012), essas imagens não têm uma função crítica, dificilmente interrogam o espectador acerca das razões da subalternização e da sujeição do corpo feminino, mas fornece instrumentos morais específicos para a configuração de um posicionamento “confortável” aos espectadores. Desprovidos de nome e de história, as duas figuras femininas (assim como o agente oficial de imposição da lei e da força) aparecem diante de nós através da mediação de uma imagem que não oferece oportunidade de dúvida ou contemplação, uma vez que juízos condenatórios ou de revolta são acionados de modo instantâneo. Desta forma, há pouca abertura, nessa imagem, para uma “indecidibilidade moral”, ou seja, para uma avaliação moral equívoca, não resolvida, que interpela à reflexão crítica e coloca em dúvida parâmetros e pressupostos que delineiam uma legibilidade e inteligibilidade ao mundo. Uma imagem que mobiliza a indecisão e questiona o lugar de “júri virtuoso” ocupado pelos receptores não aceita explicações rápidas e nem adere tão facilmente às avaliações morais dos espectadores (PERLINI, 2012). Ela escapa aos esquematismos, dialoga com outras imagens e descortina algumas das dinâmicas dos dispositivos que as fazem existir. A imagem que aqui analisamos organiza e dispõe causalmente os fatos, em vez de evocar, pelo relato, e pela falha do relato, a captura das complexidades dos modos de vida e das formas de vida de sujeitos migrantes. É uma imagem que explica e julga em Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa.v.7.e2015837, p.1-20, 2020. 12 REVISTA PAUTA GERAL ESTUDOS EM JORNALISMO 10.5212/RevistaPautaGeral.v.7.15837 vez de sugerir e preservar o difícil gesto de apreender, considerar e reconhecer as alteridades. Ela sidera, mais do que considera. A diferença entre os gestos de siderar e considerar é traçada por Marielle Macé (2018) quando ela identifica que a condição de ser reconhecido está ligada a disposições mais gerais que preparam ou modelam um sujeito para o reconhecimento a partir de sua sideração ou de sua consideração. Essa autora nos chama a atenção para a condenação das vidas que atualmente tentam se manter em meio à condição de precariedade, vulnerabilidade, mas que chegam até nós através de enquadramentos que não nos permitem perceber ou escutar seus gestos, sonhos, tentativas de melhoria e experiências (migrantes, sujeitos empobrecidos, vítimas de grandes catástrofes, vítimas de violência institucional, etc.). Os enquadramentos que regulam a aparição e a apreensão desses modos de vida são geralmente destinados a produzir a sideração, tanto daqueles que olhamos, quanto nossa própria sideração e alheamento: Siderar, deixar-se siderar é permanecer medusado, petrificado, enclausurado numa emoção que não é fácil transformar em moção, aterrado numa hipnose, numa estupefação, num enfeitiçamento em que se esgota de algum modo a re-erva de partilha, laços, gestos que poderiam ser alimentados pelo conhecimento que temos dessas situações, mas que permanece como um sofrimento à distância.[...] Considerar seria levar em conta os vivos, suas vidas efetivas, uma vez que é desse modo e não de outro que essas vidas são furtadas ao presente - levar em conta suas práticas, seus dias, e então desenclausurar o que a sideração enclausura; não designar e rotular vítimas, mas descrever tudo o que cada um põe em ação para lidar com situações de vulnerabilidade. (MACÉ, 2018, p.28) Considerar é um convite para a contemplação e para a reabertura de uma relação, de uma proximidade, de uma possibilidade de avizinhamento com a alteridade. Seria uma forma de reconhecimento que privilegia a responsabilidade pelo outro, a atenção e o cuidado com sua trajetória e com suas demandas. Uma ética da responsabilidade que desafia os quadros normativos da justiça e do direito no sentido de requerer uma outra forma de avaliarmos uns aos outros, privilegiando o acolhimento e não a condenação sumária de quem se mostra diferente. Seria uma provocação para descolarmos o reconhecimento da compaixão, da tolerância e da caridade, para produzirmos uma forma de justiça na qual não só seja possível nos surpreendermos pelo outro, mas também trabalharmos sobre uma outra possibilidade de sermos quem somos. É precisamente esse o desafio: “Como experimentar essas vidas como semelhantes e dessemelhantes? Como não singularizá-las ao extremo?” (MACÉ, 2018, Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa.v.7.e2015837, p.1-20, 2020. 13 REVISTA PAUTA GERAL ESTUDOS EM JORNALISMO 10.5212/RevistaPautaGeral.v.7.15837 p.31). A consideração implica pensar nos entrelaçamentos entre vida social, identidades, vulnerabilidades, justiça, hospitalidade e condições de existência nas quais imigrantes, refugiados e refugiadas, elaboram constantemente em suas narrativas e buscam, com elas, insistir e resistir a partir da migração e das várias desterritorializações e reterritorializações que ela exige. Argumentamos que o processo de consideração envolve duas dimensões interligadas: a problematização de enquadramentos que ainda seguem uma lógica colonial de opressão e controle, e a proposição de outras possibilidades de registro narrativo e visual das formas de vida precárias. De um lado, temos o fotojornalismo feito pela grande mídia que nos revela como os significados dominantes numa dada sociedade sufocam e tornam invisíveis as perspectivas particulares de um grupo específico. Não obstante, as fotografias também oferecem traços para pensarmos acerca de como uma forma de vida liminar encontra alternativas de habitar o mundo, de torná-lo habitável e de circular, de modo camuflado, entre os discursos legitimados e legitimantes. Quando Judith Butler (2004, 2018) nos apresenta o conceito de vida precária, ela destaca que os sujeitos e grupos estão diferentemente expostos à injúria, à agressão, à rejeição e à morte. Além disso, ela argumenta que a vulnerabilidade não é só uma condição ontológica, mas um estado contingente que pode ser modificado, alterando o estatuto de um sujeito ou grupo se considerarmos que os vínculos e condições (materiais, simbólicas, humanas) que nos permitem de viver podem ser acionados de modo a compor arranjos que promovam alternativas e potenciais possibilidades de ruptura. Vulnerabilidades não são essenciais, imutáveis, mas são situadas e resultam de uma complexa rede de múltiplas relações. A vulnerabilidade, assim compreendida, nos revela uma maneira relacional de existir que nos desafia a contemplar o outro, a desacelerarmos nossas expectativas de apreensão e categorização rápida e superficial. Compreendê-lo, portanto, requer tempo, requer abrir espaço à aproximação da alteridade, do inquietante outro que se manifesta diante de nós como rosto, como voz que nos interpela eticamente, rompendo todo e qualquer enquadramento explicativo. Para Butler (2015, p.22), “não há vida e morte sem relação com um determinado enquadramento. Ambas nos são apresentadas dentro de molduras específicas que não apenas estruturam a maneira pela qual passamos a conhecê-las e a identificá-las, mas constituem condições que lhes conferem suporte e legitimidade”. Não se trata apenas de uma operação de seleção e saliência de informações: trata-se de uma poderosa operação Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa.v.7.e2015837, p.1-20, 2020. 14 REVISTA PAUTA GERAL ESTUDOS EM JORNALISMO 10.5212/RevistaPautaGeral.v.7.15837 de julgamento e reconhecimento de sujeitos e grupos e das condições sociais e institucionais que permitem a permanência e espraiamento de códigos específicos de valorização e desvalorização desigual das vidas. Segundo Butler (2015, p.14), as convenções e as normas gerais que agem nos dispositivos de enquadre moldam, por exemplo, um ser humano em um sujeito reconhecível por meio da apreensão, isto é, uma forma de conhecimento associada ao sentir e ao perceber, muitas vezes sem utilizar conceitos (julga-se e condena-se antes de conhecer). O problema, de acordo com Butler (2015, p.20) é tornar evidente como essas normas operam para produzir legibilidades e inteligibilidades através de narrativas capazes de tornar certos sujeitos pessoas reconhecíveis e tornar outros decididamente mais difíceis de reconhecer5. Contra um enquadramento desfigurante presente muitas vezes nos enquadramentos da grande mídia, pelo fotojornalismo, por exemplo, Didier Fassin (2015), assim como Butler, propõe repensarmos as vulnerabilidades como provedoras de uma outra sintaxe ética, como uma ética da responsabilidade que não tem como objetivo condenar os sujeitos a estados crônicos de dificuldades, mas de construir relatos provendo novas ferramentas, habilidades, quadros morais e linguagens alternativas para definir injustiças e politizar injúrias. Macé (2018) se preocupa, ao refletir sobre a condição de sujeitos migrantes, com o modo através do qual as imagens e relatos auxiliam ou coíbem as possibilidades de consideração das formas que vida que ali oscilam entre representação e figuração, ou seja, entre a adequação a papéis sociais impostos e a afirmação persistente de um “como”; de astúcias que não têm relação com a auto reprodução cega e funcional de normas, mas sim com a maneira possível de habitar o mundo, contra a intolerável aceitação e normalização da desigualdade das vidas. Ela argumenta que a experiência de migrantes, as perdas, rupturas e lutos por vivenciados tem a capacidade de nos revelar a brutalidade dos poderes assimétricos, mas também instauram a possibilidade de: Por meio dos enquadramentos e enredos narrativos, a mídia cria um padrão estético e comportamental a ser adotado pela sociedade como parâmetro de julgamento moral. Esse modelo abrange, majoritariamente, pessoas brancas, com visuais e conflitos incomuns a negros. Assim, eles são forçados a adaptar-se para se encaixar em um padrão, que naturalmente não os contempla, e a omitir os seus próprios conflitos, pois não estão em pauta, e, consequentemente, não merecem espaço para discussão. Portanto existe o preconceito que eles sofrem, por serem estrangeiros, por não falarem o português, por serem pobres e principalmente, por serem negros. 5 Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa.v.7.e2015837, p.1-20, 2020. 15 REVISTA PAUTA GERAL ESTUDOS EM JORNALISMO 10.5212/RevistaPautaGeral.v.7.15837 [...] pensarmos nos limiares multiplicados, nos espaços inabitáveis e contudo habitados, nos migrantes que apreendemos por suas penas e perdas, que percebemos apenas como espectros, no impossível lado a lado, na memória fraquejante, com o sentimento de sideração que nasce disso tudo e a violência que essa sideração autoriza cotidianamente. É importante falarmos das vidas que se mantêm, que tentam se manter ou têm que se manter em pleno acampamento; de migrantes que não apreenderíamos apenas por sua invisibilidade e por sua distância em relação à maior parte de nossas vidas; mas a quem nos reportaríamos também por seus gestos, seus sonhos, suas tentativas e sua experiência. Poderíamos falar então do movimento de consideração, de observação, de atenção, delicadeza, cuidado, estima, reabertura de uma relação, de uma proximidade, de uma possibilidade (MACÉ, 2018, p.27-28). Imagens fotográficas que permitem a consideração sobre o outro são também aquelas que oferecem condições à figuração, ou seja, que “faz falar duas vezes o rosto dos anônimos”: por um lado, “como testemunhas mudas de uma condição inscrita diretamente em seus traços, suas roupas, seus modos de vida”; e, por outro, “como detentores de um segredo que nunca iremos saber, um segredo roubado pela imagem mesma que nos traz esses rostos” (RANCIÈRE, 2012, p. 23-24). Dito de outro modo, enquanto a representação tende a imobilizar e fixar sujeitos em categorias que os definem e os submetem; a figuração revela o quão difícil (e mesmo impossível) é reter os sujeitos e a complexidade de suas experiências e modos de vida em uma imagem. Na figuração, o sujeito tem que escapar à nossa tentativa incessante de tudo categorizar, avaliar, julgar e submeter ao já familiar: ele deve permanecer estranho, não familiar e, por isso mesmo, inquietante. Uma imagem não se resume a uma escolha dicotômica entre a sideração e a consideração, assim como ela não abrange só representação ou só figuração. Nos interessa justamente mostrar quando e como passagens entre essas dimensões podem acontecer e que forma assumem na materialidade da imagem, em sua relação com a implicação ou o convite ao olhar do receptor. À guisa de conclusão: por um olhar decolonial no fotojornalismo No caso da imagem aqui analisada, as dimensões interseccionais que atravessam a criação de um enunciado específico permite pensar em como os corpos, rostos, paisagens e objetos podem ser lidos e estudados a partir de um ponto de vista comunicacional, político, estético e ético. Para a produção de um olhar decolonial são necessários lampejos e curto-circuitos que interrompem a linearidade de uma possível Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa.v.7.e2015837, p.1-20, 2020. 16 REVISTA PAUTA GERAL ESTUDOS EM JORNALISMO 10.5212/RevistaPautaGeral.v.7.15837 história daqueles e daquelas que sobrevivem/sobreviveram às vulnerabilidades associadas à migração forçada e despertam, no espectador, novos modos de percepção da imagem, do corpo e do espaço da cena. O que está em jogo aqui não é uma revelação do mundo habitado por esses sujeitos migrantes, mas a possibilidade de olhar a história de novo, de desprogramar o olhar, de trazer posicionamentos variados e considerar seus efeitos no presente (POIVERT, 2010). Assim, acreditamos ser central mostrar como as imagens, dialeticamente, tornam sensíveis – acessíveis, legíveis e dignas de consideração – a vida e a sobrevivência dos sujeitos em situação de vulnerabilidade, ao mesmo tempo em que elas declaram a impotência dos sujeitos oprimidos em situações que os expõem à violência, ao silenciamento e, justamente por isso, demandam outras formas de acolhimento, consideração e hospitalidade. Em relação com a capa da revista Time, e a diversidade de contextos nos quais a fotografia (assim como as icônicas imagens do menino sírio Aylan Kurdi ou da menina vietnamita Kim Puc) compareceu produzida e elaborada para distintas finalidades artística, publicitária, jornalística -, podemos afirmar que a imagem analisada é um entre vários elementos textuais em uma rede ou diagrama em que se dispõem e se articulam diferentes enunciados, atores, forças e fluxos em circulação. A reflexão trazida à luz do pensamento decolonial oferece, portanto, uma possibilidade de repensar os modelos sistematizados e normativos que mantém a invisibilidade destes sujeitos. No campo jornalístico, a produção de figuras e representações ilustrativas, sem densidade, que nutrem ainda os distintos contextos de circulação social, reitera posições e consensos estigmatizantes, tanto quanto propõem modos normalizados e hierarquizados de olhar. E é justamente como parte de um complexo diagrama que resulta de processos de midiatização e de circulação que a figuração pode conectar formas de vida na imagem e para além dela: a biopotência minoritária pode agir sobre a biopolítica do controle, ao revelar como as vulnerabilidades situadas e desafiadas podem desenhar formas de vida para mulheres migrantes que não são facilmente classificáveis, pois são excessivas; existem entre identidades, sobrevivem e escapam à tentativa de serem capturadas. É nos gestos não capturáveis, ambíguos e que geram indecidibilidade nas formas hegemônicas de produção de legibilidades dos corpos e das vidas que pode atuar a biopotência. Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa.v.7.e2015837, p.1-20, 2020. 17 REVISTA PAUTA GERAL ESTUDOS EM JORNALISMO 10.5212/RevistaPautaGeral.v.7.15837 Na fotografia, note-se, o corpo da mulher aparece sem uma cabeça, sem sua face, sem qualquer traço de sua singularidade. No entanto, sua filha assume a vocalização do rosto, do apelo ético e biopotente de suas existências que atua na definição do que é uma vida humana e do que conta como vida sem subestimar as potencialidades, táticas, realizações, imaginários e solidariedades que lhes permitem escapar dos constrangimentos que pesam sobre elas. Essas mulheres migrantes são sobreviventes e buscam criar uma forma de vida que lhes garante um rosto a ser contemplado num jogo de enunciação e de invenção de uma cena dissensual que tematiza os danos causados pela persistente colonialidade de gênero. Referências AKOTIRENE, Carla. O que é interseccionalidade. São Paulo: Sueli Carneiro – Pólen, 2019. ANZALDÚA, Glória. “La conciencia de la mestiza: rumo a uma nova consciência”. Estudos Feministas, Florianópolis, v.13, n.3, p.704-719, 2005. BUTLER, Judith. Quadros de guerra: quando a vida é passível de luto? Rio de Janeiro: Civilização Brasileira, 2015. BUTLER, Judith. Precarious Life. London: Verso, 2004. BUTLER, Judith. Corpos em Aliança. Rio de Janeiro: Civilização Brasileira, 2018. CALLEGARO, Adriana; LAGO, María Cristina. La crónica latinoamericana: cruce entre literatura, periodismo y análisis social. Quórum Académico, vol. 9, núm. 2, juliodiciembre, p. 246-262, 2012. CARON, Carole. Humaniser le regard. Du photojournalisme humanitaire à l'usage humanitaire de la photographie. Commposite, Paris, v.1, p.1-19, 2007. CASTRO-GÓMEZ, Santiago; GROSFOGUEL, Ramón (orgs.). El giro decolonial: reﬂexiones para una diversidad epistémica más allá del capitalismo global. Bogotá: Siglo del Hombre Editores; Universidad Central, Instituto de Estudios Sociales Contemporáneos y Pontiﬁ cia Universidad Javeriana, Instituto Pensar, 2007. CRUZ, Guilherme Silva da. Narrativas do poder: o jornalismo narrativo como outra ferramenta de representação da política e do poder na América Latina. Anais Eletrônicos do Congresso Epistemologias do Sul, Foz do Iguaçu, v. 1, n. 1, p. 103-108, abr., 2017. ____________________. Jornalismo narrativo: uma reflexão sobre representações políticas e simbólicas da América Latina. Chasqui. Revista Latinoamericana de Comunicación, n.141, abril-julio, p. 349-364, 2019. Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa.v.7.e2015837, p.1-20, 2020. 18 REVISTA PAUTA GERAL ESTUDOS EM JORNALISMO 10.5212/RevistaPautaGeral.v.7.15837 FASSIN, Didier. At the heart of the State: the moral world of institutions. London: Pluto Press, 2015. FERRARESE, Estelle.; LAUGIER, Sarah. Formes de vie. Paris: CNRS Éditions, 2018. LEAL, Bruno; ANTUNES, Elton. Desafios metodológicos à pesquisa sobre gênero e comunicação: reflexões a partir de narrativas de um problema cotidiano. Compós, Porto Alegre, 2019. Disponível em http://compos.org.br/anais_texto_por_gt.php?idEncontro=Mjg. Acesso em junho 2019. LEAL, Bruno. Do corpo como texto: na mídia, na rua. Revista Fronteiras, Porto Alegre, v. 8, p.144-151, 2006. LOPES, Maria Immacolata Vassalo de. A teoria barberiana da comunicação. Revista Galáxia, vol. 12, n. 1, p. 39-63, 2018. LUGONES, Maria. Rumo a um feminismo Florianópolis, v.22, n.3, p.935-952, 2014. descolonial. Estudos Feministas, MACÉ, Marielle. Siderar, considerar: migrantes, formas de vida. Rio de Janeiro: Bazar do Tempo, 2018. MARQUES, A. C. S.; BIONDI, A. G. . O doméstico tem um gênero: figurações de mulheres empobrecidas no discurso visual do fotojornalismo. Revista Latinoamericana de Ciencias de la Comunicación, v. 16, p. 86-99, 2019. MARQUES, A. C. S.; BIONDI, A. G. . Vulnerabilidades no enquadramento biopolítico de mulheres empobrecidas em fotografias jornalísticas sobre o Programa Bolsa-Família. In: AGUIAR, Leonel; SILVA, Marcos Paulo da; MARTINEZ, Monica; (orgs.). (Org.). Desigualdades, relações de gênero e estudos de jornalismo. 1ed.São Paulo: Life Editora, 2018, v. 1, p. 225-244. MARQUES, A. C. S.; BIONDI, Angie. (In)visibilidade de mulheres sem rosto: ética e política em imagens fotográficas de Teresa Margolles. Comunicação e Sociedade, v. 32, p. 269-286, 2017. MARTINO, Luís Mauro; MARQUES, Ângela Salgueiro. Fotografias do limiar: dicotomias, fabulações e temporalidades intervalares em imagens de famílias empobrecidas durante a Depressão norte-americana dos anos 1930. Revista Ínterin, vol. 25, n. 2, p. 83-110, 2020. MBEMBE, Achille. Necropolítica. Biopoder, soberania, estado de exceção, política da morte. São Paulo: N-1 Edições, 2018. MIGNOLO, Walter. Desobediência epistêmica: a opção descolonial e o significado de identidade em política. Cadernos de Letras da UFF. Dossiê Literatura, língua e identidade, n.34, p.287-324, 2008. ______________. Desafios Decoloniais Hoje. Epistemologias do Sul. Foz do Iguaçu/PR, 1 (1), pp. 12-32, 2017. Revista Pauta Geral-Estudos em Jornalismo, Ponta Grossa.v.7.e2015837, p.1-20, 2020. 19 REVISTA PAUTA GERAL ESTUDOS EM JORNALISMO 10.5212/RevistaPautaGeral.v.7.15837 PERLINI, T. Le pacte moral comme condition d'existence du photojournalisme humanitaire. In: Imaginaires du présent: Photographie, politique et poétique de l'actualité. Cahier ReMix [online], Montreal, n° 1, mai, 2012. Disponível em http://oic.uqam.ca/fr/remix/le-pacte-moral-comme-condition-dexistence-duphotojournalisme-humanitaire Acesso em janeiro 2019. PERSICHETTI, Simonetta. A encruzilhada do fotojornalismo. Revista Discursos Fotográficos. Londrina, vol. 2, n.2, p. 179-190, 2006. POIVERT, Michel. Destin de l’image performée. In: La photographie contemporaine. Paris: Flammarion, p. 209-235, 2010. RANCIÈRE, Jacques. O espectador emancipado. São Paulo: Martins Fontes, 2010. _________________. O destino das imagens. Rio de Janeiro: Contraponto, 2012. ROSA, Ana Paula. Circulação: das múltiplas perspectivas de valor à valorização do visível. Texto apresentado no VI Colóquio Semiótica das Mídias, CISECO. Japaratinga: Alagoas, p.1-17, 2017. URANGA, Washington. Desarollo, ciudadanía, democracia: aportes desde la comunicacíon. “Integración comercial o diálogo cultural ante el desafío de la sociedad de la información”. 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https://openalex.org/W2994869153	https://bmcplantbiol.biomedcentral.com/track/pdf/10.1186/s12870-019-2143-x	English	null	Dynamic changes in physiological and biochemical properties of flue-cured tobacco of different leaf ages during flue-curing and their effects on yield and quality	BMC plant biology	2,019	cc-by	12,021	Dynamic changes in physiological and biochemical properties of flue-cured tobacco of different leaf ages during flue- curing and their effects on yield and quality Yanjie Chen1,2†, Ke Ren1,2†, Xian He1†, Jiangshiqi Gong1,3, Xiaodong Hu1, Jiaen Su1, Yan Jin1, Zhengxiong Zhao2, Yanmei Zhu1 and Congming Zou1* Chen et al. BMC Plant Biology (2019) 19:555 https://doi.org/10.1186/s12870-019-2143-x Chen et al. BMC Plant Biology (2019) 19:555 https://doi.org/10.1186/s12870-019-2143-x Open Access Abstract Background: The leaf age for harvesting flue-cured tobacco leaves is closely related to the quality of tobacco leaves, so an appropriate leaf age for harvesting is important for improving yield and quality of flue-cured tobacco, however, at present, there are few studies on effects of leaf age on physiological and biochemical changes during flue-curing and there is no clear standard of proper leaf ages for harvesting in production. Results: In the Yunnan tobacco-growing area, an experiment was carried from 2016 to 2017 and different leaf ages were set. The results demonstrate that leaf age has a significant on tissue cell gap, leaf age and flue-curing stages exert significant effects on upper epidermis, palisade and spongy tissue, and leaf thickness of tobacco leaves. The thicknesses of upper and lower epidermis as well as palisade and spongy tissues at different ages show an approximately W-shaped change trend during flue-curing. With the advance of flue-curing stages, contents of starch, chlorophyll, carotenoid, and water in tobacco leaves at different leaf ages decrease, while polyphenol and malondialdehyde (MDA) contents increase. The older the leaf, the faster the chlorophyll, carotenoid, and water contents reduce, while the faster the polyphenol and MDA content rise during flue-curing. The flue-cured tobacco leaves at 116 DAT (days after transplanting) show the highest contents of total nitrogen and nicotine, followed by 123 DAT and those at 130 DAT are the lowest; however, the contents of total sugar and reducing sugar demonstrate a contrary tendency, and the starch content at 116 DAT is much lower than those in the other two treatments. The proportion of superior tobacco, average price, yield, and output value of upper tobacco leaves at different leaf ages are the highest at 123 DAT. The highest sensory evaluation score is found at 123 DAT, while that at 130 DAT is significantly lower in comparison with the other two treatments. Conclusions: Tobacco leaves harvested at 123 DAT are mature and exhibit a low degree of membrane lipid peroxidation, moderate chemical compositions, and high economic value. 123 DAT improves availability of tobacco leaves. words: Leaf age, Curing process, Tissue structure, Physiological and biochemical change, Economic traits * Correspondence: zoucongmingzcm@163.com * Correspondence: zoucongmingzcm@163.com †Yanjie Chen, Ke Ren and Xian He contributed equally to this work. Abstract 1Yunnan Academy of Tobacco Agricultural Sciences, 33 Yuantong Street, Kunming, Yunnan 650021, People’s Republic of China 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. Background increasing leaf age. With gradient changes in leaf age, a unique gradient of nitrogen contents can be formed in leaves. In addition, because old leaves are exposed to the environment with CO2 for a longer time, non-structural carbohydrate contents of old leaves are higher than those of young leaves [10]. As the leaf ages the contents of chlorophyll and soluble sugar in leaves increase, while the protein content decreases [11]. MDA content represents the aging speed of plants: with the increase of leaf age, the MDA content rises, indicating that the older the leaf, the faster the plants age [12]. g Flue-cured tobacco is an important economic crop and timely harvesting and moderate flue-curing are key to en- suring quality. Maturity is regarded as the primary quality factor for grading and is an important index used to meas- ure tobacco quality: tobacco leaf age is one of important factors affecting maturity. Leaf age influences maturity, and further has impacts on flue-curing characteristics of tobacco leaves and yield and quality of flue-cured tobacco leaves. For tobacco, the upper leaves have high economic value but more difficult to get suitable maturity and flue- curing high-quality tobacco because the weather becomes unsuitable for its maturity later in the growing season [1]. Physical properties and chemical and physiological changes in upper tobacco leaves at different ages during flue-curing process have not been studied, so a proper leaf age of upper tobacco leaves has not been determined for harvesting and flue-curing. Leaf age has certain influences on yield and quality of plants. With the increase of leaf age, the growth time of leaves in the field increases, so the probability of being damaged by climate or insects also rises [13]. Tobacco leaves harvested at different ages exhibit inconsistent maturity. In general, output value, the proportion of su- perior tobacco, and average price of flue-cured tobacco leaves in upper, middle, and lower parts increase with leaf age. These indices reach the highest when leaves are mature and gradually reduce with increasing age [14]. Furthermore, leaf age also influences flue-curing charac- teristics of tobacco leaves. Within a certain range, to- bacco leaves become easier to flue-cure, while resistance to flue-curing worsens, with the increase of leaf age [15]. Plants harvested too early or late are more susceptible to post-harvest physiological disorders than those harvested at maturity and hence impairing product quality [16]. © 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. Chen et al. BMC Plant Biology (2019) 19:555 Chen et al. BMC Plant Biology (2019) 19:555 Page 2 of 21 Page 2 of 21 Background Leaf age affects plant growth, and leaf structure and physical properties of plants change accordingly with changes in leaf age. Leaf structure of tobacco leaves is posi- tively correlated with leaf age [2]. Leaves of different ages have differences in morphology and leaf thickness increases on the whole; moreover, the degree of hardening of old leaves is higher than that of young leaves [3]. The leaf structure of tobacco growing in the field increases with the extension of leaf age in a certain range, however, due to double stress of water and nutrient during flue-curing, cells of tobacco leaves dehydrate and shrink during flue-curing and appearance and leaf structure of tobacco leaves change to a significant extent [4]. Research has been conducted into the changes in leaf structure and physical properties of tobacco leaves during flue-curing, while tobacco of differ- ent leaf ages during flue-curing is rarely studied. Physiological and biochemical changes of the upper tobacco leaves at different ages during flue-curing are still unknown and there is no quantitative standard for har- vesting mature leaves at a proper age in production. To explore dynamic changes in structure and physiological and biochemical metabolism of upper tobacco leaves at different ages during flue-curing, we collected tobacco samples from seven flue-curing stages from 2016 to 2017. Based on this, metabolic changes to tobacco leaves at different ages during flue-curing and quality of flue-cured tobacco leaves were investigated, so as to propose a stand- ard for harvesting tobacco leaves at a proper leaf age in the tobacco-growing area of Yunnan Province, China. Leaf age also influences physiological and biochemical changes in plants, thus further affecting plant metabol- ism. Takuro et al. [5] showed that leaf age affects the photosynthetic rate of tobacco leaves, which further af- fects photosynthesis of tobacco leaves. The photosyn- thetic rate of young leaves is higher than that of over- mature old leaves. Some research demonstrates that photosynthetic rate and nitrogen utilisation efficiency show a negative correlation with leaf age, and nitrogen content per unit leaf area decreases with the increasing leaf age [6]. The increase of peroxidase activity is a reli- able index for judging maturity and senescence of plants. With increasing leaf age, peroxidase activity in tobacco leaves decreases, while the degree of lignification in- creases during maturation and senescence [7]. Effects of different leaf ages on morphological characteristics of tobacco leaves Table 1 shows that leaf age, year and their interaction do not have significant influences on tobacco leaf length and leaf width, but leaf age significantly affects tissue cell gap (P < 0.05). The leaf length, leaf width, and tissue cell gap of tobacco leaves increased with the increase of leaf age. Palisade tissue cell gaps at a leaf age of 130 DAT were sig- nificantly higher than those at leaf ages of 123 DAT and 116 DAT, while sponge tissue cell gaps in leaf at 116 DAT were significantly lower than in the other two treatments. The contents of metabolites in plants vary with leaf age and young leaves are better than old ones in preventing biological and abiotic damage [8]. Hikosaka et al. [9] proposed that the nitrogen content is the highest in newly developed young leaves of grapes and decreases with Chen et al. BMC Plant Biology (2019) 19:555 Page 3 of 21 Table 1 Effects of different leaf ages on morphological characteristics of tobacco leaves Leaf age (A) Year (Y) DF Leaf length (cm) Leaf width (cm) Palisade tissue cell gap (%) Sponge tissue cell gap (%) 116 DAT 2016 51.2a 16.3a 9.32c 17.25b 123 DAT 54.7a 17.8a 20.34b 31.47a 130 DAT 55.8a 17.5a 29.89a 34.12a 116 DAT 2017 55.6a 18.4a 9.66c 19.60b 123 DAT 61.2a 21.3a 21.81b 34.57a 130 DAT 63.7a 22.6a 28.94a 25.66a A 2 NS NS < 0.05 < 0.05 Y 1 NS NS NS NS A × Y 2 NS NS NS NS Note: Lowercase letters represent significant differences for different treatments in the same year (P < 0.05) Effects of different leaf ages on lower epidermis thickness of tobacco leaves during flue-curing Effects of different leaf ages on structure of tobacco Effects of different leaf ages on lower epidermis thickness of tobacco leaves during flue-curing Table 2 demonstrates that year and flue-curing stage as well as their interactions significantly influence lower epidermis thickness of tobacco leaves (P < 0.05). Table 2 shows that leaf age, stage, and interactions of leaf age, year, and stage significantly affect upper epider- mis thickness (P < 0.05). It can be seen from Fig. 2 that lower epidermis thickness gradually reduced with the advance of flue-curing. Effects of different leaf ages on morphological characteristics of tobacco leaves Within two years, the lower epidermis thickness at 123 DAT and 130 DAT rapidly reduced in Stages 1 to 4, while it rose slightly in Stages 4 and 5, then slowly decreased thereafter Stage 5. The decreases in Stages 1 to 4 at 123 DAT and 130 DAT in 2016 were greater than in 2017. The lower epidermis thickness at 116 DAT rose slightly before Stage 3 and then rapidly decreased in Stages 3 to 7. As shown in Fig. 1, the upper epidermis thicknesses of tobacco leaves at different ages gradually decreased during flue-curing and approximately showed a tendency to first decrease, then slightly increase, and finally decrease. In 2016, the upper epidermis thickness in 123 DAT rapidly reduced in Stages 1 to 3. The upper epidermis thickness in Stage 3 was significantly lower than that in Stage 1, while that in Stage 4 rose slightly then slowly decreased. At 130 DAT, the upper epidermis thickness gradually de- creased in Stages 1 to 4, while it rose slightly in Stages 5 and 6 and then rapidly reduced after Stage 6. The upper epidermis thickness at 116 DAT rapidly decreased in Stages 3 to 6, while it rose slightly in Stages 6 and 7. In 2017, the upper epidermis thicknesses in Stages 1 to 3 in the three treatments showed a decreasing trend, and the thickness reduced slightly by 116 DAT. The thickness rap- idly decreased and then slightly rose in Stages 4 to 6 at 123 DAT. Moreover, the thickness slowly decreased after Stage 4 in 116 DAT and 130 DAT. Effects of different leaf ages on palisade tissue thickness of tobacco leaves during flue-curing Note: different capital letters indicate the significant differences between different treatments at the same stage. Different lowercase letters denote the significant differences between different stages of the same treatment. The asterisk indicates the significant differences between different years under the same treatments and same period (same below) decreased, then increased and finally decreased in flue- curing stages: however, the thicknesses at 123 DAT and 130 DAT fell quickly in Stages 1 to 3, while slowly de- creasing after a slight increase in Stages 3 to 5, then slowly rising again in Stages 6 and 7. At 116 DAT, the thickness quickly decreased in Stages 1 and 2 and 4 and 5, then slightly increased in Stages 2 to 4 and 5 and 6. It can be observed from Fig. 4 that the spongy tissue thicknesses in the three treatments were all reduced by flue-curing in 2016. Spongy tissue thicknesses at 123 DAT and 130 DAT rapidly decreased in Stages 1 to 3 and then slowly reduced, while the decrease at 116 DAT during flue-curing was smaller than those in the other two treatments. In 2017, the spongy tissue thick- nesses in the three treatments rapidly declined in Stages 1 to 3. The thicknesses of spongy tissue in treat- ments at 123 DAT and 130 DAT rose slightly, then slowly decreased in Stages 3 to 5, while that at 116 DAT were rapidly reduced after undergoing a slight in- crease in Stages 3 and 4, and then slowly increased again in Stages 5 and 6. Effects of different leaf ages on palisade tissue thickness of tobacco leaves during flue-curing As displayed in Table 2, leaf age and flue-curing stage exert significant effects on palisade tissue thickness (P < 0.05). As demonstrated in Fig. 3, palisade tissue thicknesses at 123 DAT and 130 DAT decreased with flue-curing in 2016, of which the thickness rapidly decreased in Stages 1 to 3 and then more slowly after Stage 4. At 116 DAT, the thickness rapidly decreased in Stages 1 to 3, then rose slightly, then slowly reduced in Stages 3 and 4. In 2017, the palisade tissue thickness in different treatments first Table 2 Analysis of variance for the effects of the age of tobacco leaves, year, stage and their interactions on tissue structure Effect/contrast DF Upper epidermis Lower epidermis Palisade tissue Spongy tissue Leaf thickness -------------Probability of a greater F-value-------------- Leaf age (L) 2 0.0049 0.4107 <.0001 0.0025 0.0003 Year (Y) 1 0.0003 0.0411 0.2758 0.0533 0.1895 Stage (S) 6 <.0001 <.0001 <.0001 <.0001 <.0001 LY 2 0.093 0.2566 0.5202 0.1901 0.1859 LS 12 0.0035 0.6867 0.0077 0.4023 0.0938 YS 6 0.0796 0.015 0.6096 0.3335 0.2378 LYS 12 0.0383 0.894 0.4387 0.3115 0.273 Chen et al. BMC Plant Biology (2019) 19:555 Page 4 of 21 Fig. 1 Effects of interactions of different leaf ages, flue-curing stages, and years on upper epidermis thickness. Note: different capital letters indicate the significant differences between different treatments at the same stage. Different lowercase letters denote the significant differences between different stages of the same treatment. The asterisk indicates the significant differences between different years under the same treatments and same period (same below) Fig. 1 Effects of interactions of different leaf ages, flue-curing stages, and years on upper epidermis thickness. Note: different capital letters indicate the significant differences between different treatments at the same stage. Different lowercase letters denote the significant differences between different stages of the same treatment The asterisk indicates the significant differences between different years under the same Fig. 1 Effects of interactions of different leaf ages, flue-curing stages, and years on upper epidermis thickness. Note: different capital letters indicate the significant differences between different treatments at the same stage. Different lowercase letters denote the significant differences between different stages of the same treatment. The asterisk indicates the significant differences between different years under the same treatments and same period (same below) Fig. 1 Effects of interactions of different leaf ages, flue-curing stages, and years on upper epidermis thickness. Effects of different leaf ages on spongy tissue thickness of tobacco leaves during flue-curing Table 2 shows that leaf age and flue-curing stage as well as their interaction show significant effects on spongy tissue thickness (P < 0.05). Chen et al. BMC Plant Biology (2019) 19:555 Page 5 of 21 Fig. 2 Effects of interactions of different leaf ages, flue-curing stages, and years on lower epidermis thickness Fig. 2 Effects of interactions of different leaf ages, flue-curing stages, and years on lower epidermis thickness treatments of different leaf ages firstly reduced and then slightly rose and decreased during flue-curing. treatments of different leaf ages firstly reduced and then slightly rose and decreased during flue-curing. Effects of different leaf ages on tobacco leaf thickness during flue-curing Effects of different leaf ages on tobacco leaf thickness during flue-curing Effects of different leaf ages on tobacco leaf thickne during flue-curing Table 2 displays that leaf age and flue-curing stage sig- nificantly influence leaf thickness (P < 0.05). Effects of different leaf ages on physiological and biochemical indices of tobacco leaves during flue-curing Effects of different leaf ages on starch contents in tobacco leaves during flue-curing Table 3 demonstrates that leaf age, flue-curing stage, and year as well as their interactions have significant ef- fects on the starch content (P < 0.05). Effects of different leaf ages on physiological and biochemical indices of tobacco leaves during flue-curing Effects of different leaf ages on starch contents in tobacco leaves during flue-curing Effects of different leaf ages on physiological and biochemical indices of tobacco leaves during flue-curing Effects of different leaf ages on starch contents in tobacco leaves during flue-curing As displayed in Fig. 5, during the two years, leaf thick- nesses in the three treatments all rapidly decreased in flue-curing Stages 1 to 3, while the reduction tended to be smaller in other stages. In 2016, leaf thickness in treatments of different leaf ages showed a stable decrease during flue-curing. In 2017, the leaf thickness in Table 3 demonstrates that leaf age, flue-curing stage, and year as well as their interactions have significant ef- fects on the starch content (P < 0.05). Chen et al. BMC Plant Biology (2019) 19:555 Page 6 of 21 Fig. 3 Effects of interactions of different leaf ages, flue-curing stages, and years on palisade tissue thickness of tobacco leaves ig. 3 Effects of interactions of different leaf ages, flue-curing stages, and years on palisade tissue thickness of tobacco leave As shown in Fig. Effects of different leaf ages on spongy tissue thickness of tobacco leaves during flue-curing 6, the 123 DAT group showed the highest starch content in flue-curing Stage 1, followed by 116 DAT and 130 DAT during the two years. In Stages 1 to 3, starch contents rapidly decreased in different treatments and those of the three treatments in Stage 3 were significantly lower in comparison with Stage 1. Starch contents at 116 DAT and 123 DAT rapidly reduced again in Stages 4 to 5, while those at 130 DAT rose slightly, then slowly decreased in these stages. Effects of different leaf ages on chlorophyll contents during flue-curing Effects of different leaf ages on chlorophyll contents during flue-curing Effects of different leaf ages on chlorophyll contents during flue-curing Table 3 indicates that leaf age, flue-curing stage and year as well as their interactions exert significant effects on contents of chlorophylls a and b (P < 0.05). It can be seen from Fig. 7 that contents of chloro- phylls a and b in different treatments decreased dur- ing flue-curing. Contents of chlorophyll a in different treatments showed significant differences in flue- curing Stages 1 and 2 and the decrease in chlorophyll Chen et al. BMC Plant Biology (2019) 19:555 Page 7 of 21 Fig. 4 Effects of interactions of different leaf ages, flue-curing stages, and years on spongy tissue thickness of tobacco leaves and 4 at 116 DAT were higher than those in the other treatments. a content in Stages 1 and 2 of the three treatments in 2016 was larger than that in 2017. Contents of chlorophyll a slowly decreased in other stages after Stage 2. Contents of chlorophyll b in different treat- ments largely reduced in Stages 1 and 2 and the de- crease in 2016 was greater than that in 2017. Contents of chlorophylls a and b at 116 DAT in each stage in 2016 were higher than those in the other two treatments and the contents thereof in Stages 3 Effects of different leaf ages on carotenoid contents during flue-curing According to Table 3, leaf age, flue-curing stage, and year as well as their interactions have significant impacts on carotenoid contents (P < 0.05). Chen et al. BMC Plant Biology (2019) 19:555 Page 8 of 21 Fig. 5 Effects of interactions of different leaf ages, flue-curing stages, and years on tobacco leaf thickness Fig. 5 Effects of interactions of different leaf ages, flue-curing stages, and years on tobacco leaf thickness As shown in Fig. 8, carotenoid contents in different treatments decreased during flue-curing. Carotenoid contents at 116 DAT were greater than those in the other two treatments in each flue-curing stage in 2016. The three treatments were such that the carotenoid con- tents largely decreased in flue-curing Stages 1 to 3 in 2016 and 2017, of which the content in Stage 3 was significantly lower than those in Stage 1 and the rate of change decreased in the three treatments after Stage 3. After flue-curing, the three treatments demonstrated that carotenoid contents in Stage 7 were significantly different and the highest content was found at 116 DAT during the two years. Effects of different leaf ages on total sugar and reducing sugar in flue-cured tobacco leaves Effects of different leaf ages on total sugar and reducing sugar in flue-cured tobacco leaves In accordance with Table 4, leaf age and year as well as their interaction have significant effects on the contents of total sugar (P < 0.05), and leaf age and its interaction with year significantly affect the contents of reducing sugar (P < 0.05). Effects of different leaf ages on total nitrogen and nicotine in flue-cured tobacco leaves Table 4 shows that leaf age, year and their interaction significantly influence contents of total nitrogen and nicotine (P < 0.05). In 2016, contents of total nitrogen at 116 DAT were significantly higher than those in the other two treatments, while nicotine contents at 130 DAT were significantly lower than those elsewhere. In 2017, the contents of total nitrogen were the highest at 116 DAT, followed by that at 130 DAT and the lowest contents were found at 123 DAT: however, nicotine contents peaked at 123 DAT, followed by 130 DAT and 116 DAT. Under the same treatment, the contents of total nitrogen at 116 DAT demonstrated significant Effects of different leaf ages on MDA contents during flue- curing Effects of different leaf ages on polyphenol contents during flue-curing In 2016, the contents of total sugar and reducing sugar at 116 DAT were significantly lower than those in the other two treatments. In 2017, the contents of total sugar were the highest at 130 DAT, followed by 116 DAT, while the lowest contents were found at 123 DAT. Moreover, 123 DAT, 116 DAT, and 130 DAT were ranked from high to low according to the contents of re- ducing sugar. Under the same treatment, contents of total sugar and reducing sugar at 116 DAT were signifi- cantly different in different years and the contents of re- ducing sugar at 130 DAT showed significant differences in different years. Table 3 shows that leaf age, flue-curing stage, and year as well as their interactions significantly influence poly- phenol contents (P < 0.05). p As demonstrated in Fig. 10, polyphenol contents in differ- ent treatments gradually rose during flue-curing and the contents thereof at 123 DAT and 130 DAT were higher than that at 116 DAT on the whole. In 2016, polyphenol contents in different treatments significantly increased in Stages 1 to 2 and rose slowly rose after decreasing in Stages 3 and 4. Polyphenol contents in Stage 7 were significantly higher than those in other stages. In 2017, polyphenol con- tents in Stages 3 and 4 rapidly increased at 123 DAT and 130 DAT and were significantly higher than those in Stages 1 and 2. Moreover, the content thereof at 116 DAT rapidly increased in Stages 1 to 3 and then rose gradually after a large decrease in Stages 3 and 4. Effects of different leaf ages on water contents during flue- curing In accordance with Table 3, the interaction between leaf age and flue-curing stage as well as between year and flue-curing stage significantly affect water con- tents (P < 0.05). Chen et al. BMC Plant Biology (2019) 19:555 Page 9 of 21 Table 3 Analysis of variance for the effects of the age of tobacco leaves, year, stage and their interactions on physiological changes Effect/contrast DF Starch Chlorophyll a Chlorophyll b Carotenoid Water Polyphenol MDA -------------------------Probability of a greater F-value------------------------ Leaf age (L) 2 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 Year (Y) 1 <.0001 <.0001 <.0001 <.0001 0.0202 <.0001 <.0001 Stage (S) 6 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 LY 2 <.0001 <.0001 <.0001 <.0001 0.0674 <.0001 <.0001 LS 12 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 YS 6 <.0001 <.0001 <.0001 <.0001 0.0045 <.0001 <.0001 LYS 12 <.0001 <.0001 <.0001 <.0001 0.0023 <.0001 <.0001 Stages 3 to 5 and then the increase slowed down there- after. The rising MDA content at 116 DAT was greater than those in the other two treatments in 2016, while the amplitude at 130 DAT was larger than those in the other treatments in 2017. Stages 3 to 5 and then the increase slowed down there- after. The rising MDA content at 116 DAT was greater than those in the other two treatments in 2016, while the amplitude at 130 DAT was larger than those in the other treatments in 2017. As shown in Fig. 9, with the advance of flue-curing, the moisture content in tobacco leaves in treatments of different ages gradually reduced. Moisture was lost slowly at first, then fast, and then slowly in tobacco leaves in different treatments during flue-curing. Less moisture in tobacco leaves was lost at a slower rate in Stages 1 to 4, while tobacco leaves lost a lot of moisture and showed a rapid rate of moisture loss in Stages 4 to 6. Moreover, the moisture loss rate of tobacco leaves slo- wed down in Stages 6 and 7. In different treatments, moisture contents in Stage 6 were lower than those in Stages 1 to 5. In Stages 4 to 6, moisture was lost fastest at 123 DAT, followed by 116 DAT, and 130 DAT. curing Based on Table 3, leaf age, flue-curing stage, and year as well as their interactions exert significant influences on MDA content (P < 0.05). It can be seen from Fig. 11 that MDA contents in dif- ferent treatments gradually increased during flue-curing. MDA contents in different treatments rapidly rose in Chen et al. BMC Plant Biology (2019) 19:555 Page 10 of 21 Fig. 6 Effects of interactions of different leaf ages, flue-curing stages, and years on starch contents of tobacco leaves Fig. 6 Effects of interactions of different leaf ages, flue-curing stages, and years on starch contents of tobacco leaves contents at 123 DAT were much higher than those in the other two treatments in 2017. Under the same treat- ment, starch contents in different years were signifi- cantly different. differences in different years and nicotine contents in the three treatments were significantly different in differ- ent years. leaves As shown in Table 5, year has significant effects on yield of upper tobacco leaves (P < 0.05). In the same year, yield differences were insignificant in different treatments and the highest yield was found at 123 DAT in 2016 and 2017. Under the same treatment, yields at 123 DAT and 130 DAT were significantly different in different years and yields in the three treatments in 2016 were higher than those in 2017. Effects of different leaf ages on polyphenol contents in tobacco leaves Table 4 shows that leaf age, year, and their interaction significantly influence starch contents (P < 0.05). In 2016, starch contents at 116 DAT were significantly lower than those in the other treatments, while the Table 4 demonstrates that leaf age and year have signifi- cant influences on polyphenol contents (P < 0.05). In 2016, polyphenol contents at 123 DAT were significantly Chen et al. BMC Plant Biology (2019) 19:555 Page 11 of 21 Fig. 7 Effects of interactions of different leaf ages, flue-curing stages, and years on chlorophyll contents of tobacco leaves ig. 7 Effects of interactions of different leaf ages, flue-curing stages, and years on chlorophyll contents of tobacco leaves than those in 2016, while the score at 130 DAT in 2016 was higher than that in 2017. higher than those elsewhere, while differences in poly- phenol contents in different treatments were not sig- nificant in 2017. The polyphenol contents were the highest at 116 DAT, followed by that at 123 DAT and the lowest contents were found at 130 DAT. Under the same treatment, polyphenol contents at 116 DAT and 130 DAT were significantly different in different years. Effects of different leaf ages on economic traits of flue- cured tobacco leaves Effects of different leaf ages on sensory evaluation scores of flue-cured tobacco leaves It can be observed from Table 4 that leaf age affects sensory evaluation scores of tobacco leaves (P < 0.05). Sensory evaluation scores at 130 DAT were signifi- cantly lower than those in the other two treatments in 2016 and 2017. In both years, sensory evaluation scores were the highest at 123 DAT, followed by 116 DAT, while 130 DAT had the lowest scores. Same treatment exhibited insignificant differences of sensory evaluation scores in different years. Sensory evaluation scores at 116 DAT and 123 DAT in 2017 were higher leaves Table 5 demonstrates that average prices in different treatments in the same year differed insignificantly and they were the highest at 123 DAT in 2016 and Chen et al. BMC Plant Biology (2019) 19:555 Page 12 of 21 Fig. 8 Effects of interactions of different leaf ages, flue-curing stages, and years on carotenoid contents of tobacco leaves Fig. 8 Effects of interactions of different leaf ages, flue-curing stages, and years on carotenoid contents of tobacco leaves Fig. 8 Effects of interactions of different leaf ages, flue-curing stages, and years on carotenoid contents of tobacco leave output value of upper tobacco leaves. The highest output value was found at 123 DAT in 2016 and 2017. Output values at 123 DAT in 2016 were 1.86 and 6.28% higher than those at 130 DAT and 116 DAT and 8.22 and 4.17% higher than those at 130 DAT and 116 DAT in 2017. Output values in dif- ferent years in the same treatment showed insignifi- cant differences and the three treatments showed that output value in 2016 was higher than that in 2017. 2017. Significant differences in average prices were not found in the same treatment in different years. In 2017, average prices at 116 DAT and 123 DAT were higher than those in 2016 and 130 DAT showed the highest average prices in 2016. Effects of different leaf ages on the proportion of superior tobacco of upper tobacco leaves in different years were insignificant, while the propor- tions in the three treatments in 2016 were higher than those in 2017. It can be seen from Table 5 that leaf age, year, and their interaction have no significant influence on the proportion of superior tobacco. In the same year, the proportions of superior tobacco showed insignificant differences in different treatments and the highest proportions of superior tobacco in 2016 and 2017 were found at 123 DAT. Under the same treatment, the differences in the proportions of superior tobacco Effects of different leaf ages on output value of upper tobacco leaves As displayed in Table 5, leaf age and year, as well as their interaction did not significantly affect the Chen et al. BMC Plant Biology (2019) 19:555 Page 13 of 21 Fig. 9 Effects of interactions of different leaf ages, flue-curing stages, and years on moisture contents of tobacco leaves Fig. 9 Effects of interactions of different leaf ages, flue-curing stages, and years on moisture contents of tobacco leaves Effects of different leaf ages on the proportion of superior tobacco of upper tobacco leaves Effects of different leaf ages on the proportion of superior tobacco of upper tobacco leaves Morphological characteristics of tobacco leaves at different leaf age Different leaf age tobacco leaves have different morpho- logical characteristics. For example, the leaf length, and leaf width of old tobacco leaves exceed those of young Chen et al. BMC Plant Biology (2019) 19:555 Page 14 of 21 Fig. 10 Effects of interactions of different leaf ages, flue-curing stages, and years on polyphenol contents of tobacco leaves ig. 10 Effects of interactions of different leaf ages, flue-curing stages, and years on polyphenol contents of tobacco leaves tobacco leaves, at present, farmers, in tobacco leaf pro- duction, judge whether the tobacco leaves are mature or not mainly according to tobacco blade colour, main vein colour and villus condition. In a certain growing period, the length and width of tobacco leaves are variable and rainfall, temperature and soil condition will affect the growth of leaf length and width [17]. In this research, leaf length and leaf width did not show any significant difference between different leaf age, because tobacco leaf length and width did not increase continuously with growing time [18]. Therefore, tobacco leaf length and width can be regarded as reference indicators for to- bacco maturity but not judgement indicators. Tissue cell gap is an indirect reflection of tobacco leaf age as it increases with the senescence of tobacco [19]. The older the leaf, the bigger the tissue cell gap. In this research, for palisade tissue cell gaps, different leaf age tobacco leaves had a significant difference, 130 DAT leaf Chen et al. BMC Plant Biology (2019) 19:555 Page 15 of 21 Fig. 11 Effects of interactions of different leaf ages, flue-curing stages, and years on MDA contents of tobacco leaves Fig. 11 Effects of interactions of different leaf ages, flue-curing stages, and years on MDA contents of tobacco leaves Structural changes in tobacco leaves at different ages during flue-curing The structure of tobacco leaves firstly depends on leaf age for harvesting fresh tobacco leaves, because the age of fresh tobacco leaves determines maturity, while maturity sets the framework of the structure of to- bacco leaves. Moreover, structure is limited by flue- curing [20, 21]. Changes of leaf thickness and struc- ture are mainly related to dehydration and inclusion Structural changes in tobacco leaves at different ages during flue-curing Structural changes in tobacco leaves at different ages during flue-curing Structural changes in tobacco leaves at different ages during flue-curing 1 to 6, the thinning amplitudes of palisade and spongy tis- sues are much greater than those of the upper and lower epidermis during flue-curing, indicating that changes in leaf thickness mainly result from palisade and spongy tissues. During flue-curing, palisade and spongy tissues in the three treatments rapidly shrink in Stages 1 to 3 because peak shrinkage of palisade and spongy tissues during flue-curing is found be- tween 35 and 38 °C, during which the acceleration of drying of tobacco leaves results in rapid shrinkage of palisade and spongy tissues [24]. During flue-curing, leaf thickness slightly increases in Stages 6 and 7, because leaf thickness slightly increases after tobacco leaves regain moisture and absorb water. Leaf changes. During flue-curing, dehydration of tobacco leaves and inclusion changes mainly occur in the yel- lowing stage and leaf-drying stage [22]. In this experi- ment, reduction of leaf thickness is mainly shown in Stages 2 to 5 during flue-curing, that is, the yellow- ing, and early leaf-dying stages. The lower epidermis thickness at 116 DAT increases before Stage 3. Some studies [23] also show that the lower epidermis thick- ness of immature tobacco leaves increases before ris- ing to 38 °C during flue-curing. Based on Figs. 1 to 6, the thinning amplitudes of palisade and spongy tis- sues are much greater than those of the upper and lower epidermis during flue-curing, indicating that changes in leaf thickness mainly result from palisade and spongy tissues. During flue-curing, palisade and spongy tissues in the three treatments rapidly shrink in Stages 1 to 3 because peak shrinkage of palisade and spongy tissues during flue-curing is found be- tween 35 and 38 °C, during which the acceleration of drying of tobacco leaves results in rapid shrinkage of palisade and spongy tissues [24]. During flue-curing, leaf thickness slightly increases in Stages 6 and 7, because leaf thickness slightly increases after tobacco leaves regain moisture and absorb water. Leaf Note: Lowercase letters represent significant differences for different treatments in the same year (P < 0.05); * indicates significant di treatment in different years (P < 0.05); ** denotes the significant difference of each factor and their interaction on the index Structural changes in tobacco leaves at different ages during flue-curing age treatments have the maximum value of palisade tis- sue cell gap in 2016 and 2017. For sponge tissue cell gap, a leaf age of 116 DAT was significantly lower than other two leaf age treatments, and a leaf age of 123 DAT and 130 DAT did not make a significant difference be- cause, when the tobacco leaf is younger, their cell tissue structure is tight, and the tissue cell gap is small. While when the tobacco leaf ages, the cells shrink, and the tis- sue cell gap decreases [20]. The structure of tobacco leaves firstly depends on leaf age for harvesting fresh tobacco leaves, because the age of fresh tobacco leaves determines maturity, while maturity sets the framework of the structure of to- bacco leaves. Moreover, structure is limited by flue- curing [20, 21]. Changes of leaf thickness and struc- ture are mainly related to dehydration and inclusion Chen et al. Structural changes in tobacco leaves at different ages during flue-curing BMC Plant Biology (2019) 19:555 Page 16 of 21 Table 4 Effects of the leaf age, year, and their interactions on chemical compositions and sensory evaluation score Leaf age (A) Year (Y) DF Total sugar content (%) Reducing sugar content (%) Nitrogen content (%) Nicotine content (%) Starch content (%) Polyphenol content (mg/g) Sensory evaluation score 123 DAT 27.33 a 24.41 a 2.46 b 3.57 a* 6.3 a* 26.21 a 90.5 a 130 DAT 2016 28.19 a 26.08 a* 2.05 b 3.09 b* 6.62 a* 19.81 b* 85 b 116 DAT 2017 29.56 a* 22.21 a* 2.32 a* 2.19 a* 2.65 b* 29.87 a* 90 a 123 DAT 26.19 a 22.45 a 2.18 a 2.38 a* 4.43 a* 31.98 a 91 a 130 DAT 29.73 a 20.03 a* 2.26 a 2.32 a* 2.81 b* 28.94 a* 85 b A 2 Y 1 NS NS A × Y 2 ** NS NS Note: Lowercase letters represent that differences are significant in different treatments in the same year (P < 0.05); * indicates the significant difference in the same treatment in different years (P < 0.05); ** denotes the significant differences of each factor and their interactions on this index e leaf age, year, and their interactions on chemical compositions and sensory evaluation score Note: Lowercase letters represent that differences are significant in different treatments in the same year (P < 0.05); * indicates the significant difference in the same treatment in different years (P < 0.05); ** denotes the significant differences of each factor and their interactions on this index thickness is a sensitive to water conditions of plants. When leaves lose water and shrink, leaf thickness re- duces, while it moderately rises when leaves absorb water and expand [25]. changes. During flue-curing, dehydration of tobacco leaves and inclusion changes mainly occur in the yel- lowing stage and leaf-drying stage [22]. In this experi- ment, reduction of leaf thickness is mainly shown in Stages 2 to 5 during flue-curing, that is, the yellow- ing, and early leaf-dying stages. The lower epidermis thickness at 116 DAT increases before Stage 3. Some studies [23] also show that the lower epidermis thick- ness of immature tobacco leaves increases before ris- ing to 38 °C during flue-curing. Based on Figs. epresent significant differences for different treatments in the same year (P < 0.05); * indicates significant differences for the same rs (P < 0.05); ** denotes the significant difference of each factor and their interaction on the index Physiological and biochemical changes of tobacco leaves at different ages during flue-curing In the whole flue- curing stage in 2016, the polyphenol content was the highest at 130 DAT, followed by that at 123 DAT, while that at 116 DAT showed the lowest content; however, 123 DAT, 130 DAT, and 116 DAT were ranked from high to low according to polyphenol content in 2017. This is because more mature the to- bacco leaf in the field, the higher the polyphenol con- tent; however, when the tobacco leaves are completely mature, polyphenol contents reduce and are affected by environmental factors [31]. During flue-curing, due to thermolysis of phenolic glycoside and enzymatic decomposition, phenolic substances of flue-cured to- bacco change to significant extents; because of pyro- lytic conversion of pyrolysates, such as lignin and cellulose in tobacco leaves, polyphenol contents in different treatments generally rise during flue-curing, while they fall slightly in Stages 3 to 5. The reason for this that this stage is the sensitive period of browning reaction and polyphenol substances are eas- ily oxidised to quinones [32]. Starch metabolism, as the primary metabolism in tobacco leaves, affects the internal quality of tobacco leaves. Starch contents in flue-cured tobacco leaves in different treatments rise with increasing leaf age, because starch contents in tobacco leaves gradually increase with greater maturity. The maturation stage of flue-cured tobacco is a period of rapid synthesis and accumulation of starches in tobacco leaves, and accumulation of starches in tobacco leaves with long leaf age is larger than that at short leaf age. Starch in tobacco leaves peaks at physiological maturity stage and then begins to decrease [37, 38]. Polyphenol, as an important index of measuring quality of tobacco, influences colour and lustre as well as aroma and taste of tobacco leaves. Phenylalan- ine ammonialyase is the key enzyme used to catalyse the synthesis of polyphenols, while polyphenol oxidase is the key enzyme used to catalyse the degradation of polyphenols. The activities of such two enzymes dur- ing flue-curing determine polyphenol contents [39]. Polyphenol contents in tobacco leaves rise with ma- turity and polyphenol accumulation begins to de- crease when reaching physiological maturity [40]. Polyphenol content in flue-cured tobacco leaves at 123 DAT is higher than those at 116 DAT and 130 DAT, indicating that 123 DAT is moderately mature and a lot of polyphenols are accumulated in fresh to- bacco leaves. Physiological and biochemical changes of tobacco leaves at different ages during flue-curing Starch metabolism, as the primary metabolism in tobacco leaves, affects the internal quality of tobacco leaves. Starch contents in flue-cured tobacco leaves in different treatments rise with increasing leaf age, because starch contents in tobacco leaves gradually increase with greater maturity. The maturation stage of flue-cured tobacco is a period of rapid synthesis and accumulation of starches in tobacco leaves, and accumulation of starches in tobacco leaves with long leaf age is larger than that at short leaf age. Starch in tobacco leaves peaks at physiological maturity stage and then begins to decrease [37, 38]. by climatic conditions, such as temperature, light, and rainfall [28], while the region showed different cli- matic conditions in 2016 and 2017. In different treat- ments, a lot of water is lost quickly in Stages 4 to 6, which is the late yellowing stage and leaf-drying stage during flue-curing. During this period, a lot of chloro- phylls in tobacco leaves degrade, dehydrate and dry, and decrease rate of water in this period is fast [29]. sugar of tobacco leaves after flue-curing in the two years, while 116 DAT, 123 DAT, and 130 DAT were ranked from large to small in accordance with content of total nitrogen. In comparison with 116 DAT and 123 DAT, 130 DAT has an older leaf age, so its maturity is higher. With increasing maturity, the content of reducing sugar increases, while content of total nitrogen decreases [35]. With the increase of maturity, tobacco leaves gradually change from C-metabolism and accumulation to N- metabolism, so sugar contents in flue-cured tobacco leaves reduce, while total nitrogen and nicotine rise [36]. The senescence of plant tissue is always accompan- ied by the damage to intracellular membrane struc- tures and the MDA content indicates the extent of damage to the membrane. In flue-curing Stages 3 to 6, MDA contents of tobacco leaves at different ages increase greatly, while water contents of tobacco leaves decrease in the same period. Some research shows that the changes in MDA contents during flue- curing are closely correlated with dehydration rate. Rapid dehydration of tobacco leaves is the direct cause of drastic membrane lipid peroxidation and in- crease of MDA contents [30]. Physiological and biochemical changes of tobacco leaves at different ages during flue-curing Moreover, phenylalanine ammonialyase of tobacco leaves with a moderate maturity during flue-curing has a high activity and is not easily oxi- dised into quinone by polyphenol oxidase [41]. During flue-curing, much of the starch is degraded in flue-curing Stages 1 to 3 and 5 and 6: the degradation of starches mainly results from combined action of amylase and amylophosphorylase. During flue-curing, starches are transferred into reducing sugar in the yellowing stage and early stem-drying stage and activities of amylase and amylophosphorylase show two peaks in middle yellowing stage and early leaf-drying stage. At the two peaks, starches are largely degraded [33, 34]. Physiological and biochemical changes of tobacco leaves at different ages during flue-curing Plastid pigments mainly include chlorophyll and ca- rotenoid, which are important aroma precursors influ- encing appearance and quality of tobacco leaves. In flue-curing Stage 1, chlorophyll and carotenoid con- tents are the highest at 116 DAT, followed by 123 DAT, while the lowest contents are found at 130 DAT, because pigment contents reduce during the maturation process [26]. Degradation rates of chloro- phylls at 116 DAT during flue curing are faster than those at 123 DAT and 130 DAT, which results from the too-young leaf age, so that chlorophyll degrad- ation in tobacco leaves with a low maturity is higher than that of tobacco leaves with a high maturity [27]. It can be found (Figs. 7 and 8) that chlorophyll and carotenoid contents are significantly different in different years. The reason is that degradation and transformation of plastid pigments are greatly affected Table 5 Effects of the leaf age, year, and their interactions on economic traits of upper leaves Leaf age (A) Year (Y) DF Yield (kg/ha) Average price (dollar/kg) Output values (dollar/ha) Proportions of superior tobacco (%) 123 DAT 860.56 a* 3.54 a 3036.63 a 66.42 a 130 DAT 2016 850.67 a* 3.5 a 2981.07 a 65.58 a 116 DAT 2017 743.3 a 3.53 a 2631.96 a 62.68 a 123 DAT 754.9 a* 3.63 a 2741.61 a 65.64 a 130 DAT 728.19 a* 3.47 a 2533.44 a 62.34 a A 2 NS NS NS NS Y 1 ** NS NS NS A × Y 2 NS NS NS NS Note: Lowercase letters represent significant differences for different treatments in the same year (P < 0.05); * indicates significant differences for the same Page 17 of 21 Page 17 of 21 Chen et al. BMC Plant Biology (2019) 19:555 sugar of tobacco leaves after flue-curing in the two years, while 116 DAT, 123 DAT, and 130 DAT were ranked from large to small in accordance with content of total nitrogen. In comparison with 116 DAT and 123 DAT, 130 DAT has an older leaf age, so its maturity is higher. With increasing maturity, the content of reducing sugar increases, while content of total nitrogen decreases [35]. With the increase of maturity, tobacco leaves gradually change from C-metabolism and accumulation to N- metabolism, so sugar contents in flue-cured tobacco leaves reduce, while total nitrogen and nicotine rise [36]. Economic traits of upper tobacco leaves at different ages after flue-curing Mature tobacco leaves at a proper age have higher quality and economic value. If the leaf is too young, then such short leaves are not matured adequately and their weights are insufficient after picking, which reduces yield and causes economic loss. If a leaf is too long, vegetables such as lettuce, escarole, spinach, Swiss chard are difficult to store and process [13]. In the experiment, the proportion of superior tobacco, average price, yield and output value of upper tobacco Chemical quality of tobacco leaves at different ages after flue-curing 130 DAT, 123 DAT, and 116 DAT were roughly ranked from large to small according to the content of reducing Chen et al. BMC Plant Biology (2019) 19:555 Page 18 of 21 Page 18 of 21 Page 18 of 21 Chen et al. BMC Plant Biology (2019) 19:555 leaves at 123 DAT show good economic traits and the highest sensory evaluation score. leaves in 123 DAT are superior to those in the other two treatments. In comparison with specimens at 123 DAT, 116 DAT shows a shorter leaf age and lower maturity while 130 DAT demonstrates longer leaf age and over-mature tobacco leaves. In the three treat- ments, the yield of upper leaves at 116 DAT is low- est, because the dry weight of crops is highly correlated with leaf area and the increase of leaf area can increase the dry weight of crops to some extent [42]. Leaf age is an important factor in controlling leaf area index to improve yield [43]. At 116 DAT, the tobacco leaves are young and smaller in length and width, so that less dry matter is accumulated on a small area of leaf. The research demonstrates that upper tobacco leaves at 123 DAT were harvested at a leaf age with an appropriate maturity and have an optimal chemical composition after flue-curing. Moreover, their eco- nomic value is maximised and the availability of to- bacco leaves is improved. For upper tobacco leaves that are too young, pigments and starches degrade slowly during flue-curing and contents of total nitro- gen and nicotine are high in such flue-cured tobacco leaves. While flue-curing tobacco leaves at too great an age, water is fast lost and pigments are quickly de- graded, so polyphenol and MDA contents in flue- cured tobacco leaves increase. Experimental location and materials This study was conducted at Yiliang, Kunming, Yun- nan, China (24°91′N 103°14′W). The soil was a sandy red soil (silty loam), which is the dominant soil type for flue-tobacco production in Yunnan. This research site has an altitude of 1539 m, with annual average temperature of 16.3 °C, an annual average total pre- cipitation of 912 mm and 2177 h of annual average sunshine. Details of soil nutrient levels in the research site are provided in Table 6 and a summary of the mean monthly temperatures and total monthly pre- cipitation is provided in Table 7. Economic traits of upper tobacco leaves at different ages after flue-curing To sum up, 123 DAT can be selected as the quantitative standard for prop- erly harvesting mature tobacco leaves in production. Previous studies found that tobacco leaves with a high maturity have increasing average price and yield, are golden yellow and contain sufficient oil after flue-curing [44]. In the research, yield, output value, and average price of upper tobacco leaves at 123 DAT were superior to those in the other two treatments, demonstrating that tobacco leaves at 123 DAT were harvested at an appro- priate maturity. Conclusion Age of tobacco leaves lays the foundation of leaf structure and chemical compositions and harvesting at an optimal leaf age is an important precondition to ensure quality of tobacco leaves during flue-curing. This study compared physiological and biochemical metabolism of tobacco leaves in treatments of differ- ent leaf ages during flue-curing and found that tissue cell gap increased with the increase of leaf age, the thicknesses of upper and lower epidermis as well as palisade and spongy tissues show a W-shaped trend during flue-curing. The older the leaf, the faster the thickness of its structure decreases. With the advance of flue-curing, starch, chlorophyll, carotenoid, and water contents in tobacco leaves at different ages de- crease, while polyphenol and MDA contents increase. Furthermore, the older the leaf, the faster the chloro- phyll, carotenoid, and water contents reduce during flue-curing, while the faster the polyphenol and MDA contents increase. With the increase of leaf age, con- tents of total sugar, reducing sugar, and starch in flue-cured tobacco leaves rise, while contents of total nitrogen and nicotine reduce. Flue-cured tobacco Table 6 Contents of soil nutrients in Yiliang tobacco-growing area Soil Type pH Organic matter (g·kg−1) Total nitrogen (g·kg−1) Total phosphorus (g·kg−1) Total potassium (g·kg−1) Soil available nitrogen (mg·kg−1) Soil available phosphorus (mg·kg−1) Soil available potassium (mg·kg−1) Red soil 6.42 23.96 0.24 0.18 1.92 119.37 29.53 168.74 Requirement for field management q g Tobacco varieties K326 were provided by Zhongyan Tobacco Seed Co., Ltd., China. For the tobacco plants assessed in the experiment, young seedlings growing under film were transplanted to the field on 15 April 2016 and 18 April 2017, respectively. Pure nitrogen at 90 kg/ha was applied during transplanting and fertil- iser at N: P2O5: K2O (1:1:2.5) was applied. More fer- tiliser from the bottom of the ponds was applied and 50% of the total amount thereof was used as base fer- tiliser, while 25% was used as seedling promoting fer- tiliser, and the other 15% was additional fertiliser. All fertiliser was applied 25 d after transplantation. When the tobacco plants grew to 23 to 25 leaves, they were topped: at time of topping (removal of flowers from Table 6 Contents of soil nutrients in Yiliang tobacco-growing area Soil Type pH Organic matter (g·kg−1) Total nitrogen (g·kg−1) Total phosphorus (g·kg−1) Total potassium (g·kg−1) Soil available nitrogen (mg·kg−1) Soil available phosphorus (mg·kg−1) Soil available potassium (mg·kg−1) Red soil 6.42 23.96 0.24 0.18 1.92 119.37 29.53 168.74 Page 19 of 21 Chen et al. BMC Plant Biology (2019) 19:555 Table 7 The average monthly air temperature and total monthly precipitation (2016 to 2017) Year Precipitation April May June July August September Growing season —————————————mm————————————— 2016 14.4 58.5 57.6 47.7 136.6 115.2 430 2017 40.5 21.8 178.4 318.4 145.5 125.4 830 ————————————Temperature, °C —————————— 2016 20.2 22.2 22.7 22.7 22.5 20.0 130.3 2107 18.3 21.2 23.1 21.3 22.5 21.8 128.2 bulb temperature of 54 °C), and Stage 7 (after flue- curing). bulb temperature of 54 °C), and Stage 7 (after flue- curing). Economic traits Table 8 Experiment design and experiment code Year Transplanting date Harvested date for upper leaves Treatment code 2016 15 April 9 August 116 DAT (Days after transplanting) 16 August 123 DAT (Days after transplanting) 23 August 130 DAT (Days after transplanting) 2017 18 April 12 August 116 DAT (Days after transplanting) 19 August 123 DAT (Days after transplanting) 26 August 130 DAT (Days after transplanting) Table 8 Experiment design and experiment code We had the tobacco samples rated in accordance with National Standard (GB2635–92) and recorded the grad- ing results. According to national purchasing data and supervision and inspection data of grade quality during industry-commerce handover in that year, the average price and proportion of superior tobacco could be esti- mated. Moreover, based on weight of the tobacco sam- ples, yield, and output value of tobacco leaves were calculated. Analysis of physiological indices y y g Contents of chlorophyll, carotenoid, water, starch, polyphenol, and MDA of the tobacco samples in the seven flue-curing stages were determined. Chlorophyll and carotenoid were determined by using spectropho- tometry, while starch was determined by utilising a continuous flow method [45]. Moreover, high- performance liquid chromatography was used to de- termine polyphenol and MDA contents. All chemical composition determination methods are determined according to the Chinese tobacco industry standard method, and the measurement results are recognised by the tobacco industry. Experimental design The experiment was designed with three different treatments (Table 8), 116 DAT, 123 DAT, and 130 DAT. Different treatments at each time of harvest in- cluded approximately 1000 pieces tobacco leaves. The samples of tobacco leaves at different ages were flue- cured with the same flue-curing process. The samples were piecewise collected in seven stages (Fig. 12), i.e. Stage 1 (before flue-curing), Stage 2 (dry-bulb temperature of 35 °C), Stage 3 (dry-bulb temperature of 38 °C), Stage 4 (dry-bulb temperature of 42 °C), Stage 5 (dry-bulb temperature of 48 °C), Stage 6 (dry- Determination items and methods Structure analysis Different treatments used nine fresh tobacco leaves and we measured leaf length and leaf width, and the sample leaves were fixed with FAA (50% alcohol) fixative, and specimens were quickly embedded in paraffin and sliced, observed under Motic optical microscope, and statistically analysed for palisade tis- sue cell gap. The tobacco samples obtained in the seven flue-curing stages were sampled at a position 10 mm from the chief vein in the middle of a leaf by using a hole punch with the diameter of 10 mm and the sampled leaves were placed into formalin–aceto– alcohol (FAA) stationary liquid. A section was made by using paraffin. The section with thickness of 10 μm was dyed with hematoxylin and then thick- nesses of upper and lower epidermis as well as palis- ade and spongy tissues were measured by using a Motic optical microscope. Based on this, structural differences in tobacco leaves at different ages during flue-curing were compared. the top of the plant), two or three useless basal leaves were removed and 20 effective leaves per plant were reserved. After topping on 20 July in 2016 and 2017, chemical suckercides “Maleic hydrazide” (the active ingredient: N-sec-Butyl-4-(2-methyl-2-propanyl)-2,6- dinitroanilin) was applied to suppress buds, so that there were no tobacco flowers on the top and branches on the middle part. Other cultivation mea- sures were applied according to the technical require- ments of high-quality tobacco production in Kunming city, Yunnan Province, China. the top of the plant), two or three useless basal leaves were removed and 20 effective leaves per plant were reserved. After topping on 20 July in 2016 and 2017, chemical suckercides “Maleic hydrazide” (the active ingredient: N-sec-Butyl-4-(2-methyl-2-propanyl)-2,6- dinitroanilin) was applied to suppress buds, so that there were no tobacco flowers on the top and branches on the middle part. Other cultivation mea- sures were applied according to the technical require- ments of high-quality tobacco production in Kunming city, Yunnan Province, China. Analysis of chemical compositions Contents of total sugar, reducing sugar, starch, and pro- tein were determined by using the Pulse-3000 continu- ous flow analyser, and total nitrogen was determined by Chen et al. BMC Plant Biology (2019) 19:555 Page 20 of 21 Fig. 12 Dry- bulb and wet-bulb temperature in flue-curing stages Fig. 12 Dry- bulb and wet-bulb temperature in flue-curing stages utilising the perchloric-sulphuric acid digestion method. Furthermore, an ultraviolet (UV) spectrophotometric method was applied to determine nicotine contents. for supporting Congming Zou. The funders did not play any role in the design, collection, analysis, interpretation of the relevant data, or in writing the manuscript. for supporting Congming Zou. The funders did not play any role in the design, collection, analysis, interpretation of the relevant data, or in writing the manuscript. Availability of data and materials All data generated or analysed during this study are included in this published article [and its supplementary information files]. All data generated or analysed during this study are included in this published article [and its supplementary information files]. Competing interests The authors declare that they have no competing interests. All authors approved the final manuscript. Acknowledgements c o edge e ts The authors are thankful to Dr. Junying Li for his valuable assistance and advice in the preparation of this paper. The authors are thankful to Dr. Junying Li for his valuable assistance and advice in the preparation of this paper. Received: 18 October 2019 Accepted: 18 November 2019 Received: 18 October 2019 Accepted: 18 November 2019 Abbreviations DAT: Days after transplanting; MDA: Malondialdehyde Consent for publication Not applicable. Consent for publication Not applicable. Author details 1Y A d 1Yunnan Academy of Tobacco Agricultural Sciences, 33 Yuantong Street, Kunming, Yunnan 650021, People’s Republic of China. 2College of Tobacco Science, Yunnan Agricultural University, Kunming 650201, Yunnan, China. 3Faculty of Life Science and Technology, Kunming University of Science and Technology, 727 South Jingming Road, Kunming 650500, Yunnan, China. 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https://openalex.org/W2777982137	https://europepmc.org/articles/pmc5910613?pdf=render	English	null	MEGAN-LR: New algorithms allow accurate binning and easy interactive exploration of metagenomic long reads and contigs	bioRxiv (Cold Spring Harbor Laboratory)	2,017	cc-by	13,368	Abstract Background: There are numerous computational tools for taxonomic or functional analysis of microbiome samples, optimized to run on hundreds of millions of short, high quality sequencing reads. Programs such as MEGAN allow the user to interactively navigate these large datasets. Long read sequencing technologies continue to improve and produce increasing numbers of longer reads (of varying lengths in the range of 10k-1M bps, say), but of low quality. There is an increasing interest in using long reads in microbiome sequencing, and there is a need to adapt short read tools to long read datasets. Methods: We describe a new LCA-based algorithm for taxonomic binning, and an interval-tree based algorithm for functional binning, that are explicitly designed for long reads and assembled contigs. We provide a new interactive tool for investigating the alignment of long reads against reference sequences. For taxonomic and functional binning, we propose to use LAST to compare long reads against the NCBI-nr protein reference database so as to obtain frame-shift aware alignments, and then to process the results using our new methods. Results: All presented methods are implemented in the open source edition of MEGAN, and we refer to this new extension as MEGAN-LR (MEGAN long read). We evaluate the LAST+MEGAN-LR approach in a simulation study, and on a number of mock community datasets consisting of Nanopore reads, PacBio reads and assembled PacBio reads. We also illustrate the practical application on a Nanopore dataset that we sequenced from an anammox bio-rector community. Reviewers: This article was reviewed by Nicola Segata together with Moreno Zolfo, Pete James Lockhart and Serghei Mangul. Conclusion: This work extends the applicability of the widely-used metagenomic analysis software MEGAN to long reads. Our study suggests that the presented LAST+MEGAN-LR pipeline is sufficiently fast and accurate. Keywords: Microbiome, Long reads, Sequence analysis, Taxonomic binning, Functional binning, Algorithms, Software, Nanopore, PacBio Correspondence: daniel.huson@uni-tuebingen.de 1Center for Bioinformatics, University of Tübingen, Sand 14, 72076 Tübingen, Germany 2Life Sciences Institute, National University of Singapore, 28 Medical Drive, Singapore 117456, Singapore Full list of author information is available at the end of the article Correspondence: daniel.huson@uni-tuebingen.de 1Center for Bioinformatics, University of Tübingen, Sand 14, 72076 Tübingen, Germany 2Life Sciences Institute, National University of Singapore, 28 Medical Drive, Singapore 117456, Singapore Full list of author information is available at the end of the article © The Author(s). MEGAN-LR: new algorithms allow accurate binning and easy interactive exploration of metagenomic long reads and contigs Daniel H. Huson1,2* , Benjamin Albrecht1, Caner Ba˘gcı1,5, Irina Bessarab4, Anna Górska1,5, Dino Jolic3,5 and Rohan B. H. Williams4 Huson et al. Biology Direct (2018) 13:6 https://doi.org/10.1186/s13062-018-0208-7 Huson et al. Biology Direct (2018) 13:6 https://doi.org/10.1186/s13062-018-0208-7 Long read taxonomic binning The naïve LCA (lowest common ancestor) algorithm is widely used for binning short reads onto the nodes of a given taxonomy (such as the NCBI taxonomy), based on alignments [5]. Consider a read r that has significant alignments a1, . . . , ak to reference sequences associated with taxa t1, . . . , tk. The naïve LCA assigns r to the low- est taxonomic node that lies above the set of all nodes representing t1, . . . , tk. The set of significant alignments is defined to consist of those alignments whose score lies close to the best score achieved for the given read, defined, say, as those that have a bit score that lies within 10% of the best bit score. Current long read sequencing technologies, such as pro- vided by Oxford Nanopore Technologies (ONT) or Pacific Biosciences (PacBio), produce smaller numbers (in the range of hundreds of thousands) of longer reads (of vary- ing lengths in the range of 10 kb – 300 kb, say) of lower quality (error rates around 10%) [11, 12]. There is increas- ing interest in using long reads in microbiome sequencing and there is a need to adapt short read tools to long read datasets. There are a number of tools that are applica- ble to long reads, such as WIMP [13], Centrifuge [14] or Kaiju [15]. While the two former are based on comparing against DNA references, the latter can also use a protein reference database. The naïve LCA algorithm is fast, easy to implement and the results are easy to interpret. When applied to pro- tein alignments, an implicit assumption of the algorithm is that any read aligns to only one gene and so all associ- ated taxa are “competing” for the same gene; this justifies the above definition of significant alignments. While reads that are only a few hundred base pairs long usually ful- fill this assumption, longer reads or assembled contigs often overlap with more than one gene and so the naïve algorithm is not suitable for them. To make the naïve algorithm applicable to protein align- ments on a long read or contig r, a simple idea is to first determine “conserved genes” as regions along the read where alignments accumulate. The second step is to apply the naïve LCA to each of these regions indi- vidually. Abstract 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Huson et al. Biology Direct (2018) 13:6 Page 2 of 17 Background taxonomic profiling and correlation studies will usually require knowledge of the functional content. There are numerous computational tools for taxonomic or functional binning or profiling of microbiome samples, optimized to run on hundreds of millions of short, high quality sequencing reads [1–4]. Alignment-based taxo- nomic binning of reads is often performed using the naïve LCA algorithm [5], because it is fast and its results are easy to interpret. Functional binning of reads usually involves a best-hit strategy to assign reads to functional classes. Here we present a new classification pipeline for taxonomic and functional analysis of long reads and contigs, based on protein alignments. The pipeline, LAST+MEGAN-LR, consists of first running the align- ment tool LAST and then processing the resulting DNA- to-protein alignments using new algorithms provided in MEGAN-LR. We perform a simulation study to evalu- ate the performance of the method in the context of the taxonomic assignment and compare it with Kaiju, one of the few other tools that use protein references. We also investigate the performance of the pipeline using mock-community datasets and illustrate its application on Nanopore reads sequenced from an anammox enrich- ment bio-rector. Software or websites for analyzing microbiome shotgun sequencing samples usually provide some level of interac- tivity, such as MG-RAST [2]. The interactive microbiome analysis tool MEGAN, which was first used in 2006 [6], is explicitly designed to enable users to interactively explore large numbers of microbiome samples containing hun- dreds of millions of short reads [1]. Illumina HiSeq and MiSeq sequencers allow researchers to generate sequencing data on a huge scale, so as to ana- lyze many samples at a great sequencing depth [7–9]. A wide range of questions, in particular involving the pres- ence or absence of particular organisms or genes in a sample, can be answered using such data. However, there are interesting problems that are not easily resolved using short reads. For example, it is often very difficult to deter- mine whether two genes that are detected in the same microbiome sample also belong to the same genome, even if they are located close to each other in the genome, despite the use of metagenomic assembly in combination with contig binning techniques and paired-end reads [10]. Long read functional binning and annotation Functional binning of short reads is usually performed by assigning each read to a class in a functional classification system such as InterPro [19], eggNOG [20] or KEGG [21], based on its alignments. This is often done using a simple best-hit strategy, as fol- lows. For a short read r, let a denote the highest-scoring alignment of r to a reference protein for which the func- tional class c is known. Assign r to the functional class c. For example, c might be an InterPro family or an eggNOG cluster. In short read analysis, each read is assigned to at most one class in any given functional classification. Many reads remain unclassified because all the reference proteins that they align to are unclassified. In the second step, for each taxon t that is associated with any of the alignments, let I(t) denote the union of all intervals for which there exists some significant align- ment ai associated with taxon t. In a post-order traversal, for each higher-rank taxonomic node s we compute I(s) as the union of the intervals covered by the children of s. In result, every node of the taxonomy is labeled by a set of intervals. Note that, during the computation of the union of interval sets, we merge any overlapping intervals into a single interval. A long read may contain multiple genes, and for each gene, there may be many alignments involving differ- ent taxa. To avoid redundancy in functional assignments when processing alignments between the long read and different taxa, we consider the “dominance” of individual alignments (as defined below). Let r be a long read and let a1, . . . , ak be a set of DNA-to- protein alignments from r to a suitable protein reference sequences. Note that this set will often include alignments between the read and the same homologue in different taxa. The read r is then placed on the taxon s that has the property that its set of intervals I(s) covers 80% (by default) of the total aligned or covered portion of the read, while none of its children does (see Fig. 1). In MEGAN-LR this threshold is referred to as the percentToCover parameter. Note that it is possible that there are multiple nodes that have this property, in To reduce the number of redundant functional classes associated with r, we introduce the following concept. Long read functional binning and annotation We say that an alignment ai dominates an alignment aj, if (1) Fig. 1 To illustrate the interval-union LCA algorithm, here we show eight hypothetical species A, B, . . . , H separated into two genera, P and Q, belonging to the same family R. Alignments from the read r to proteins associated with the species are indicated by arrows on the right and cover between 80% (for A) and 20% (for H) of the aligned read. Using arrows, on the left we depict the sets of intervals computed for nodes P, Q, R as the union of the sets of intervals of the children of each node. Nodes R and P each cover 100% of the aligned read. The read r is placed on A as it is the lowest taxonomic node with ≥80% coverage. Note that, if A only covered 60% of the aligned read, then the read would be assigned to the higher taxon P (and this would remain the case even if one of the taxa below Q had 60% coverage) Fig. 1 To illustrate the interval-union LCA algorithm, here we show eight hypothetical species A, B, . . . , H separated into two genera, P and Q, belonging to the same family R. Alignments from the read r to proteins associated with the species are indicated by arrows on the right and cover between 80% (for A) and 20% (for H) of the aligned read. Using arrows, on the left we depict the sets of intervals computed for nodes P, Q, R as the union of the sets of intervals of the children of each node. Nodes R and P each cover 100% of the aligned read. The read r is placed on A as it is the lowest taxonomic node with ≥80% coverage. Note that, if A only covered 60% of the aligned read, then the read would be assigned to the higher taxon P (and this would remain the case even if one of the taxa below Q had 60% coverage) Fig. 1 To illustrate the interval-union LCA algorithm, here we show eight hypothetical species A, B, . . . , H separated into two genera, P and Q, belonging to the same family R. Long read taxonomic binning The placement of the read is finally determined using the LCA of all these gene-based LCAs. There are two problems here. First, because protein alignments around the same location can have quite different lengths, delineating different “conserved genes” can be difficult in practice. Second, because a large proportion of genes on a long read or contig may be conserved to different extents across different taxonomic groups, the placement In this paper, we focus on protein-alignment-based approaches. One reason for this is that existing DNA reference databases cover only a small fraction of the genome sequences believed to be present in the envi- ronment [16], although much work has been done on sequencing human-associated microbes [17]. This prob- lem can be ameliorated, to a degree, by using protein alignments, because amino acid sequences are more con- served than DNA sequences. Moreover, work on bac- terial pangenomes suggest that the association between species level taxonomic assignment and coding gene con- tent can be weak [18]. Finally, questions going beyond Huson et al. Biology Direct (2018) 13:6 Page 3 of 17 of the read will often be to a high-level (or “unspecific”) taxon. which case the read is assigned to the LCA of all such nodes. To address these issues, we present a new taxonomic binning for long reads that we call the interval-union LCA algorithm. This algorithm processes each read r in turn, in two steps. First, the read is partitioned into a set of inter- vals v1, . . . , vm that have the property that every alignment associated with r starts and ends at the beginning or end of some interval, respectively. In other words, a new inter- val starts wherever some alignment begins or ends. We say that an alignment ai is significant on an interval vj, if its bit score lies within 10% (by default) of the best bit score seen for any alignment that covers vj. In MEGAN-LR this threshold is referred to as the topPercent parameter. Long read functional binning and annotation Alignments from the read r to proteins associated with the species are indicated by arrows on the right and cover between 80% (for A) and 20% (for H) of the aligned read. Using arrows, on the left we depict the sets of intervals computed for nodes P, Q, R as the union of the sets of intervals of the children of each node. Nodes R and P each cover 100% of the aligned read. The read r is placed on A as it is the lowest taxonomic node with ≥80% coverage. Note that, if A only covered 60% of the aligned read, then the read would be assigned to the higher taxon P (and this would remain the case even if one of the taxa below Q had 60% coverage) Page 4 of 17 Huson et al. Biology Direct (2018) 13:6 Huson et al. Biology Direct (2018) 13:6 bases are pushed up the taxonomy until a taxon is reached that has enough aligned bases to be reported. ai covers more than 50% of the read that is covered by aj, (2) if the bit score of ai is greater than that of aj, and (3) both alignments lie on the same strand of r. Option- ally, one might also require that the taxonomic identity of each protein reference sequence under consideration is compatible with the taxonomic bin assigned to the read r. Long read alignment In this paper, we focus on taxonomic and functional binning of long reads using DNA-to-protein alignments. Currently long read sequencing technologies (Oxford Nanopore and PacBio) exhibit high rates of erroneous insertions and deletions [11, 12]. Consequently, programs such as BLASTX [22] are not suitable for such reads as they cannot handle frame-shifts. The set of functional classes associated with a long read r is then given by the functional classes associated with those alignments of r that are not dominated by some other alignment of r. Each read can be binned to all functional classes associated with it. Moreover, the set of associated classes can be used to provide simple, functional annotation of the read or contig. The LAST program [23, 24] uses a frame-shift aware algorithm to align DNA to proteins and produces long protein alignments on long reads, even in the pres- ence of many frame-shifts. Initial indexing of the NCBI– nr database (containing over 100 million sequences) by LAST takes over one day on a server. However, once completed, alignment of reads against the NCBI- nr database using the index is fast; the alignment of Nanopore reads takes roughly one hour per gigabase on a server. To exploit that latter, we provide a dialog for exporting taxonomic and functional annotations in GFF3 format. It can be applied to any selection of taxonomic or func- tional classification nodes, or to a set of selected reads in the new long read inspector, which is described in more detail below. The user chooses a classification, and then each alignment to a reference sequence associated with that classification is exported as a CDS item. By default, only those alignments that are not dominated by another alignment are exported. In addition, the user can decide to export only those items for which the taxon associated with the corresponding reference sequence is compatible with the taxon assigned to the read. The DIAMOND program [25] is widely used in micro- biome analysis to compute alignments of short metage- nomic reads against a protein reference database such as NCBI–nr. A new frame-shift aware alignment mode is currently under development and DIAMOND will pro- vide an alternative to LAST in the future. Reporting counts In taxonomic or functional binning of short reads, it usu- ally suffices to report the number of reads assigned to a specific classification node, because all reads are of a very similar length and all alignments have much the same length as the reads. For long reads or contigs, the lengths and alignment coverage can vary widely. Moreover, the number of reads contained in a contig, or contig coverage, is an additional factor to be considered. To address this, in MEGAN-LR each node can be labeled by one of the following: Long read analysis LAST produces output in a simple text–based mul- tiple alignment format (MAF). For performance rea- sons, LAST processes all queries and all reference sequences in batches and alignments associated with a given query are not reported consecutively, but rather in batches. In addition, the size of a MAF file is often very large and subsequent sorting and parsing of alignments can be time consuming. To address these issues, we have imple- mented a new program called “MAF2DAA” that takes MAF format as input, either as a file or piped directly from LAST, and produces a DAA (“Diamond alignment archive”) file as output [25]. The program processes the input in chunks, first filtering and compressing each chunk of data on-the-fly, and then interleaving and fil- tering the results into a single DAA file that contains all reads with their associated alignments. During filtering, MAF2DAA removes all alignments that are strongly dom- inated by some other alignment, to reduce a large number of redundant alignments. 1. the number of reads assigned, 2. the total length of all reads assigned 3. the total number of aligned bases of all reads assigned, or 4. in the case of contigs, the total number of reads contained in all assigned contigs. contained in all assigned contigs. For long reads, by default, MEGAN–LR reports (3), the number of aligned bases, rather than (2), as this down- weights any long stretches of unaligned sequence. In addi- tion, we use this value to determine the minimum support required for a taxon to be reported. By default, a taxon is only reported if it obtains at least 0.05% of all aligned bases. In MEGAN-LR, this is called the minSupport parameter. If the number of aligned bases assigned to a taxon t does not meet this threshold, then the assigned In more detail, for a given read r, we say that an align- ment a of r strongly dominates an alignment b for r, if it covers most of b (by default, we require 90% coverage) and if its bit score is significantly larger (by default, we require that 0.9 × bitscore(a) > bitscore(b)). Page 5 of 17 Page 5 of 17 Huson et al. Biology Direct (2018) 13:6 Huson et al. Long read analysis Biology Direct (2018) 13:6 A DAA file obtained in this way can then be processed by MEGAN’s Meganizer program that performs taxo- nomic and functional binning, and indexing, of all reads in the DAA file. This program does not produce a new file but appends the results to the end of the DAA file, and any such “meganized” DAA file can be directly opened in MEGAN for interactive analysis. We have modified MEGAN so that it supports frame-shift containing align- ments. The final DAA file is usually around ten times smaller than the MAF file produced by LAST. In this tool, each long read or contig r is represented by a horizontal line and all corresponding aligned reference sequences are shown as arrows above (forward strand alignments) or below (reverse strand alignments) the line. The user can select which annotations to display in the view. For example, if the user requests Taxonomy and InterPro annotations, then all reference sequences will be labeled by the associated taxonomic and InterPro classes. The user can search for functional attributes in all loaded reads. Let a be an arrow representing an alignment of r to a reference sequence associated with taxon s. We use a hierarchical coloring scheme to color such arrows. Ini- tially, we implicitly assign a color index to each taxon, e.g., using the hash code of the taxon name. For each arrow a with associated reference taxon s we distinguish between three different cases. First, if s = t, then we use the color assigned to t to color a. Second, if s is a descendant of t, then t has a unique child u that lies on the path from t down to s and we use the color of u to color a. Otherwise, we color a gray to indicate that the taxon associated with a is either less specific or incompatible with t. Long read visualization Interactive analysis tools for short read microbiome sequencing data usually focus on representing the tax- onomic and functional classifications systems used for binning or profiling the reads, for example reporting the number of reads assigned to each class. In addi- tion, some tools provide a reference-centric visualization that displays how the reads align against a given refer- ence sequence. However, visualizations of the short reads themselves are usually not provided. For long read or contigs, there is a need for visual- ization techniques that make it easy to explore the tax- onomic and functional identity of reference sequences to which the reads align. To address this, we have designed and implemented a long read inspector (using JavaFX) that allows one to investigate all long reads assigned to a given taxonomic or functional class (see Fig. 2). For example, if a read r is assigned to the genus Can- didatus Brocadia and has an alignment to the strain Candidatus Brocadia sinica JPN1, then we color the cor- responding arrow a using the color that represents the species Candidatus Brocadia sinica. This is a useful strategy when used in combination with the taxonomic binning procedure described above: a read Fig. 2 This screen shot of the MEGAN-LR long read inspector shows three contigs assigned to the genus Candidatus Brocadia, with alignments to more specific taxa. Alignments to reference protein sequences are shown as arrows, colored by species of the references; blue for Candidatus Brocadia sinica, brown for Candidatus Brocadia sp. 40 and pink for Candidatus Brocadia fulgida. Alignments are labeled by taxonomic and functional classes associated with the corresponding reference proteins Fig. 2 This screen shot of the MEGAN-LR long read inspector shows three contigs assigned to the genus Candidatus Brocadia, with alignments to more specific taxa. Alignments to reference protein sequences are shown as arrows, colored by species of the references; blue for Candidatus Brocadia sinica, brown for Candidatus Brocadia sp. 40 and pink for Candidatus Brocadia fulgida. Alignments are labeled by taxonomic and functional classes associated with the corresponding reference proteins Page 6 of 17 Huson et al. Biology Direct (2018) 13:6 Nanopore Technologies, Oxford, UK) for 2D sequenc- ing. Nanopore sequencing To obtain a Nanopore dataset, we sequenced the genomic DNA of the Microbial Mock Community B (even, high concentration, catalog nr. HM-276D, BEI Resources). Library preparation was performed using a Low Input by PCR Genomic Sequencing Kit SQK-MAP006 (Oxford Long read visualization Briefly, 100 ng of genomic DNA was sheared in a Covaris g-TUBE (Covaris, Inc., Woburn, MA, USA) at 6000 rpm, treated with PreCR (New England Biolabs, Ipswich, MA, USA) and used as input for adapter lig- ation according to the ONT protocol. Adapter-ligated DNA was further amplified with the LongAmp Taq 2X Master Mix (NEB) using the following program: 95◦C 3 min; 18 cycles of 95◦C 15 sec, 62◦C 15 sec, 65◦C 10 min; 65◦C 20 min. Sequencing was performed using an early access MinION device (ONT) on a FLO-MAP003 flowcell (ONT). Raw fast5 files were obtained with MinKNOW (v0.50.2.15, ONT) using a 48 h genomic sequencing proto- col, basecalled with ONT’s proprietary Metrichor cloud- based basecalling service and the 2D Basecalling for SQK-MAP006 v1.34 workflow. r is binned to the lowest taxon t that covers 80% (by default) of the aligned read and the taxonomy-based col- oring makes it easy to see how the different taxonomic classes below t contribute. For example, if all arrows on one half of the read have one color and all arrows on the other half have some other color, then this may indicate a chimeric read or misassembled contig. As discussed above, an alternative approach is to export reads and their alignments in GFF3 format and then to use a genome browser such as IGB [26] to explore them (see Fig. 3). LAST+MEGAN-LR In summary, we propose to use the following pipeline to analyze metagenomic long reads and contigs (see Fig. 4): • Align all reads against a protein reference database (such as NCBI-nr) using LAST, producing MAF output. Genomic DNA from the lab scale Anammox enrich- ment reactor described in Liu et al. [28] was extracted using the FastDNA SPIN Kit for Soil with 4x homogeniza- tion on the FastPrep instrument (MP Bio). The DNA was further purified using Genomic DNA Clean and Concen- trator -10 Kit (Zymo Research). Approximately 1700 ng of extracted DNA was used for library preparation using a Ligation Sequencing Kit SQK-LSK108 (Oxford Nanopore Technologies, Oxford, UK) for 1D sequencing according to the manufacturer protocol. Sequencing was performed using an early access MinION device (ONT) on a SpotON FLO-MIN106 flowcell (R9.4). The run was stopped after 22 h due to low number of active pores. Fast5 files were obtained with MinKNOW (v1.3.30, ONT) using a 48 h genomic sequencing protocol. Basecalling was performed using Metrichor (Instance ID:135935, 1D Basecalling for FLO-MIN106 450 bps_RNN (rev.1.121)). • Either pipe the output of LAST directly to MAF2DAA, or apply MAF2DAA to the MAF file generated by LAST, to obtain a much smaller output file in DAA format. • Meganize the DAA file either using the Meganizer command-line tool or interactively in MEGAN. • Open the meganized DAA file in MEGAN for interactive exploration using the long-read inspector. Export annotated reads in GFF3 format for further investigation, e.g. using a genome browser such as IGB [26] or Artemis [27]. Parameters The MEGAN-LR approach employs a number of different user-specified parameters. The main effect of changing Fig. 3 Example of long read data exported from MEGAN-LR and imported into the IGB genome browser [26] Fig. 3 Example of long read data exported from MEGAN-LR and imported into the IGB genome browser [26] Page 7 of 17 Huson et al. Biology Direct (2018) 13:6 Fig. 4 The LAST+MEGAN-LR pipeline. Long reads or contigs are aligned against the NCBI-nr database using LAST and the resulting MAF file (multiple alignment format) is converted to DAA format (Diamond alignment format), including filtering of dominated alignments. Taxonomic and functional binning of the reads or contigs is then performed using the Meganizer program and the results are appended to the DAA file. The meganized DAA file can then opened and interactively analyzed in MEGAN-LR Fig. 4 The LAST+MEGAN-LR pipeline. Long reads or contigs are aligned against the NCBI-nr database using LAST and the resulting MAF file (multiple alignment format) is converted to DAA format (Diamond alignment format), including filtering of dominated alignments. Taxonomic and functional binning of the reads or contigs is then performed using the Meganizer program and the results are appended to the DAA file. The meganized DAA file can then opened and interactively analyzed in MEGAN-LR any of these is usually a shift in the trade-off between false positive and false negative taxonomic assignments. What balance of false positives and false negatives is ideal depends on the biological question at hand, and so the parameters may have to be adjusted by the user. and TopPercentScoreToStronglyDominate=90% to filter reads. When reporting functional classes of intervals of a long read, a key problem is which alignments to report on. In practice, using all alignments found for a read produces too many redundant gene calls. Here MEGAN-LR uses a parameter MinPercentCoverToDominate = 50% to filter the alignments that are reported. The minSupport parameter (default setting 0.05%) sets the “level of detection”, that is, it is used to decide whether a taxonomic node has been assigned enough weight (such as number of reads or number of aligned bases, say) so as to appear in the displayed tree. If the threshold is not met, then the weights are pushed up the tree until enough weight has been accumulated. Parameters Lowering this threshold will improve sensitivity for low-abundance species while increasing the risk of false positives induced by the erroneous assignment of individual reads, i.e., due to random hits or database errors. Increasing this threshold will decrease false positives while causing more low-abundance taxa to be missed. In the “Results” section, we illustrate the effect of vary- ing most of these parameters on the performance of MEGAN-LR on mock community data. Simulation study To evaluate the performance of the proposed LAST+MEGAN-LR approach and, in particular, of the interval-union LCA algorithm, we undertook a simu- lation study to estimate the sensitivity and precision of the algorithm, following the protocol reported in [15], as defined below. We attempted to model two major obsta- cles in metagenomic studies, namely sequencing errors and the incompleteness of reference databases. The topPercent parameter (default value 10%) is used to determine which alignments on the same interval of a read are considered significant. An alignment is only considered significant if its bitscore lies within the given percentage of the bitscore for the best alignment. Setting this threshold too small will result in false positive assign- ments based on chance differences in alignment score, whereas setting this threshold too large will result in false negatives on lower taxonomic ranks due to assignment to higher taxonomic classes. Our simulation study is based on a set P of 4282 prokaryotic genomes from NCBI for which both anno- tated genomes and annotated sets of proteins are available, downloaded in March 2017. In addition, we identified a subset Q of 1151 genomes that consists of all those organisms in P whose genus contains at least 2 and at most 10 organisms in P, and for which a full taxonomic classification is given. Note that Q can be parti- tioned into nine different categories, based on the number 2 −10 of organisms in Q that the corresponding genus contains. The percentToCover parameter (default value 80%) influences at what rank of the taxonomy a long read will be placed. Setting this parameter too high or too low will usually result in less specific assignments. LAST alignment of long reads against the NCBI-nr database can produce very large files due to large num- bers of alignments covering the same segment of reads. The concept of strong-domination was developed to address this issue. By default, MEGAN-LR uses a setting of MinPercentCoverToStronglyDominate = 90% For each target species t in Q, we performed the follow- ing “leave-one-out” evaluation: • First, we collected a set of R of 2000 simulated reads from the genome sequence of t using NanoSim [29], a read simulator that produces synthetic reads that Huson et al. Biology Direct (2018) 13:6 Page 8 of 17 achieved at genus level by LAST+MEGAN-LR and Kaiju, for both the R7.3 and R9 datasets. Results We have implemented the interval-union LCA algorithm and the modified functional binning algorithm. In addi- tion, we have implemented a new long read interactive viewer. We provide methods for exporting long read anno- tations in GFF3 format. Our code has been integrated into the open source edition of MEGAN. In addition, we have modified MEGAN (and all tools bundled with MEGAN) so as to support DNA-to-protein alignments that contain frame-shifts. We use the term MEGAN-LR (MEGAN long read) to refer to this major extension of MEGAN. Kaiju classified 280,465 of these reads, assigning 128,774 to a species or lower rank node with a true positive rate of 76.9%. 209,435 reads were assigned to a genus or lower rank node with a true positive rate of 84.5%. PacBio reads on HMP mock community PacBio reads on HMP mock community To test LAST+MEGAN-LR on a publicly available PacBio mock community dataset, we downloaded “HMP dataset 7” from the PacBio website https://github.com/ PacificBiosciences/DevNet/wiki/Human_Microbiome_ Project_MockB_Shotgun in April 2017. This dataset contains 319,703 reads of average length 4,681 bp. It was sequenced using the P5 polymerase and C3 chemistry. To compare the performance of MEGAN-LR and Kaiju, we calculated the sensitivity and precision of taxonomic assignments at the genus, family and order levels. In more detail, following the approach used in [15], we define sen- sitivity as the percentage of reads in R that are assigned either to the correct taxon or to one of its descendants. We define precision as the percentage of reads that are assigned correctly, out of all reads that were binned to any node that is not an ancestor of the correct taxon. q g p y y LAST alignment against the NCBI-nr database (down- loaded January 2017) resulted in protein alignments for 284,728 reads (89% of all reads). MEGAN-LR analysis using the interval-union LCA algorithm assigned 1054 megabases (Mb) aligned bases to taxonomic nodes. Of these, 945.3 Mb were assigned to bacterial genera, with no false positives. A total of 758.4 Mb of aligned sequences were assigned to bacterial species, of which 755 Mb were assigned to true positive species (that is, species known to be contained in the mock-community), whereas approx- imately 3.4 Mb (0.4%) were assigned to false positive species. The 20 bacterial species in the mock commu- nity received between 2.8 Mb (0.37%) and 145 Mb (19%) aligned bases assigned at the species level, whereas the highest false positive species obtained 1.1 Mb (0.14%). Simulation study In all cases, LAST+MEGAN-LR shows better sensitivity and precision than Kaiju. As expected, both methods are less sensi- tive on the R7.3 data, as many reads remain unclassified. However, the difference in performance between the two methods is larger on the R7.3 data, and we suspect that this is due to the ability of LAST to perform frame-shift aware alignments and thus to accommodate erroneous insertions and deletions. reflect the characteristic base-calling errors of ONT reads, running in linear mode. g • Second, we constructed a protein reference database Dˆt that contained all proteins associated with all organisms in P except for t (“leave one out”). • Third, we performed taxonomic binning of all reads in R using LAST+MEGAN-LR as follows. We first build a LAST reference index on Dˆt, then aligned all reads in R against Dˆt using LAST, with a frameshift cost of 15, and then performed taxonomic binning of all reads in MEGAN using the interval-union LCA algorithm (default parameters). Per-dataset performance analysis of LAST+MEGAN- LR and Kaiju is presented in Fig. 6. This shows that LAST+MEGAN-LR outperforms Kajiu on a vast majority of the simulated datasets, with Kajiu sometimes showing better performance when the sensitivity or precision is very low. • Fourth, for comparison, we also ran the taxonomic binning program Kaiju[15] on R and Dˆt, building a custom Kaiju index on Dˆt. We performed taxonomic binning of simulated reads using Kaiju’s greedy mode, with the maximum number of allowed substitutions set to 5. Kaiju is many times faster than LAST+MEGAN-LR. However, the latter approach computes and uses all rele- vant protein alignments, and these are also used to per- form functional analysis of the reads or contigs. Hence, we suggest to use Kaiju to obtain a fast, first taxonomic profile for a set of long reads or contigs, and then to use LAST+MEGAN-LR to perform a more accurate and detailed subsequent analysis. To be precise, we ran each of the four steps twice to pro- duce two simulation datasets, each containing 2,000 reads per target species. The first dataset was produced using the ecoli_R73_2D (R7.3) simulator profile, whereas the second was produced using the ecoli_R9_2D (R9) profile. Both profiles were downloaded from the NanoSim FTP address (http://ftp.bcgsc.ca/supplementary/NanoSim/) in April 2017. The R7.3 profile introduces more errors in reads and should make it harder for analysis methods to identify appropriate reference sequences. Simulation study To investigate the use of LAST+MEGAN-LR on assem- bled reads, we assembled this set of reads using minimap The results of our simulation study are shown in Fig. 5, where we summarize the sensitivity and precision scores Page 9 of 17 Huson et al. Biology Direct (2018) 13:6 a b c d Fig. 5 Violin plots comparing the performance of LAST+MEGAN-LR and Kaiju for two simulation studies, one based on a R7.3 Nanopore chemistry profile and the other based on a R9 Nanopore chemistry profile. In both cases, we report the sensitivity (percentage of reads assigned to the correct taxon) and precision (percentage of reads assigned correctly out of all reads not binned to an ancestor of the correct taxon) of taxonomic assignments. This is done at the genus level for nine different categories of genera (reflecting the number of species in the genus from which the target species was removed), and for all. Results for the R7.3 profile are shown in a and b, and results for the R9 profile are shown in c and d a b b c d d c d c Fig. 5 Violin plots comparing the performance of LAST+MEGAN-LR and Kaiju for two simulation studies, one based on a R7.3 Nanopore chemistry profile and the other based on a R9 Nanopore chemistry profile. In both cases, we report the sensitivity (percentage of reads assigned to the correct taxon) and precision (percentage of reads assigned correctly out of all reads not binned to an ancestor of the correct taxon) of taxonomic assignments. This is done at the genus level for nine different categories of genera (reflecting the number of species in the genus from which the target species was removed), and for all. Results for the R7.3 profile are shown in a and b, and results for the R9 profile are shown in c and d the species level was 36.8 Mb, all of which was assigned to true positive species. (options -Sw5 -L100 -m0 -t8) and miniasm (version 0.2, default options) [30] and obtained 1130 contigs, with a mean length of 43,976 and maximum length of 1,272,994. LAST alignment against the NCBI-nr database resulted in 41.8 Mb of aligned sequences. Of this, 41.1 Mb and 38.6 Mb, were assigned to bacterial genus and species nodes, respectively, with no false positives and only one false negative species. PacBio reads on Singer et al. mock community On this data, Kaiju assigned 47,056 reads at the species level, with a true positive rate of 98.7%. Our analysis of PacBio reads recently published on a mock-community containing 26 bacterial and archaeal species [31] gave rise to results of similar quality. Of 53,654 reads of average length 1,041 and maximum length 16,403, exactly 51,577 received LAST alignments against NCBI-nr. Of 49.5 Mb of aligned sequences, 45.8 Mb were assigned to prokaryotic genera, with no assignments to false positive species. The amount of sequence assigned at Simulation study Of the 26 species in the mock community, two are not reported in the analysis and therefore constitute false negative species. These make up approximately 0.01% (Nocardiopsis dassonvillei) and 0.1% (Salmonella bongori) of the community and are thus on the borderline of detec- tion using the default settings of MEGAN-LR. By default, MEGAN-LR requires that a taxon receives at least 0.05% of all aligned bases before it is reported. Nanopore reads on HMP mock community To perform the first test of our new methods on Nanopore data, we sequenced the content of the Genomic DNA from Microbial Mock Community B, as described in the “Methods” section. We obtained 124,911 pass reads of Huson et al. Biology Direct (2018) 13:6 Page 10 of 17 a b c d Fig. 6 Here we plot the sensitivity and precision at genus level for Kaiju versus LAST+MEGAN-LR on the R7.3 samples in a and b, and on the R9 samples in c and d, respectively a b a b b a d c d c Fig. 6 Here we plot the sensitivity and precision at genus level for Kaiju versus LAST+MEGAN-LR on the R7.3 samples in a and b, and on the R9 samples in c and d, respectively Performance To illustrate the computational resources required by the LAST+MEGAN-LR approach, we measured the wall- clock time and memory consumption on the four datasets discussed above. In addition, we considered a further unpublished Nanopore dataset obtained from cheese, consisting of 34 million reads of average length 1460 and maximum length 229,439 (unpublished data pro- vided by the Dutton Lab, UCSD, during the Santa Barbara Advanced School of Quantitative Biology 2017). The pro- grams were run on a Linux server with 32 cores and 512 GB of main memory. We ran LAST using a volume size setting (parame- ter -s) of 20 GB (the maximum value), and recorded the peak memory used by the program. We set the maximum memory limit of MEGAN to between 5 GB and 10 GB, depending on the input size. We summarize our mea- surements in Table 3. The LAST alignment of reads was performed against the entire NCBI-nr protein database and the total size of the LAST index was 215 GB. This step took between a few minutes and a few hours, depending on the size of the input file. The subsequent two steps of conversion and meganization took less than half as long as alignment. By using a smaller LAST volume size, the whole pipeline can also be run on a computer with 16 GB main memory, such as a laptop. in Table 2 we list the number of alignments to genes for the main KEGG categories of metabolism. MEGAN-LR also makes it possible to investigate function in detail. For example, the anammox process relies on the extremely reactive intermediate hydrazine, produced by the enzyme hydrazine synthase, comprised of the three protein sub- units HSZ-α, HZS-β and HZS-γ [33]. Using MEGAN-LR, we identified eight reads that together contain all three subunits, see Fig. 7. Performance To illustrate the use of LAST+MEGAN-LR on assem- bled reads, we assembled this set of reads using min- imap (options -Sw5 -L100 -m0 -t8) and miniasm (default options) [30] and obtained 31 contigs, with a mean length Table 2 For each of the main KEGG categories of metabolism, we report the number of alignments against KEGG Orthology reference sequences for the given category, and the number of different KEGG Orthology groups (KOs) involved in such alignments KEGG metabolism categories # Alignments # KOs Carbohydrate metabolism 9691 347 Amino acid metabolism 8519 371 Energy metabolism 4909 225 Metabolism of cofactors and vitamins 2826 197 Nucleotide metabolism 2675 124 Lipid metabolism 2564 95 Xenobiotics biodegradation and metabolism 1738 116 Glycan biosynthesis and metabolism 1684 114 Metabolism of other amino acids 1344 73 Metabolism of terpenoids and polyketides 1156 63 Biosynthesis of other secondary metabolites 1076 54 These results are based on a LAST+MEGAN-LR analysis of Nanopore reads from an anammox enrichment bioreactor Application to anammox data average length 2870, including all template-, complement- and 2D reads. To illustrate the utility of our new methods in a research context, we applied Nanopore sequencing to a sam- ple obtained from a laboratory bio-reactor enriched for anaerobic ammonium oxidizing bacteria (AnAOB) [32], as described in the “Methods” section. We obtained 71,411 reads of average length 4658 and maximum length 30,846. The LAST alignment against the NCBI-nr database resulted in protein alignments for 57,026 reads (45.6% of all reads). MEGAN-LR analysis assigned a total of 110 Mb aligned bases. Of these, 100 Mb were assigned to bacterial genera, with a false positive assignment rate of 0.1%. Approximately 71.9 Mb of aligned sequences were assigned at the species level, with a false positive rate of 0.9%. The 20 bacterial species in the mock commu- nity received between 0.36 Mb (0.5%) and 12.2 Mb (17%) aligned bases assigned at the species level, whereas the highest false positive species obtained 0.21 Mb (0.3%). Around 66 kb of all aligned sequences (0.05%) were falsely assigned to Eukaryota. LAST alignment against the NCBI-nr database resulted in protein alignments for 64,097 reads (90% of all reads). MEGAN-LR analysis assigned a total of 212 Mb aligned bases. Of these, 94 Mb were assigned to bacterial genera and 112 Mb to bacterial species. The reason why there are more assignments to species than there are to gen- era is that some of the species present do not have a genus designation in the NCBI taxonomy. The top ten bacterial species assignments are shown in Table 1. This indicates that the most abundant organism in the sample is Candidatus Brocadia sinica, a known AnAOB species. Kaiju exhibited a higher false positive rate than LAST+MEGAN-LR on these Nanopore reads, namely 19.8% and 12.6% at the species and genus level, respec- tively. The program assigned 22,433 reads at the species level and 39,173 reads at the genus level. Functional binning in MEGAN-LR allows one to sum- marize counts at different levels of detail. For example, Page 11 of 17 Huson et al. Biology Direct (2018) 13:6 Table 1 The ten top bacterial species identified in a Nanopore dataset taken from an anammox enrichment bioreactor, by the number of bases aligned to corresponding reference proteins Species Aligned (Mb) Candidatus Brocadia sinica 84.9 Armatimonadetes bacterium OLB18 8.8 Bacteroidetes bacterium OLB12 4.8 Rhodocyclaceae bacterium UTPRO2 2.9 Chloroflexi bacterium OLB13 2.7 Nitrospira sp. Application to anammox data OLB3 1.5 Streptomyces sp. SolWspMP-5a-2 1.1 Anaerolineae bacterium UTCFX5 0.6 Pseudorhodoplanes sinuspersici 0.4 For Candidatus Brocadia sinica, this suggests at least ten-fold coverage of the genome of 129,601 and maximum length of 750,799. LAST align- ment against the NCBI-nr database resulted in 2.98 Mb of aligned sequences. The interval-union LCA algorithm assigned 13 contigs and 96% of all aligned bases to Candi- datus Brocadia sinica. Discussion mock community. The figures for PacBio reads on the HMP and the Singer et al. mock community are available in the supplementary material. We also decided to omit the minSupport parameter in the figures as it showed little to no variability for any value above 0. Turning off minSupport causes spurious assignments of some reads (up to 4% at species level). The application of long read sequencing technologies to microbiome samples promises to provide a much more informative description of the genetic content of environ- mental samples. The alignment of long reads against a protein reference database is a key step in the functional analysis of such data. Here we show that such protein alignments can also be used to perform accurate taxo- nomic binning using the interval-union LCA algorithm. As depicted in Fig. 8, increasing the percentToCover parameter improves the specificity of the true positive assignments (i.e. more reads are binned at lower ranks), but also increases the rate of false positives. Our simulation study suggests that LAST+MEGAN-LR performs taxonomic binning more accurately than Kaiju. The reported results on mock community datasets indi- cate a high level of accuracy down to the species level when the corresponding species are represented in the protein reference database. In addition, the computed protein alignments can be used to identify genes and MEGAN-LR provides a useful visualization of the anno- tated sequences. Using a higher value of the topPercent parameter results in more alignments being considered by the LCA algorithm and thus results in a more conservative or less specific binning of reads. We would like to emphasize that the datasets tested for the effects of parameters in this study are mock communities of species whose proteins are well repre- sented in the reference database. While Fig. 8 suggests setting TopPercent to 5 % and percentToCover to 90 %, we suggest that in practice both values should be relaxed slightly, to 10 and 80 %, respec- tively, so as to account for the fact that environmental microbes are usually not so well represented by reference sequences. The main motivation for developing these new methods is to assist our work on the study of microbial communi- ties in enrichment bio-rectors, where long read sequenc- ing promises to provide access to near-complete genome sequences of the dominating species. Parameters To investigate the effect of setting particular parame- ter values, we analyzed the three mock communities employing a range of different values for minSupport, topPercent and percentToCover. We used the val- ues 0, 0.025, 0.05, 0.075 and 0.1 for minSupport; 0, 5, 10 and 20 for topPercent; and 50, 60, 70, 80, 90 and 100 for percentToCover, respectively. Starting with the DAA file containing the LAST alignments of the reads against NBCI-nr, we ran the classification step of the MEGAN- LR pipeline on all possible combinations of values for the three parameters, with all other parameters set to their default values. We turned off the strong-domination filter for the cases in which topPercent equals 20, because that filter removes any alignment whose score lies 10% below that of the best overlapping hit. For all combinations of parameters, we calculated the rate of true positives and false positives for the number of assigned bases at the species and genus ranks, as well as for the number of assigned bases at any rank above genus. Figure 8 shows these values for Nanopore reads on HMP Huson et al. Biology Direct (2018) 13:6 Page 12 of 17 Fig. 7 Long read inspector showing nine reads in the anammox sample that together contain all three subunits of the hydrazine synthase gene, labeled hydrazine synthase subunit A, partial, hydrazine synthase subunit B and hydrazine synthase subunit C Fig. 7 Long read inspector showing nine reads in the anammox sample that together contain all three subunits of the hydrazine synthase gene, labeled hydrazine synthase subunit A, partial, hydrazine synthase subunit B and hydrazine synthase subunit C Discussion The simple assembly of the anammox data presented in this paper places the dominant species into 11 contigs of Page 13 of 17 Huson et al. Biology Direct (2018) 13:6 Table 3 Performance of the LAST+MEGAN-LR pipeline Step Input Output Runtime Memory PacBio reads on HMP mock community Align Reads file (1.5 GB) MAF file 119 min 23 GB Convert MAF file (49 GB) DAA file 29 min 5 GB Classify DAA file (4.2 GB) Meganized DAA file (4.5 GB) 6 min 5 GB PacBio reads on Singer et al. mock community Align Reads file (56 MB) MAF file 10 min 22 GB Convert MAF file (8.9 GB) DAA file 5 min 5 GB Classify DAA file (197 MB) Meganized DAA file (415 MB) 1 min 5 GB Nanopore reads on HMP mock community Align Reads file (191 MB) MAF file 10 min 22 GB Convert MAF file (6.1 GB) DAA file 3 min 5 GB Classify DAA file (553 MB) Meganized DAA file (644 MB) 1 min 5 GB Anammox data Align Reads file (336 MB) MAF file 31 min 24 GB Convert MAF file (8.5 GB) DAA file 4 min 5 GB Classify DAA file (371 MB) Meganized DAA file (500 MB) 2 min 5 GB Cheese data Align Reads file (5.1 GB) MAF file 251 min 24 GB Convert MAF file (93 GB) DAA file 90 min 10 GB Classify DAA file (3.1 GB) Meganized DAA file (3.5 GB) 5 min 10 GB For each of five long read datasets, we report the wall-clock time and main memory required by LAST to align against the NCBI-nr database, for MEGAN to convert the LAST MAF output files into DAA format, and then for MEGAN to classify the reads so as to meganize the DAA file, respectively. The computations were performed on a Linux server with 32 cores and 512GB memory Table 3 Performance of the LAST+MEGAN-LR pipeline Step Input Table 3 Performance of the LAST+MEGAN-LR pipeline For each of five long read datasets, we report the wall-clock time and main memory required by LAST to align against the NCBI-nr database, for MEGAN to convert the LAST MAF output files into DAA format, and then for MEGAN to classify the reads so as to meganize the DAA file, respectively. Reviewers’ comments Reviewer’s report 1: Nicola Segata and Moreno Zolfo Reviewer’s comments: The authors present here a novel computational pipeline to address the issue of taxo- nomical and functional classification of long reads. The authors correctly underline that long reads from emerging sequencing technologies are currently a computational challenge in the field of metagenomics. Indeed, not much attention has been dedicated to the taxonomic identifica- tion of long reads, and the author developed an extension of the previously published MEGAN software, which they call MEGAN-LR. The pipeline works with long nucleotide reads which are mapped against a protein database using All datasets discussed in this paper are available here: http://ab.inf.uni-tuebingen.de/software/downloads/ megan-lr. Discussion The computations were performed on a Linux server with 32 cores and 512GB memory length greater than 100 kb, containing about 2.8 Mb of aligned sequence and 3.7 Mb of total sequence. This sug- gests that a more careful assembly, assisted by a set of high quality MiSeq reads, should result in a nearly complete genome. an extension of the widely-used metagenomic analysis software MEGAN to long reads. With MEGAN-LR, we provide new algorithms for taxonomic binning, func- tional annotation and easy interactive exploration of metagenomic long reads and contigs, based on DNA- to-protein alignments. Our work suggests that the pre- sented LAST+MEGAN-LR pipeline is sufficiently fast and accurate. Our simulation study did not incorporate chimerism or similar artifacts. Because Kaiju uses a heuristic based on the longest match found, we suspect that Kaiju will per- form poorly on chimeric reads or misassembled contigs, assigning such a read to one of the source taxa. In contrast, the interval-union LCA algorithm requires by default that 80% of the aligned read is assigned to a taxon and so in practice, such reads will often be placed on a higher taxonomic node. Conclusions There is increasing interest in using long reads in micro- biome sequencing and there is a need to adapt short read tools to long read datasets. In this paper we present Huson et al. Biology Direct (2018) 13:6 Page 14 of 17 Fig. 8 The effect of changing the topPercent and percentToCover parameters for the analysis of the Nanopore HMP mock community. True positive and false positive rates are reported for each combination of parameters at the levels of species and genus, and for the sum of ranks above genus. The rate is calculated as the number of correctly assigned bases divided by the total number of bases assigned at the respective taxonomic level Fig. 8 The effect of changing the topPercent and percentToCover parameters for the analysis of the Nanopore HMP mock community. True positive and false positive rates are reported for each combination of parameters at the levels of species and genus, and for the sum of ranks above genus. The rate is calculated as the number of correctly assigned bases divided by the total number of bases assigned at the respective taxonomic level this threshold was chosen as default: was it the result of a parameter- optimization of some sort? Author’s response: We have added a section on “Parameters” to Methods. LAST, it accounts for read that align against more than one protein, and is frameshift aware. The authors pro- vide convincing evidences on the accuracy and precision of MEGAN-LR on synthetic data and mock communities sequenced ad-hoc. This review was performed by Nicola Segata and Moreno Zolfo 3. Similarly, one could test the impact of the threshold that is used to determine whether a LAST alignment is strongly dominated by another alignment. Since this value is set by default to 90%, it would be interesting to see the behaviour of the mapper at different thresholds. As summarized in my comments above, I think this is a well written and clear paper. I do not think there are many major issues, but there are several points that the authors should at least consider addressing to improve the paper: different thresholds. Author’s response: We have added a section on “Parameters” to Methods. 1. It would be useful for the general comprehension of the frameset in which MEGAN-LR is set, to understand why the authors decided to focus on protein-based taxonomic assignment. “Parameters” to Methods. “Parameters” to Methods. 4. The fact that some alignments in the MAF file are eliminated if they are strongly dominated by another alignment can affect the correct placement of a read. How did the authors decide the default thresholds by which this mechanism is implemented in MEGAN-LR? Author’s response: We have added a section on “Parameters” to Methods. Conclusions Most of the other existing algorithms use nucleotide-based approaches. I would suggest to add a paragraph exploring the advantages and disadvantages of the two approaches. Author’s response: We have added a section on “Parameters” to Methods. Author’s response: We have added a paragraph discussing this to the Background section. Author’s response: We have added a paragraph discussing this to the Background section. 5. Overall, a precise estimate on the memory and CPU requirements of MEGAN-LR is not provided. I think this point should be reported more clearly, by providing the computational resources used by MEGAN-LR in the analysis. Specifically, I think it would be useful to report how much CPU time and memory were required in each of the validations step. Moreover, it would be also useful to have an estimate 2. The default threshold to report the presence for a taxon is set to 0.05% of the total aligning bases. Since the overall performance of the algorithm could be dramatically affected by this parameter, it would be nice to see how the precision and specificity of MEGAN- LR vary when changing the threshold. Also, I think that the authors should clarify on how Huson et al. Biology Direct (2018) 13:6 Page 15 of 17 Identify genome functional characteristics. The latter will include e.g. virulence and pathogenicity, and pro- vides a means e.g. for assessing health risk posed by micro- organisms in metagenomics samples. I have indi- cated some minor points of communication that should be considered. on the order of magnitude of time required to analyse a whole average PacBio/Nanopore metagenome. Author’s response: We have added a section on “Performance” to Results. on the order of magnitude of time required to analyse a whole average PacBio/Nanopore metagenome. Author’s response: We have added a section on “Performance” to Results. on the order of magnitude of time required to analyse a whole average PacBio/Nanopore metagenome. Author’s response: We have added a section on “Performance” to Results. 6. Figure 5, the performances of Kaiju and LAST+MEGAN-LR are binned by the number of species in the genus. It would be interesting to see in the same box plot also the summed (i.e. overall) distributions for each subplot. 6. Figure 5, the performances of Kaiju and LAST+MEGAN-LR are binned by the number of species in the genus. It would be interesting to see in the same box plot also the summed (i.e. overall) distributions for each subplot. 1. Also a number of default thresholds are indicated for different stages of analysis, e.g. 80% threshold for the LCA assignment, 50% for the alignment dominance criterion, 0.05% for MEGAN-LR reporting. Author’s response: We have added a section on “Parameters” to Methods. It would help potential users to have more insight into the thinking behind these values , and whether or not additional threshold values should be considered. Author’s response: We have added a section on “Parameters” to Methods. Author’s response: To each subplot, we have added a category that summarizes all datasets. Author’s response: To each subplot, we have added a category that summarizes all datasets. 7. The comparison between Kaiju and MEGAN-LR is performed only on the simulated dataset. I would suggest to run Kaiju also on the PacBio and Nanopore reads from the mock communities, if the genomes of the species present in the communities are available and well annotated. This should provide further support to the higher specificity and precision of MEGAN-LR. Author’s response: We have added true positive and false positive rates of Kaiju’s assignments for mock communities against NCBI-nr to their respective sections. 7. The comparison between Kaiju and MEGAN-LR is performed only on the simulated dataset. I would suggest to run Kaiju also on the PacBio and Nanopore reads from the mock communities, if the genomes of the species present in the communities are available and well annotated. This should provide further support to the higher specificity and precision of MEGAN-LR. Reviewer’s report 3: Serghei Mangul Reviewer’s comments: Author’s response: We have added true positive and false positive rates of Kaiju’s assignments for mock communities against NCBI-nr to their respective sections. Author’s response: We have added true positive and false positive rates of Kaiju’s assignments for mock communities against NCBI-nr to their respective sections. 1. The authors propose protein based alignment. Is there an advantage to use protein-based alignment versus nucleotide-based alignment? Author’s response: We have added a paragraph discussing this to the Background section. 8. Another computational tool that is addressing the problem of long-reads mapping is MinHash (Jain et al., https://doi.org/10.1101/103812). It is understandable that the validation was conducted only on Kaiju (as it is the only tool using protein-alignments). Nevertheless, it would be interesting to see the other approaches compared. Author’s response: A comparison against DNA-based analysis approaches is beyond the scope of this paper. 2. The nucleotide- based methods (for example Centrifuge) have been excluded from the comparison. Including those methods (by using the comparable database with nucleotide sequences ) can be valuable. Also, this will provide a general comparison of nucleotide-based versus protein based performance of metagenomic tools. Author’s response: While we agree that such a comparison would be useful, such a comparison against DNA-based analysis approaches is beyond the scope of this paper. 9. There is no much on the task of “functional classification” in the “Results” section. Estimating the functional potential of a microbiome is an important task, and it would be very nice if the authors provide some details, validation, and application on real data for this. ror example could the authors provide some comments on the functional landscape detectable with MEGAN-LR of the anammox dataset? Author’s response: We have added a high-level summary genes assigned to KEGG metabolic categories and also a detailed inspection of the key hydrazine syntase subunits for the anammox sample. 3. 3. p.9, line 46. More information about the leave-one- out experiment is required. What is the motivation for the experiment? Does it refer to removing one reference genome, from which reads were simulated? Such experiment can quantify, the possibility of misassignment of reads to the close-related genome, due to the incompleteness of the reference. Author’s response: Yes, all genes associate with the source genome are removed from the reference database. 3. p.9, line 46. More information about the leave-one- out experiment is required. What is the motivation for the experiment? Reviewer’s report 3: Serghei Mangul Reviewer’s comments: Does it refer to removing one reference genome, from which reads were simulated? Such experiment can quantify, the possibility of misassignment of reads to the close-related genome, due to the incompleteness of the reference. Author’s response: Yes, all genes associate with the source genome are removed from the reference database. MEGAN-LR: long read extension of the metagenome analysis tool MEGAN MEGAN-LR: long read extension of the metagenome analysis tool MEGAN g 5. Huson DH, Auch AF, Qi J, Schuster SC. MEGAN analysis of metagenomic data. Genome Res. 2007;17(3):377–86. https://doi.org/10.1101/gr. 5969107. Consent for publication Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. 6. p.10. The paper report sensitivity and precision on the read level. It would be interesting to know such performance on different taxa levels. In such, case sensitivity, for example, would be the percentage of taxa correctly identified. Author’s response: We have added supplementary plots for higher taxonomic levels to the companion website. 6. p.10. The paper report sensitivity and precision on the read level. It would be interesting to know such performance on different taxa levels. In such, case sensitivity, for example, would be the percentage of taxa correctly identified. Acknowledgements We thank Dr Gayathri Natarajan and Dr Ying Yu Law for their assistance with obtaining samples from the anammox bioreactor. 6. Poinar HN, Schwarz C, Qi J, Shapiro B, Macphee RDE, Buigues B, Tikhonov A, Huson DH, Tomsho LP, Auch A, Rampp M, Miller W, Schuster SC. Metagenomics to paleogenomics: large–scale sequencing of mammoth DNA. Science. 2006;311(5759):392–4. https://doi.org/10.1126/ science.1123360. 6. Poinar HN, Schwarz C, Qi J, Shapiro B, Macphee RDE, Buigues B, Tikhonov A, Huson DH, Tomsho LP, Auch A, Rampp M, Miller W, Schuster SC. Metagenomics to paleogenomics: large–scale sequencing of mammoth DNA. Science. 2006;311(5759):392–4. https://doi.org/10.1126/ science.1123360. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author details Author’s response: We have added supplementary plots for higher taxonomic levels to the companion website. 1Center for Bioinformatics, University of Tübingen, Sand 14, 72076 Tübingen, Germany. 2Life Sciences Institute, National University of Singapore, 28 Medical Drive, Singapore 117456, Singapore. 3Max-Planck Institute for Developmental Biology, 72076 Tübingen, Germany. 4Singapore Centre for Environmental Life Sciences Engineering, National University of Singapore, 28 Medical Drive, Singapore 117456, Singapore. 5IMPRS ‘From Molecules to Organisms’, Tübingen, Germany. 7. p.11. The contribution of LAST algorithms to the superiority of MEGAN-LR in comparison to other methods needs to be quantified. One way to do so is to compare the performance of Kaiju with LAST instead of current alignment algorithm. Author’s response: As an aligner, LAST does not perform taxonomic binning and so a comparison of Kaiju with LAST without MEGAN-LR is not possible. 7. p.11. The contribution of LAST algorithms to the superiority of MEGAN-LR in comparison to other methods needs to be quantified. One way to do so is to compare the performance of Kaiju with LAST instead of current alignment algorithm. Received: 19 October 2017 Accepted: 29 March 2018 Funding h h The authors acknowledge support by the High Performance and Cloud Computing Group at the Zentrum für Datenverarbeitung of the University of Tübingen, the state of Baden-Württemberg through bwHPC and the German Research Foundation (DFG) through grant no INST 37/935-1 FUGG and grant no HU 566/12-1. 7. Mackelprang R, Waldrop M, DeAngelis K, David M, Chavarria K, Blazewicz S, Rubin E, Jansson J. Metagenomic analysis of a permafrost microbial community reveals a rapid response to thaw. Nature. 2011;480(7377):368–71. https://doiorg/101038/nature10576. This work supported in part by the Singapore National Research Foundation and Ministry of Education under the Research Centre of Excellence Programme, and by a program grant from the Environment and Water Industry Programme Office (EWI), project number 1301–RIS–59. This research was supported in part by the National Science Foundation under grant no NSF PHY-1748958. This work supported in part by the Singapore National Research Foundation and Ministry of Education under the Research Centre of Excellence Programme, and by a program grant from the Environment and Water Industry Programme Office (EWI), project number 1301–RIS–59. This research as s pported in part b the National Science Fo ndation nder This work supported in part by the Singapore National Research Foundation and Ministry of Education under the Research Centre of Excellence Programme, and by a program grant from the Environment and Water Industry Programme Office (EWI), project number 1301–RIS–59. 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Reviewer’s report 2: Pete James Lockhart 4. p.10, line 18. What is the maximum number of mismatches allowed by MEGAN-LR? The effect of this parameter on the performance of both Megan-LR and Kaiju needs to be explored. Author’s response: While the number of mismatches is an important parameter for DNA-DNA alignments, it does not usually play a role in amino-acid alignments. 4. p.10, line 18. What is the maximum number of mismatches allowed by MEGAN-LR? The effect of this parameter on the performance of both Megan-LR and Kaiju needs to be explored. Author’s response: While the number of mismatches is an important parameter for DNA-DNA alignments, it does not usually play a role in amino-acid alignments. Reviewer’s comments: The manuscript by Huson et al. describes and evaluates a novel approach for analyzing long sequence reads and these to taxa and functional cat- egories. 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https://openalex.org/W4378602931	https://www.researchsquare.com/article/rs-2979849/latest.pdf	English	null	EnMSP: An Elastic-net Multi-step Screening Procedure for High-dimensional Regression	Research Square (Research Square)	2,023	cc-by	9,978	EnMSP: An Elastic-net Multi-step Screening Procedure for High-dimensional Regression Yushan Xue (  cnxueyushan@163.com ) Version of Record: A version of this preprint was published at Statistics and Computing on February 16th, 2024. See the published version at https://doi.org/10.1007/s11222-024-10394-9. EnMSP: An Elastic-net Multi-step Screening Procedure for High-dimensional Regression Rui Chen1, Jie Ren2, Jing Chen1∗, Jie Huang1, Bin Yang1, and Yushan Xue1† 1School of Statistics and Mathematics, Central University of Finance and Economics, Beijing, China. 2Cinda Securities Co., Ltd., Beijing, China. †Corresponding Author: cnxueyushan@163.com. ∗R. C. and J. C. contributed equally to this work. Research Article Posted Date: May 29th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2979849/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 Statistics and Computing on February 16th, 2024. See the published version at https://doi.org/10.1007/s11222-024-10394-9. Abstract To improve the estimation eﬃciency of high-dimensional regression problems, penalized regularization is routinely used. However, accurately estimating the model is still challenging when there are correlated eﬀects that irrelevant variables are strongly correlated with relevant variables. In this paper, we propose the Elastic-net Multi-step Screening Procedure (EnMSP), an iterative algorithm designed to recover sparse linear models in correlated data. EnMSP uses a small repeated penalty strategy to identify truly relevant covariates in a few iterations. Speciﬁcally, in each iteration, EnMSP enhances the adaptive lasso method by adding a weighted l2 penalty, which improves the selection of relevant variables. The method is shown to select the true model and achieve the l2-norm error bound under certain conditions. Numerical comparisons and applications demonstrate the eﬀectiveness of EnMSP. Keywords: High-dimensional data, Correlated eﬀects, Elastic-net, Iterative algorithm, EnMSP 1 1 Introduction The development of data collection makes it more common to encounter large-scale datasets. One feature of these datasets is that the number of covariates is always larger or much larger than the number of samples. Correlations among variables are common. For example, chemical and ﬁnancial data usually have a limited number of samples and highly correlated variables. Model selection in high-dimensional settings has gained much attention in recent years. Extracting useful information from massive and complex cor- related data would improve the interpretation of the model and reduce the diﬃculty of data analysis. Many methods have been proposed for the high-dimensional sparse regression prob- lems. Lasso (Tibshirani, 1997) for instance, minimizes the regularized squared loss func- tion with the l1 penalty and has the functions of variable shrinkage and selection. Elastic- net (Zou and Hastie, 2005) minimizes the regularized squared loss function with l1 and l2 penalties. Other traditional regularization variable selection methods (Fan and Li, 2001; Yuan and Lin, 2006; Zou, 2006; Candes and Tao, 2007; Zhang, 2010a) are also ef- fective in performing model selection and parameter estimation simultaneously when the irrepresentable condition is met (Zhao and Yu, 2006; Kim et al., 2009; Fan and Lv, 2011). Many methods have been proposed for the high-dimensional sparse regression prob- lems. Lasso (Tibshirani, 1997) for instance, minimizes the regularized squared loss func- tion with the l1 penalty and has the functions of variable shrinkage and selection. Elastic- net (Zou and Hastie, 2005) minimizes the regularized squared loss function with l1 and l2 penalties. Other traditional regularization variable selection methods (Fan and Li, 2001; Yuan and Lin, 2006; Zou, 2006; Candes and Tao, 2007; Zhang, 2010a) are also ef- fective in performing model selection and parameter estimation simultaneously when the irrepresentable condition is met (Zhao and Yu, 2006; Kim et al., 2009; Fan and Lv, 2011). However, it is common to see relevant covariates highly correlated with irrelevant co- variates, which makes the irrepresentable condition fail, especially in high-dimensional settings. Some modiﬁed variable selection methods have been proposed to incorporate highly correlated covariates, but most of them lack of consistency in selecting variables (B¨uhlmann, 2013; Maier and Rodr´ıguez-Salas, 2017; Hilafu and Yin, 2017), or require strict assumptions such as the exact number of relevant covariates being known (Javan- mard and Montanari, 2014). 1 Introduction Another category of methods is based on partial correlations (B¨uhlmann et al., 2010; Cho and Fryzlewicz, 2012; Jin et al., 2014), however, limited by bounded range of partial correlation, they may diminish the strong signal of some of the relevant covariates in the estimation process. However, it is common to see relevant covariates highly correlated with irrelevant co- variates, which makes the irrepresentable condition fail, especially in high-dimensional settings. Some modiﬁed variable selection methods have been proposed to incorporate highly correlated covariates, but most of them lack of consistency in selecting variables (B¨uhlmann, 2013; Maier and Rodr´ıguez-Salas, 2017; Hilafu and Yin, 2017), or require strict assumptions such as the exact number of relevant covariates being known (Javan- mard and Montanari, 2014). Another category of methods is based on partial correlations (B¨uhlmann et al., 2010; Cho and Fryzlewicz, 2012; Jin et al., 2014), however, limited by bounded range of partial correlation, they may diminish the strong signal of some of the relevant covariates in the estimation process. In high-dimensional settings, the irrepresentable condition is often not necessary for achieving variable selection consistency with nonconvex penalization procedures (Fan and Li, 2001; Zhang, 2010a), such as SCAD (Fan and Li, 2001), MCP (Zhang, 2010b) and 2 SSL (Roˇckov´a and George, 2018). However, these methods can still present numerical challenges when ﬁtting models, and also have diﬃculty in selecting the right model with ﬁnite samples, as shown in Sections 4 and 5. Nowadays, multi-step methods get more and more attention. For example, Adaptive Lasso (Zou, 2006), considered as a two- step method, assigns diﬀerent weights in l1 penalty and is shown to be consistent for variable selection. Zou and Li (2008) proposed an iterative algorithm based on local linear approximation (LLA) which turns concave penalized problems into nonconcave penalized likelihood problems. Zhang (2010c) proposed the capped-l1 penalty, analyzing a convex relaxation scheme for solving problems with non-convex objective functions and proved the oracle property. Yang et al. (2020) provided a Multi-step Screening Procedure (MSP), which ﬁt an adaptive lasso regression with a small penalty within the reduced feature space obtained from the previous step, leading to the recovery of the true sparse linear models even when the irrepresentable condition is relaxed. It is shown that a multi- step strategy potentially improves the estimation accuracy when irrelevant covariates are correlated to the relevant covariates. 1 Introduction Yet, limited results are studied for the correlated data under this framework. This paper focuses on the high-dimensional sparse regression problem with correlated covariates. Motivated by the idea of MSP and Elastic-net, we apply iterative weights for both shrinkage and correlated eﬀect. To improve the estimation accuracy, we pro- pose an Elastic-net Multi-step Screening Procedure (EnMSP). The method has three key characteristics: (1) EnMSP applies the strength of MSP. We impose adaptive weight which is obtained in the previous step and updated in every step to reduce the estimation bias induced by the l1 penalty. The procedure guarantees consistency in model selection. The weight imposed in the l2 penalty helps to stabilize the solution paths and improve the prediction. (2) EnMSP is eﬃcient in dealing with high-dimensional data. Compared with other methods that eliminate variables at the ﬁrst step, EnMSP calculates the weights from an Elastic-net regression at the initialization step. In this step we keep all variables, which can avoid permanently deleting the variables erroneously at the beginning. At each step thereafter, the variables whose coeﬃcients have been shrunk to zero will be deleted. With this screening, the dimension decreases during the iteration, which lets EnMSP take less 3 computation time. (3) EnMSP has advantages when the variables are highly correlated. When the data is strongly correlated in high dimensions, Elastic-net is possible for every variable to be selected whereas Lasso tends to randomly select one of the correlated variables but does not care which one is chosen. Compared with other methods based on Lasso, our algorithm inherits the strength of Elastic-net to deal with the problem of multicollinearity. It also considers the complex relationship between variables, proposing a weighted l2 penalty according to the correlation coeﬃcient between variables. The rest structure of this paper is as follows. Section 2 deﬁnes EnMSP and discusses the advantages of this method. Section 3 shows its theoretical results. Simulation results comparing the method with existing methods are presented in Section 4. We apply our method to real data in section 5. Proofs for the theoretical results of the EnMSP are provided in the Appendix. 2 Model and Method We consider the linear regression problem: y = Xβ + ϵ, where y is an n × 1 response vector, with n being the sample size, X is an n × p matrix, β is the coeﬃcients vector, and ϵ is the random error vector, with ϵ ∼N(0, σ2). We are interested in the problems when p ≫n and the covariates are correlated. The existing methods, such as Lasso(Tibshirani, 1996) and Elastic-net(Zou and Hastie, 2005), face challenges when the irrepresentable condition fails or when selecting the right model with ﬁnite samples in high-dimensional sparse regression problems with correlated covariates. Some modiﬁed methods lack consistency in variable selection or require strict constraints(B¨uhlmann, 2013; Maier and Rodr´ıguez-Salas, 2017; Hilafu and Yin, 2017), while partial correlation-based methods have a limited range and can reduce the signal of relevant covariates(B¨uhlmann et al., 2010; Cho and Fryzlewicz, 2012; Jin et al., 2014). To overcome these challenges, we propose the Elastic-net Multi-step Screening Procedure (EnMSP). Speciﬁcally, EnMSP applies an adaptive weight obtained in the previous step 4 to reduce estimation bias induced by the l1 penalty, guaranteeing consistency in model selection. EnMSP also eﬃciently deals with high-dimensional data and highly correlated variables by avoiding the permanent deletion of variables at the beginning and proposing a weighted l2 penalty according to the correlation coeﬃcient between variables. The EnMSP method thus overcomes the limitations of existing methods and improves the accuracy and consistency of variable selection in high-dimensional sparse regression problems with correlated covariates. The details of the EnMSP algorithm are as follows: The details of the EnMSP algorithm are as follows: • Initialize k = 0. Attain an Elastic-net solution: • Initialize k = 0. Attain an Elastic-net solution: ˆβ[0] := arg min 1 2∥y −Xβ∥2 2 + λ01∥β∥1 + 1 2λ02∥β∥2 2 . • Then k = 1. • Then k = 1. ˆβ[1] := arg min ( 1 2∥y −Xβ∥2 2 + λ1 p X j=1 ˆw[0] j \|βj\| + 1 2λ2 p X j=1 ˆvjβ2 j ) , where the w and v are p-dimensional weight vectors, w measures the importance of variables by the size of the regression coeﬃcients. That is: ˆw[0] j =    1/ ˆβ[0] j , ˆβ[0] j ̸= 0 n, ˆβ[0] j = 0 , v measures how collinear each particular variable with the others is. That is: ˆvj = 1/γrj. 2 Model and Method where rj is the number of other variables whose correlation coeﬃcient with the jth variable is greater than γ, which is a tunning parameter. where rj is the number of other variables whose correlation coeﬃcient with the jth variable is greater than γ, which is a tunning parameter. We let A[1] represent the non-zero index set of ˆβ[1], i.e. A[1] = n j ∈{1, . . . , p} : ˆβ[1] j ̸= 0 o We let A[1] represent the non-zero index set of ˆβ[1], i.e. A[1] = n j ∈{1, . . . , p} : ˆβ[1] j ̸= 0 o • Repeat the following steps until convergence: k ←−k + 1, ˆβ[k] := arg min β(A[k−1])c=0    1 2∥y −Xβ∥2 2 + λ1 X j∈A[k−1] ˆw[k−1] j \|βj\| + 1 2λ2 X j∈A[k−1] ˆvjβ2 j   . 5 In every step, we update the ˆw[k−1] j = 1/ ˆβ[k−1] j , and the non-zero set A[k], i.e. A[k] = {j ∈{1, ..., p} : ˆβ[k] j ̸= 0}. At convergence, we denote the non-zero set by A and the coeﬃcients by ˆβ. Note that the non-zero set in each step is contained in the one in the last step, i.e. A[1] ⊇A[2] ⊇· · · ⊇A[k] ⊇· · · ⊇A. Remark 1. EnMSP commences with k = 0, a step referred to as the initialization step. During this step, we obtain an initial estimate of βj, denoted as ˆβ[0] j . Although some ˆβ[0] j values may be equal to 0, we do not ﬁlter out these variables at this step. Instead, we set the penalty coeﬃcient for these variables to a large value in the subsequent step (k = 1). As a result, real variable screening commences from k = 1. The approach’s advantage is that the algorithm avoids erroneously ﬁltering out essential variables at the outset, thereby enhancing the consistency of variable screening. Remark 2. EnMSP draws inspiration from MSP(Yang et al., 2020) but diﬀers in that it adds a weighted l2 penalty. Its primary objective is to penalize highly correlated variables. Since the value of γ falls between 0 and 1, a higher value of rj implies that more variables are strongly correlated with jth variable, leading to a larger ˆvj. Therefore, the penalty coeﬃcient for jth variable increases proportionally, while the l1 penalty assists in ﬁltering out relevant covariates. 2 Model and Method As the number of iterations increases, w adjusts the penalty coeﬃcient of essential variables, gradually decreasing it. Conversely, the penalty coeﬃcient for less important variables grows larger, thereby facilitating variable screening. At each iteration step, the algorithm performs an estimation similar to Elastic-net. The simulation study revealed that EnMSP not only screens the true set but also achieves high estimation accuracy. Remark 3. In the initialization step, the values of λ01 and λ02 may diﬀer slightly from those of λ1 and λ2 as their respective value ranges are speciﬁed in the theoretical results. To select the appropriate values for λ, we suggest performing cross-validation using either a ﬁve-fold or ten-fold approach. The threshold γ represents the correlation coeﬃcient and is used to apply l2 penalty to variables that exhibit mutual correlation above γ. Typically, γ is set between 0.5 and 1. The choice of correlation coeﬃcient calculation 6 method is ﬂexible and not explicitly required in EnMSP. Our simulation study involved using Cosine similarity, Pearson correlation coeﬃcient(Pearson, 1895), Spearman corre- lation coeﬃcient(Spearman, 1987), distance correlation coeﬃcient(Sz´ekely et al., 2007), and XICOR(Chatterjee and Holmes, 2020) to calculate the correlation coeﬃcient, and all ﬁve methods achieved comparable prediction accuracy without any noticeable diﬀerences between them. 3 Theoretical Results We ﬁrst make some preliminary assumptions. The data X are standardized: Pn i=1 Xij = 0 and Pn i=1 X2 ij/n = 1 for j = 1, . . . , p. The q denotes the number of covariates that are relevant to the model and is much smaller than both n and p. The dimensionality of the data is: p = O(enc1) and q = O(nc2) where 0 < c1 + c2 < 1. Let S = {j : βj ̸= 0}, thus \|S\| = q. Besides, let C = XTX/n, W = XTϵ/√n, u = √n(ˆβ −β). We also have the following assumptions: Condition 1. Restricted Eigenvalue (RE) condition: there exists a positive constant K2 that Condition 1. Restricted Eigenvalue (RE) condition: there exists a positive constant K2 that vTCv ⩾K2∥v∥2 2, for all v ∈G(S), where G(S) := {v ∈Rp : ∥vSc∥1 ⩽7∥vS∥1}. Condition 1 is a frequently employed method to limit the l2 error between coeﬃcients and estimates (Meinshausen and Yu, 2009; Negahban et al., 2012), and is considered to be the least restrictive of its kind. Numerous studies have demonstrated that condition 1 holds with high probability for a broad range of Gaussian matrices, even when the predictors exhibit high correlation. In contrast, more stringent conditions such as the restricted isometry property may not be satisﬁed in such situations (Raskutti et al., 2010). Proposition 1. Given the Condition 1 holds, and set λ01 = 4σ√n log p, λ1 = 4σ√n log n, λ02 ⩽ λ01/4∥β∥∞, λ2 ⩽λ11/(4∥β∥∞max vj) then if there exists a positive constant K1 > 7/K2 7 such that min j∈S \|βj\| > K1 √qλ0/n, (1) min j∈S \|βj\| > K1 √qλ0/n, (1) then the set A[1] satisﬁes P(S ⊆A[1]) ⩾1 −1/n. (2) (2) Remark 4. (1) suggests that a slight diﬀerence exists between βj∈S and 0, which enables the algorithm to diﬀerentiate between βj∈S and βj /∈S. Moreover, (1) has been utilized to establish model selection consistency in various literature, such as Lasso(Zhao and Yu, 2006), Capped-l1(Zhang, 2010a), LLA(Fan et al., 2014) and MSP(Yang et al., 2020). Remark 5. (2) indicates that the active set chosen in the ﬁrst step has a high likelihood of including the true set, similar to the ﬁnding in MSP. Both ensure the consistency of the approach. However, EnMSP has an advantage over MSP in that it includes an initialization step. 3 Theoretical Results This step enables the algorithm to retain all variables at the outset, without the possibility of eliminating some crucial variables by chance in the beginning. Consequently, the subsequent variable screening step is more reasonable. Theorem 1. Under the same setting of Proposition 1. Set λ1 = 4σ√n log n and λ2 ⩽ λ1/(4∥βA[k−1]∥∞maxj∈A[k−1] vj) for k = 2, 3, . . . . Set 1 > c3 ≥c1 + c2. Then the following error bound for the estimates holds with probability at least 1 −1/n, ∥ˆβ −β∥2 ⩽ 8σ K2 · K3 (q log n nc3 )1/2, (3) (3) where K3 < K1. Moreover, we have, where K3 < K1. Moreover, we have, P(sign(ˆβ) = sign(β)) ⩾1 −1/n. (4) (4) Remark 6. The error bound given in (3) for EnMSP demonstrates that nc3/2 dominates the denominator of the error bound for EnMSP. When c3 approaches 1, the l2-norm error bound becomes close to the rate of (qlogn/n)1/2. Remark 6. The error bound given in (3) for EnMSP demonstrates that nc3/2 dominates the denominator of the error bound for EnMSP. When c3 approaches 1, the l2-norm error bound becomes close to the rate of (qlogn/n)1/2. Remark 7. (4) establishes not only that ˆβ and β are highly likely to share the same sign but also that the active set A selected by EnMSP has a high probability of matching 8 the true set S. While Theorem 1’s ﬁndings are similar to MSP, which also ensures the method’s consistency in ﬁltering out the true set, EnMSP has an advantage over MSP. EnMSP penalizes irrelevant covariates that are correlated with relevant covariates through a weighted l2 penalty, thus helping to stabilize the solution paths and enhance prediction performance. This improvement is evident in the simulation study. 4 Simulation Study We let (β1, β2, β3, β4, β5, β6) = (8, 8, 4, 4, 0, -4), others are set to 0. The predictors X in group A and C are generated as: The predictors X in group A and C are generated as: X ∼N(0, Σ). The predictor X in group B is generated as: X5 = 2X4. We compute L2-error (∥ˆβ−β∥2), L1-error (∥ˆβ−β∥1), NZ (the number of nonzero coef- ﬁcients), TPR (true positive rate) and FPR (false positive rate) to measure the prediction eﬀect of the methods. TPR and FPR are respectively deﬁned as: TPR = \|j ∈{1, . . . , p} : ˆβj ̸= 0 and βj ̸= 0\| \|j ∈{1, . . . , p} : βj ̸= 0\| , FPR = \|j ∈{1, . . . , p} : ˆβj ̸= 0 and βj = 0\| \|j ∈{1, . . . , p} : βj = 0\| . Table 1 summarizes the results, Cor1, Cor2, Cor3, Cor4, and Cor5 are corresponding to the algorithm that uses Pearson correlation coeﬃcient, Cosine similarity, Distance correlation coeﬃcient, XICOR, and Spearman correlation to calculate the weight v. We replicated each simulation 100 times and calculated the standard deviation as shown in brackets. 4 Simulation Study In this section, we conduct a simulation study to test the performance of algorithm En- MSP. We also compare it with Lasso (Tibshirani, 1997), Elastic-net (Zou and Hastie, 2005), Capped (Zhang, 2010c), Adaptive Lasso (Zou, 2006), LLA (Zou and Li, 2008) and MSP (Yang et al., 2020). We employed a value of γ = 0.8 to impose a weighted l2 penalty only on variables whose correlation coeﬃcient exceeds 0.8. To determine the appropriate threshold for γ, we calculated the correlation coeﬃcient between variables beforehand and examined their distribution. Typically, the value of γ falls within the range of 0.5 to 1 since variables with pairwise correlation coeﬃcients surpassing 0.5 are usually regarded as strongly correlated. For other tuning parameters, we set λ01 = λ1 , λ02 = λ2 , which are selected by ten-fold cross-validation. We present two examples in this section, and ﬁx (n, p) = (200, 400) in each example. Consider the following linear regression model for simulation research: yi = p X j=1 xijβj + ϵi, i = 1, . . . , n ϵi ∼N(0, 1). The example settings are shown as following, variables are divided into three groups: A, B and C in each example: The example settings are shown as following, variables are divided into three groups: A, B and C in each example: (1) In Example 1, each group containing 1, 5, 394 variables, the pairwise correlation in group B is corr(i, j) = 0.9\|i−j\|, and in group C is 0.9. X1 in group A is highly correlated with the variables in group B. We let (β1, β2, β3, β4, β5, β6) = (0, 2, 4, 4, 4, 4), others are set to 0. The predictors X in group B and C are generated as: The predictors X in group B and C are generated as: X ∼N(0, Σ). 9 The predictor X in group A is generated as: The predictor X in group A is generated as: The predictor X in group A is generated as: The predictor X in group A is generated as: X1 = 7 8X2 + 3 8X3 + 1 8X4 + 1 8X5 + 1 8ei ei ∼N(0, 1). (2) In Example 2, each group containing 5, 1, 394 variables, the pairwise correlation in group A is corr(i, j) = 0.9\|i−j\|, in group C is 0.9. X5 in group B is highly correlated with X4. 4.1 Performance comparison Table 1 summarizes the performance of EnMSP. As can be seen that L2-error and L1- error of EnMSP are lower than other methods, which means EnMSP is competitive in estimation accuracy and can guarantee the interpretation of the model. The outperfor- mance in TPR and FPR makes EnMSP identify all the true positives and pick up false 10 positives as few as possible. EnMSP also eliminates some variables to obtain a sparse model, NZ of EnMSP is about 68% lower than Lasso and Capped, it nearly performs best in sparsity. Although MSP enjoys better performance in NZ, it is inferior to EnMSP in selecting the important variables, so the overall performance of the EnMSP appears to be the best. It is noteworthy that EnMSP demonstrates strong performance across all ﬁve correlation coeﬃcients, with no signiﬁcant diﬀerences observed between them. Therefore, practitioners need not be overly concerned with the choice of correlation coeﬃcient in practical applications. For the sake of consistency in subsequent comparisons, we will utilize EnMSP based on the Pearson correlation coeﬃcient. MSP EnMSP 0 1 2 3 X1 X2 X3 X4 X5 X6 X estimation error (a) MSP Lasso EnMSP 0 1 2 3 X1 X2 X3 X4 X5 X6 X estimation error (b) Lasso Capped EnMSP 0 1 2 3 X1 X2 X3 X4 X5 X6 X estimation error (c) Capped Alasso EnMSP 0 1 2 3 X1 X2 X3 X4 X5 X6 X estimation error (d) Alasso ElasticNet EnMSP 0 1 2 3 X1 X2 X3 X4 X5 X6 X estimation error (e) Elastic-net LLA EnMSP 0 1 2 3 X1 X2 X3 X4 X5 X6 X estimation error (f) LLA Figure 1: Results of the estimation errors under Example 1. The black dotted lines stand for EnMSP; the red dotted lines stand for MSP (a), Lasso (b), Capped (c), Alasso (d), Elastic-net (e), LLA (f). 4.1 Performance comparison Capped EnMSP 0 1 2 3 X1 X2 X3 X4 X5 X6 X estimation error Lasso EnMSP 0 1 2 3 X1 X2 X3 X4 X5 X6 X estimation error MSP EnMSP 0 1 2 3 X1 X2 X3 X4 X5 X6 X estimation error 1 2 estimation error (c) Capped LLA EnMSP 0 1 2 3 X1 X2 X3 X4 X5 X6 X estimation error (f) LLA (c) Capped (b) Lasso (a) MSP Alasso EnMSP 0 1 2 3 X1 X2 X3 X4 X5 X6 X estimation error (d) Alasso (b) Lasso ElasticNet EnMSP 0 1 2 3 X1 X2 X3 X4 X5 X6 X estimation error (e) Elastic-net 1 2 estimation error (f) LLA (f) LLA (e) Elastic-net (d) Alasso Figure 1: Results of the estimation errors under Example 1. The black dotted lines stand for EnMSP; the red dotted lines stand for MSP (a), Lasso (b), Capped (c), Alasso (d), Elastic-net (e), LLA (f). Next, we compare estimation errors (\| ˆβj −βj\|) between the estimated parameter and the true parameter of each covariate in groups A and B. Figure 1 shows the estimation 11 Table 1: Performance comparison under two examples. Data Method L2-error NZ L1-error TPR FPR Example 1 Elastic-net 1.949(0.610) 6.200(0.603) 3.755(1.082) 0.988(0.020) 0.003(0.001) Lasso 2.814(1.861) 17.550(15.031) 6.414(1.877) 0.886(0.099) 0.033(0.037) MSP 1.968(2.861) 5.160(0.394) 3.613(2.249) 0.882(0.098) 0.002(0.001) Capped 2.813(3.333) 17.130(14.049) 6.351(1.731) 0.886(0.099) 0.032(0.034) Alasso 2.050(2.633) 6.600(2.084) 3.929(2.054) 0.884(0.099) 0.006(0.004) LLA 3.016(0.104) 5.450(0.641) 5.598(0.623) 0.800(0.00) 0.003(0.002) EnMSP(cor1) 0.632(0.922) 5.440(0.782) 1.232(1.140) 0.994(0.034) 0.001(0.002) EnMSP(cor2) 0.588(0.413) 5.900(1.072) 1.182(0.871) 1.000(0.000) 0.002(0.002) EnMSP(cor3) 0.513(0.401) 5.760(1.078) 1.036(0.860) 1.000(0.000) 0.002(0.003) EnMSP(cor4) 0.574(0.386) 5.620(1.017) 1.034(0.837) 1.000(0.000) 0.002(0.002) EnMSP(cor5) 0.547(0.406) 5.760(1.021) 0.961(0.875) 1.000(0.000) 0.002(0.002) Example 2 Elastic-net 1.507(0.305) 6.080(0.706) 2.640(0.540) 1.000(0.000) 0.003(0.002) Lasso 4.512(0.056) 22.000(9.469) 8.115(0.946) 0.800(0.000) 0.045(0.023) MSP 4.474(0.056) 5.000(0.000) 6.636(0.312) 0.800(0.000) 0.003(0.000) Capped 4.521(0.064) 22.270(9.819) 8.251(1.085) 0.800(0.000) 0.046(0.024) Alasso 4.483(0.054) 5.220(0.416) 6.614(0.275) 0.800(0.000) 0.003(0.001) LLA 0.518(0.250) 5.880(0.945) 1.031(0.573) 1.000(0.000) 0.002(0.002) EnMSP(cor1) 0.429(0.185) 5.270(0.782) 0.831(0.401) 1.000(0.000) 0.000(0.002) EnMSP(cor2) 0.427(0.182) 5.290(0.742) 0.829(0.395) 1.000(0.000) 0.000(0.002) EnMSP(cor3) 0.428(0.182) 5.310(0.747) 0.831(0.394) 1.000(0.000) 0.000(0.002) EnMSP(cor4) 0.426(0.178) 5.250(0.575) 0.819(0.374) 1.000(0.000) 0.000(0.001) EnMSP(cor5) 0.430(0.185) 5.290(0.742) 0.834(0.399) 1.000(0.000) 0.000(0.002) Table 1: Performance comparison under two examples. 4.1 Performance comparison 12 MSP EnMSP 0.0 1.2 2.5 3.8 X1 X2 X3 X4 X5 X6 X estimation error (a) MSP Lasso EnMSP 0.0 1.2 2.5 3.8 X1 X2 X3 X4 X5 X6 X estimation error (b) Lasso Capped EnMSP 0.0 1.2 2.5 3.8 X1 X2 X3 X4 X5 X6 X estimation error (c) Capped Alasso EnMSP 0.0 1.2 2.5 3.8 X1 X2 X3 X4 X5 X6 X estimation error (d) Alasso ElasticNet EnMSP 0.0 1.2 2.5 3.8 X1 X2 X3 X4 X5 X6 X estimation error (e) Elastic-net LLA EnMSP 0.0 0.2 0.4 X1 X2 X3 X4 X5 X6 X estimation error (f) LLA Figure 2: Results of the estimation errors under Example 2. The black dotted lines stand for EnMSP; the red dotted lines stand for MSP (a), Lasso (b), Capped (c), Alasso (d), Elastic-net (e), LLA (f). MSP EnMSP 0.0 1.2 2.5 3.8 X1 X2 X3 X4 X5 X6 X estimation error Lasso EnMSP 0.0 1.2 2.5 3.8 X1 X2 X3 X4 X5 X6 X estimation error MSP EnMSP 0.0 1.2 2.5 3.8 X1 X2 X3 X4 X5 X6 X estimation error Capped EnMSP 0.0 1.2 2.5 3.8 X1 X2 X3 X4 X5 X6 X estimation error (a) MSP Alasso EnMSP 0.0 1.2 2.5 3.8 X1 X2 X3 X4 X5 X6 X estimation error (d) Alasso (b) Lasso ElasticNet EnMSP 0.0 1.2 2.5 3.8 X1 X2 X3 X4 X5 X6 X estimation error (e) Elastic-net (c) Capped LLA EnMSP 0.0 0.2 0.4 X1 X2 X3 X4 X5 X6 X estimation error (f) LLA (f) LLA (e) Elastic-net (d) Alasso Figure 2: Results of the estimation errors under Example 2. The black dotted lines stand for EnMSP; the red dotted lines stand for MSP (a), Lasso (b), Capped (c), Alasso (d), Elastic-net (e), LLA (f). 13 errors under Example 1. As we can see, EnMSP has smaller estimation errors than other methods in all six variables. The estimation errors of β3, β4, β5, β6 of other methods are very similar and close to EnMSP. However, EnMSP performs distinctly better in estimating β1 and β2, especially compared with Lasso, Capped, and LLA. Figure 2 shows the estimation errors under Example 2. The improvement of β4 and β5 of EnMSP is signiﬁcant compared with MSP, Lasso, Capped, and Adaptive Lasso. 4.1 Performance comparison The estimation errors of ˆβ4 and ˆβ5 of Elastic-net are lower than the above four methods, but the error in ˆβ6 have signiﬁcantly increased. Although LLA estimates β4 and β5 with lower errors than the other ﬁve methods, EnMSP still performs slightly better than LLA. (a) MSP (b) Lasso (c) Capped (d) Alasso (e) LLA (f) EnMSP Figure 3: Results of model selection with diﬀerent λ1 in Example 1. The black solid line stands for X1, the black dashed line stands for X2, the black dotted lines stand for X3, X4, X5, X6. X1 is irrelevant but highly correlated with the relevant variables. When λ1 is 5.65, EnMSP can choose the correct model which only X2 involved but other methods retain X1 and X2 at the same time. ( ) MSP (b) Lasso (e) LLA (c) Capped (b) Lasso (c) Capped (f) EnMSP (d) Alasso (f) EnMSP (f) EnMSP (e) LLA (d) Alasso Figure 3: Results of model selection with diﬀerent λ1 in Example 1. The black solid line stands for X1, the black dashed line stands for X2, the black dotted lines stand for X3, X4, X5, X6. X1 is irrelevant but highly correlated with the relevant variables. When λ1 is 5.65, EnMSP can choose the correct model which only X2 involved but other methods retain X1 and X2 at the same time. 14 14 (a) MSP (b) Lasso (c) Capped (d) Alasso (e) LLA (f) EnMSP Figure 4: Results of model selection with diﬀerent λ1 in Example 2. The black solid line stand for X4, the black dashed line stand for X5, the black dotted lines stand for X1, X2, X3, X6. EnMSP selects X4 and X5 correctly and estimates the coeﬃcients with lower errors when λ1 is less than 123.1. (a) MSP (d) Alasso (c) Capped (b) Lasso (e) LLA (c) Capped (e) L Figure 4: Results of model selection with diﬀerent λ1 in Example 2. The black solid line stand for X4, the black dashed line stand for X5, the black dotted lines stand for X1, X2, X3, X6. EnMSP selects X4 and X5 correctly and estimates the coeﬃcients with lower errors when λ1 is less than 123.1. 4.2 Model selection This subsection details the performance of our method in model selection. In Figure 3, we show the results of model selection under Example 1. We focus on the performance of estimating X1 and X2. X1 is irrelevant but highly correlated with the relevant variables, all the methods except for EnMSP keep it in the model constantly. As a comparison, EnMSP shrinks the coeﬃcient of X1 to zero when λ1 is large. As λ1 decreases, EnMSP selects the variable X2 into the model, and in an appropriate λ1 (5.65 in this case), EnMSP can choose the correct model which only X2 is involved, but other methods retain X1 and X2 at the same time. Figure 4 shows the results of model selection under Example 2, in this case, X5 is an 15 irrelevant variable. Lasso, Capped, and Alasso estimates the coeﬃcient of X4 to zero and select X5 as an important variable over a wide range of λ1. MSP performs slightly better for it can eliminate X5 sometimes but is not able to choose X4 correctly. As a comparison, EnMSP and LLA select X4 and X5 correctly when the λ1 is appropriate (less than 123.1 in this case), but we know from Table 1 that EnMSP estimates the coeﬃcients with lower errors. We see that EnMSP can select the right model and estimate the coeﬃcients more accurately, so EnMSP is good at dealing with the problem of multicollinearity. 5 Empirical Analysis We apply the proposed EnMSP method to the index tracking problem in ﬁnancial mod- eling. Index tracking aims to replicate and track the trend of a ﬁnancial index through building a portfolio, such as selecting a set of stocks. But the number of stocks that investors can choose from is usually hundreds or thousands. And they can only observe tens or hundreds of days. Further, due to the limited transactional cost, investors only wish to choose a few stocks. Thus, this is a high-dimensional sparse model. We consider the dataset of the S&P500 index. Due to the limitations of data acqui- sition, we only collected 471 days of observations from January 2018 to December 2019 and 482 stocks. We consider the linear model: yt = Pp i=1 βixit + εt, which yt is the return of S&P500 index on day t, xit is the return of stock i on day t. We divide the dataset into 19 rolling periods. Each period consists of 6 months, the ﬁrst ﬁve months as training set and the sixth month as testing set. The training set is used to select stocks and estimate βi and the testing set is used to evaluate the performance of the model. We compare 4 methods: EnMSP, Capped, LLA and MSP. We use the tracking error (Meade and Salkin, 1989) to compare the performance of diﬀerent methods. Tracking error is often used to assess the tracking performance in the ﬁnancial industry. It is deﬁned as or year = √ 252 × qX (errt −mean(err))2 /(T −1), TrackingError year = √ 252 × qX (errt −mean(err))2 /(T −1), TrackingError year = √ 252 × qX (errt −mean(err))2 /(T −1), where errt = yt −ˆyt, yt and ˆyt denotes the real return and ﬁtted return of the index on day t respectively. where errt = yt −ˆyt, yt and ˆyt denotes the real return and ﬁtted return of the index on day t respectively. where errt = yt −ˆyt, yt and ˆyt denotes the real return and ﬁtted return of the index on day t respectively. 16 16 We use EnMSP based on the Pearson correlation coeﬃcient and set γ = 0.8, other tuning parameters are selected by ten-fold cross-validation. Figure 5 shows the tracking errors of 19 training sets and testing sets. 5 Empirical Analysis As is shown, EnMSP always has the lower and more stable tracking errors, except for one period of testing sets, where EnMSP performed slightly worse than MSP. Besides, Figure 6 shows the selected frequency of constituent stock in the S&P500 index. As can be seen, only 5 constituent stocks have a frequency that is over 60%. From high to low is AAPL.O, AMZN.O, FB.O, XOM.N, and CSCO.O. The AAPL.O stock has the highest selected frequency, up to 89.5%, it was selected 17 times in 19 periods, and 70 stocks were selected twice, 97 stocks were selected only one time, 196 stocks were not selected once. 6 Conclusion In this paper, we propose the EnMSP algorithm for model selection in high-dimensional situations. We impose an adaptive l1 penalty which is updated in every step to reduce the estimation bias and guarantee consistency in model selection, we also impose a weighted l2 penalty according to the correlation coeﬃcient to deal with the problem of multicollinear- ity. Since the unimportant variables are shrunk to zeros by iteration and are deleted, the computation time has been eﬀectively reduced. We also prove that EnMSP enjoys oracle properties. We consider ﬁve correlation coeﬃcients corresponding to EnMSP to test the robustness in the simulation study. The results show that EnMSP enjoys better performance in estimation accuracy and model selection. EnMSP estimates parameters with lower errors and always selects the right model when p ≫n and the variables are highly correlated. We also apply the proposed EnMSP method to the index tracking problem in ﬁnancial modeling and obtain outperformance results. 17 17 0 2 4 6 8 Date Fitted tracking error(%) 2018.1 2018.4 2018.7 2018.1 2019.1 2019.4 2019.7 EnMSP MSP Capped LLA 0 2 4 6 8 10 Date Predicted tracking error(%) 2018.6 2018.9 2018.12 2019.3 2019.6 2019.9 2019.12 EnMSP MSP Capped LLA Figure 5: Tracking errors for diﬀerent methods in the training sets (left) and test sets (right). 0 2 4 6 8 Date Fitted tracking error(%) 2018.1 2018.4 2018.7 2018.1 2019.1 2019.4 2019.7 EnMSP MSP Capped LLA 0 2 4 6 8 10 Date Predicted tracking error(%) 2018.6 2018.9 2018.12 2019.3 2019.6 2019.9 2019.12 EnMSP MSP Capped LLA Figure 5: Tracking errors for diﬀerent methods in the training sets (left) and test sets (right). 0 100 200 300 400 500 0.0 0.2 0.4 0.6 0.8 constituent stocks frequency Figure 6: Selected frequency of constituent stock from S&P500 index. 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Given the deﬁnition of the ˆβ[0], the following inequality holds 1 2∥y −X ˆβ[0]∥2 2 + λ01∥ˆβ[0]∥1 + 1 2λ02∥ˆβ[0]∥2 2 ⩽1 2∥y −Xβ∥2 2 + λ01∥β∥1 + 1 2λ02∥β∥2 2. (5) We also have −X ˆβ[0]∥2 2 + λ01∥ˆβ[0]∥1 + 1 2λ02∥ˆβ[0]∥2 2 ⩽1 2∥y −Xβ∥2 2 + λ01∥β∥1 + 1 2λ02∥β∥2 2. (5) 1 2∥y −X ˆβ[0]∥2 2 + λ01∥ˆβ[0]∥1 + 1 2λ02∥ˆβ[0]∥2 2 ⩽1 2∥y −Xβ∥2 2 + λ01∥β∥1 + 1 2λ02∥β∥2 2. (5) (5) We also have We also have ∥ˆβ[0]∥0 = ∥ˆβ[0] S ∥0 + ∥ˆβ[0] S ∥0, (6) (6) ∥β∥1 ⩽∥ˆβ[0] S ∥1 + ∥ˆβ[0] S −βS∥1, (7) (7) ∥ˆβ[0]∥2 2 −∥β∥2 2 = (ˆβ[0] −β)T(ˆβ[0] −β) + βT(ˆβ[0] −β), (8) (8) 1 2∥y −X ˆβ[0]∥2 2 −1 2∥y −Xβ∥2 2 = 1 2(ˆβ[0] −β)TXTX(ˆβ[0] −β) −ϵX(ˆβ[0] −β). (9) Take formulas (6), (7), (8) and (9) into (5), we have 1(ˆβ[0] β)T(XTX + λ I)(ˆβ[0] β) + λ βT(ˆβ[0] β) ⩽X(ˆβ[0] β) + λ (∥ˆβ[0] β ∥ ∥ˆβ ∥ Take formulas (6), (7), (8) and (9) into (5), we have Take formulas (6), (7), (8) and (9) into (5), we have Take formulas (6), (7), (8) and (9) into (5), we have 1 2(ˆβ[0] −β)T(XTX + λ02I)(ˆβ[0] −β) + λ02βT(ˆβ[0] −β) ⩽ϵX(ˆβ[0] −β) + λ01(∥ˆβ[0] S −βS∥1 −∥ˆβSc∥1) (10) −β)T(XTX + λ02I)(ˆβ[0] −β) + λ02βT(ˆβ[0] −β) ⩽ϵX(ˆβ[0] −β) + λ01(∥ˆβ[0] S −βS∥1 − (10) 22 Then as Then as 1 2(ˆβ[0] −β)T(XTX + λ02I)(ˆβ[0] −β) ⩾0, (11) (11) and assume 4λ02 ⩽λ01/∥β∥∞, then we can have and assume 4λ02 ⩽λ01/∥β∥∞, then we can have and assume 4λ02 ⩽λ01/∥β∥∞, then we can have λ02 ˆβ(ˆβ[0] −β) ⩾−1 4λ01∥ˆβ[0] S −βS∥1 −1 4λ01∥ˆβ[0] Sc∥1, (12) (12) and as 2∥W∥∞⩽ λ0 n1/2 , we have and as 2∥W∥∞⩽ λ0 n1/2 , we have ϵX(ˆβ[0] −β) ⩽1 2λ01∥ˆβ[0] −β∥1 ⩽1 2λ01∥ˆβ[0] S −βS∥1. Appendix (13) (13) Take (11, 12, 13) into (10), we have Take (11, 12, 13) into (10), we have −1 4λ01∥ˆβ[0] S −βS∥1 −1 4λ01∥ˆβ[0] Sc∥1 ⩽1 2λ01∥ˆβ[0] S −βS∥1 + 1 2λ01∥ˆβ[0] Sc∥1 + λ01∥ˆβ[0] S −βS −1 4λ01∥ˆβ[0] S −βS∥1 −1 4λ01∥ˆβ[0] Sc∥1 ⩽1 2λ01∥ˆβ[0] S −βS∥1 + 1 2λ01∥ˆβ[0] Sc∥1 + λ01∥ˆβ[0] S −βS∥1 −λ01∥ˆβ[0] Sc∥1. −1 4λ01∥ˆβ[0] S −βS∥1 −1 4λ01∥ˆβ[0] Sc∥1 ⩽1 2λ01∥ˆβ[0] S −βS∥1 + 1 2λ01∥ˆβ[0] Sc∥1 + λ01∥ˆβ[0] S −βS∥1 −λ01∥ˆβ[0] Sc∥1. Hence, we have ∥ˆβ[0] Sc∥1 ⩽∥ˆβ[0] S −βS∥1 ∥ˆβ[0] Sc∥1 ⩽∥ˆβ[0] S −βS∥1 Lemma 2. Set λ01 = 4σ(n log p)1/2, and suppose Condition 1 holds. Then we have with probability at least 1 −1/p : ∥ˆu[0]∥2 ⩽14σ K2 p q log p. ∥ˆu[0]∥2 ⩽14σ K2 p q log p. ∥ˆu[0]∥2 ⩽14σ K2 p q log p. Proof of lemma2. According to the Gaussian tail bound, we have for t ⩾σ, Proof of lemma2. According to the Gaussian tail bound, we have for t ⩾σ, P(\|ϵ\| > t) < exp(−t2 2σ2), P(\|ϵ\| > t) < exp(−t2 2σ2), then we have P(∥W∥∞> 2σ p log p) < p · exp(−4σ2 log p 2σ2 ) = 1 p. So with probability 1 −1/p, we have ∥W∥∞⩽2σ√log p holds. Set So with probability 1 −1/p, we have ∥W∥∞⩽2σ√log p holds. Set F(β) = 1 2∥y −Xβ∥2 2 + λ01∥β∥1 + λ02∥β∥2 2 2 . So with probability 1 −1/p, we have ∥W∥∞⩽2σ√log p holds. Set F(β) = 1 2∥y −Xβ∥2 2 + λ01∥β∥1 + λ02∥β∥2 2 2 . F(β) = 1 2∥y −Xβ∥2 2 + λ01∥β∥1 + λ02∥β∥2 2 2 . 23 Deﬁne V (u) = F(ˆβ[0]) −F(β). Then we have V (u) = 1 2uT(C + λ02 n I)u −uTW + λ02 √nuTβ + λ01 X j∈S (\|βj + uj √n\| −\|βj\|) + λ0 X j∈Sc \|βj + uj √n\|. (14) 2 n √n j∈S √n j∈Sc √n (14) (14) As u[0] = q (ˆβ[0] −β), hence As u[0] = q (ˆβ[0] −β), hence = q (ˆβ[0] −β), hence u[0] = arg min V (u). Since when u = 0, V (u) is also 0. So we have V (ˆu[0]) ⩽0. Obviously, as uTCu ⩾K2∥u∥2 2 holds, uT(C + λ02 n I)u ⩾K2∥u∥2 2 also holds. As 4λ02 ⩽λ01/∥β∥∞, we have λ02 √nuTβ ⩾−λ01 4√n(∥uS∥1 + ∥uSc∥1), (15) (15) and conditional on 2∥W∥∞⩽λ01/√n, we have and conditional on 2∥W∥∞⩽λ01/√n, we have 2∥WS∥2 ⩽√qλ01/√n. Appendix (16) 2∥WS∥2 ⩽√qλ01/√n. (16) Take (15) and (16) into (14), we have Take (15) and (16) into (14), we have V (u) ⩾∥uS∥2 K2 2 ∥u∥2 −7 4 √q λ01 √n + 3λ01 4√n −∥WSc∥∞ ∥uSc∥1. Apparently 3λ01 4√n −∥WSc∥∞> 0, Apparently Apparently 3λ01 4√n −∥WSc∥∞> 0, 3λ01 4√n −∥WSc∥∞> 0, 3λ01 4√n −∥WSc∥∞> 0, thus when ∥ˆu[0] S ∥2 ⩾7λ01√q 2K2 √n = 14σ K2 p q log p, ∥ˆu[0] S ∥2 ⩾7λ01√q 2K2 √n = 14σ K2 p q log p, we have V (ˆu) > 0, so the following bound should hold we have V (ˆu) > 0, so the following bound should hold ∥ˆu[0]∥2 ⩽14σ K2 p q log p. ∥ˆu[0]∥2 ⩽14σ K2 p q log p. 24 24 Proof of Proposition 1. We have λ11 = 4σ√n log n, then we can get P(2∥W∥∞⩽λ11 √n) ⩾ 1 −1 n. The target of step 1 is Proof of Proposition 1. We have λ11 = 4σ√n log n, then we can get P(2∥W∥∞⩽λ11 √n) ⩾ 1 −1 n. The target of step 1 is 1 −1 n. The target of step 1 is ˆβ[1] := arg min ( 1 2∥y −Xβ∥2 2 + λ11 p X j=1 ˆw[0] j \|βj\| + λ12 2 p X j=1 vjβ2 j ) , where ˆw[0] j =    1/ ˆβ[0] j , ˆβ[0] j ̸= 0 n, ˆβ[0] j = 0 , ˆw[0] j =    1/ ˆβ[0] j , ˆβ[0] j ̸= 0 n, ˆβ[0] j = 0 , and we can deﬁne a matrix Vn×p, i.e. and we can deﬁne a matrix Vn×p, i.e. and we can deﬁne a matrix Vn×p, i.e. V =   √v1 0 · · · 0 0 √v2 0 ... ... ... ... 0 0 · · · √vp   . Thus, we can write λ12 Pp j=1 vjβ2 j as λ12∥V β∥2 2. Now we have Thus, we can write λ12 Pp j=1 vjβ2 j as λ12∥V β∥2 2. Appendix Now we have 1 2∥y −X ˆβ[1]∥2 2 + λ11 p X j=1 ˆw[0] j \|ˆβ[1] j \| + λ12 2 ∥V ˆβ[1]∥2 2 ⩽1 2∥y −Xβ∥2 2 + λ11 p X j=1 ˆw[0] j \|βj\| + λ12 2 ∥V β∥2 2 1 2∥y −X ˆβ[1]∥2 2 + λ11 p X j=1 ˆw[0] j \|ˆβ[1] j \| + λ12 2 ∥V ˆβ[1]∥2 2 ⩽1 2∥y −Xβ∥2 2 + λ11 p X j=1 ˆw[0] j \|βj\| + λ12 2 ∥V β∥2 2 Then we can easily have Then we can easily have Then we can easily have 1 2(ˆβ[1] −β)(XTX + λ12V TV )(ˆβ[1] −β) + λ12(V β)TV (ˆβ[1] −β) + λ11 X j∈Sc ˆw[0] j \|ˆβ[1] j \| ⩽ϵTX(ˆβ[1] −β) + λ1 1 2(ˆβ[1] −β)(XTX + λ12V TV )(ˆβ[1] −β) + λ12(V β)TV (ˆβ[1] −β) + λ11 X j∈Sc ˆw[0] j \|ˆβ[1] j \| ⩽ϵTX(ˆβ[1] −β) + λ1 1 2(ˆβ[1] −β)(XTX + λ12V TV )(ˆβ[1] −β) + λ12(V β)TV (ˆβ[1] −β) + λ11 X j∈Sc ˆw[0] j \|ˆβ[1] j \| ⩽ϵTX(ˆβ[1] −β) + λ1 (17) /(4∥β∥∞max vj), thus Set λ12 ⩽λ11/(4∥β∥∞max vj), thus Set λ12 ⩽λ11/(4∥β∥∞max vj), thus Set λ12 ⩽λ11/(4∥β∥∞max vj), thus λ12(V β)T(V ˆβ[1] −V β) ⩾−λ11 4 ∥ˆβ[1] S −βS∥1 −λ11 4 ∥ˆβ[1] Sc∥1 (18) (18) Take (18) into (17), we have Take (18) into (17), we have λ11 X j∈Sc ˆw[0] j \|ˆβ[1] j \| ⩽3λ11 4 ∥ˆβ[1] S −βS∥1 + 3λ11 4 ∥ˆβ[1] Sc∥1 + λ11 X j∈S ˆw[0] j \|ˆβ[1] j −βj\|. (19) (19) Conditional on Conditional on √n∥ˆβ[0] −β∥2 ⩽14σ K2 p q log p, √n∥ˆβ[0] −β∥2 ⩽14σ K2 p q log p, 25 then for j /∈S, we have then for j /∈S, we have \|ˆβ[0] j /∈S\| ⩽14σ K2 r q log p n . (20) \|ˆβ[0] j /∈S\| ⩽14σ K2 r q log p n . (20) (20) As q = O(nc2), p = O(exp(nc1)), 0 ⩽c1 + c2 < 1, when n is large enough we have As q = O(nc2), p = O(exp(nc1)), 0 ⩽c1 + c2 < 1, when n is large enough we have max j /∈S \|ˆβ[1] j \| < 1. (21) max j /∈S \|ˆβ[1] j \| < 1. Appendix (21) (21) For j ∈S, as For j ∈S, as r j ∈S, as For j ∈S, as j , min j∈S \|βj\| > K1 λ01√q n = K1 4σ√q log p √n , min j∈S \|βj\| > K1 λ01√q n = K1 4σ√q log p √n , and max j∈S \|ˆβ[0] j −βj\| ⩽∥ˆβ[0] −β∥∞⩽∥ˆβ[0] −β∥2 ⩽14σ K2 r q log p n . Provided K1 > 7/K2, we have min j∈S \|ˆβ[0] j \| > (4K1σ −14σ K2 ) r q log p n > 14σ K2 r q log p n ⩾max j /∈S \|ˆβ[0] j \|, (22) (22) and thus there exists a positive constant K3 < K1, and c3 ⩾c1 + c2, which let min j∈S \|ˆβ[0] j \| > K3n(c3−1)/2. (23) min j∈S \|ˆβ[0] j \| > K3n(c3−1)/2. (23) min j∈S \|ˆβ[0] j \| > K3n(c3−1)/2. (23) Then we can write (19) as Then we can write (19) as Then we can write (19) as λ11 X j∈Sc \|ˆβ[1] j \| maxj /∈S \|ˆβ[0] j \| ⩽ 3λ11 4 maxj /∈S (∥ˆβ[1] S −βS∥1 + ∥ˆβ[1] Sc∥1) + λ11 X j∈S \|ˆβ[1] j −βj\| minj∈S \|ˆβ[0] j \| . (24) (24) Take (21) and (22) into (24), we have Take (21) and (22) into (24), we have ∥ˆβ[1] Sc∥1 ⩽7∥ˆβ[1] S −βS∥1, ∥ˆβ[1] Sc∥1 ⩽7∥ˆβ[1] S −βS∥1, that is that is ∥ˆu[1] Sc∥1 ⩽7∥ˆu[1] S ∥1. ∥ˆu[1] Sc∥1 ⩽7∥ˆu[1] S ∥1. Then we have RE condition holds (ˆu[1] Sc)TCˆu[1] ⩾K2∥ˆu[2]∥2 2. (ˆu[1] Sc)TCˆu[1] ⩾K2∥ˆu[2]∥2 2. (ˆu[1] Sc)TCˆu[1] ⩾K2∥ˆu[2]∥2 2. 26 26 We write ˆβ[1] as ˆβ[1] = β + ˆu[1] √n, where ˆu[1] = arg min V (u). Appendix Set F(β) = 1 2∥y −Xβ∥2 2 + λ11 p X j=1 ˆw[0] j \|βj\| + λ12 2 p X j=1 vjβ2 j , and let V (u) = F(ˆβ[1]) −F(β), thus ˆu[1] = arg min V (u), and and let V (u) = F(ˆβ[1]) −F(β), thus ˆu[1] = arg min V (u), and V (u) = 1 2uT(C + λ12 n V TV )u −uTW + λ12 √nβTV TV u + λ11 X j∈S ( ˆw[0]\|βj + uj √n\| −ˆw[0]\|βj\|) + λ11 X j∈Sc ( ˆw[0] j \|βj + uj √n\|) ⩾L1 + L2 V (u) = 1 2uT(C + λ12 n V TV )u −uTW + λ12 √nβTV TV u + λ11 X j∈S ( ˆw[0]\|βj + uj √n\| −ˆw[0]\|βj\|) + λ11 X j∈Sc ( ˆw[0] j \|βj + uj √n\|) where L1 = K2 2 ∥u∥2 2 −uT S W T S −λ11 √n ∥uS∥1 minj∈S \|ˆβ[0] j \| −λ11 4√n∥uS∥1, (25) (25) and L2 = λ11 √n ∥uSc∥1 maxj /∈S \|ˆβ[0] j \| −uT ScW T Sc −λ11 4√n∥uSc∥1. (26) (26) Take (23), ∥W∥∞⩽2σ√log n into (25), we have Take (23), ∥W∥∞⩽2σ√log n into (25), we have Take (23), ∥W∥∞⩽2σ√log n into (25), we have L1 ⩾K2 2 ∥u∥2 2 −2σ p q log n∥uS∥2 −4σ√log n K3n(c3−1)/2 √q∥uS∥2 −σ p log n√q∥uS∥2 ⩾∥uS∥2 K2 2 ∥u∥2 −3σ p q log n −4σ K3 p qn(1−c3) log n . Take (20), ∥W∥∞⩽2σ√log n into (26), we have Take (20), ∥W∥∞⩽2σ√log n into (26), we have Take (20), ∥W∥∞⩽2σ√log n into (26), we have L2 ⩾4σ√log n∥uSc∥1 14σ K2 q q log p n −2σ p log n∥uSc∥1 −σ p log n∥uSc∥1 L2 ⩾4σ√log n∥uSc∥1 14σ K2 q q log p n −2σ p log n∥uSc∥1 −σ p log n∥uSc∥1 = (2K2 7 s n log n q log p −3σ p log n)∥uSc∥1 = (2K2 7 s n log n q log p −3σ p log n)∥uSc∥1 > 0. When ∥u∥2 > 8σ K2K3 p qn(1−c3) log n, 27 V (u) > 0, but we have V (u) ⩽0. Therefore, with probability of 1 −1/n, we have ∥ˆu[1]∥2 ⩽ 8σ K2K3 p qn(1−c3) log n. Appendix According to (5), we have ∥ˆβ[1] −β∥2 = 8σ K2K3 p qn−c3 log n ≪K14σ r q log p n = K1λ01√q n < min j∈S \|βj\|, ∥β β∥2 = K2K3 p qn 3 log n ≪K14σ r n = n < min j∈S \|βj\|, thus, we have P(S ⊆A[1]) ⩾1 −1/n. thus, we have thus, we have thus, we have Proof of Theorem 1. Conditional on λ21 = 4σ√n log n, with probability, we can easily have P(2∥W∥∞⩽λ21 √n) = 1 n. P(2∥W∥∞⩽λ21 √n) = 1 n. The target of step 2 is ˆβ[2] := arg min β(A[1])c=0    1 2∥y −Xβ∥2 2 + λ21 X j∈A[1] \|βj/ˆβ[1] j \| + λ22 2 X j∈A[1] vjβ2 j   , By the deﬁnition, we have By the deﬁnition, we have 1 2∥y −X ˆβ[2]∥2 2 + λ21 X j∈A[1] \| ˆβ[2] j ˆβ[1] j \| + λ22 2 ∥V ˆβ[2]∥2 2 ⩽1 2∥y −Xβ∥2 2 + λ21 X j∈A[1] \| βj ˆβ[1] j \| + λ22 2 ∥V β∥2 2, which can be written as which can be written as 1 2(ˆβ[2] −β)T(XTX + λ22V TV )(ˆβ[2] −β) + λ22(V β)T(V ˆβ[2] −V β) + λ21 X j∈A[1]/S \| ˆβ[2] j ˆβ[1] j \| 1 2(ˆβ[2] −β)T(XTX + λ22V TV )(ˆβ[2] −β) + λ22(V β)T(V ˆβ[2] −V β) + λ21 X j∈A[1]/S \|β[2] j ˆβ[1] j \| ⩽ϵTX(ˆβ[2] −β) + λ21 X j∈S \| ˆβ[2] j −βj ˆβ[1] j \|. (27) (27) Conditional on Conditional on Conditional on ∥ˆβ[1] −β∥2 ⩽ 8σ K2K3 p qn−c3 log n, 28 we have for j /∈S we have for j /∈S ∥ˆβ[1] j /∈S∥∞⩽ 8σ K2K3 p qn−c3 log n, (28) ∥ˆβ[1] j /∈S∥∞⩽ 8σ K2K3 p qn−c3 log n, (28) nd when n is large enough, we have max j /∈S \|ˆβ[1] j \| ⩽1. (29) max j /∈S \|ˆβ[1] j \| ⩽1. (29) max j /∈S \|ˆβ[1] j \| ⩽1. (29) For j ∈S, we have min j∈S \|ˆβ[1] j \| > K3n(c3−1)/2, (30) min j∈S \|ˆβ[1] j \| > K3n(c3−1)/2, (30) min j∈S \|ˆβ[1] j \| > K3n(c3−1)/2, (30) and min \|β[1]\| > max \|ˆβ[1]\| (31) j∈S j and and and min j∈S \|β[1] j \| > max j /∈S \|ˆβ[1] j \|. (31) min j∈S \|β[1] j \| > max j /∈S \|ˆβ[1] j \|. Appendix (31) min j∈S \|β[1] j \| > max j /∈S \|ˆβ[1] j \|. (31) j∈S j /∈S Take (29) into (27), we have Take (29) into (27), we have λ21 maxj∈A[1]/S \|ˆβ[1] j \| ∥ˆβ[2] j∈A[1]/S∥1 ⩽ 3λ21 4 maxj /∈S \|ˆβ[1] j \| (∥ˆβ[2] S −βS∥1 + ∥ˆβ[2] A[1]/S∥1) + λ21 minj∈S \|ˆβ[1] j \| ∥ˆβ[2] j −βj∥1, (32) maxj∈A[1]/S \|ˆβ[1] j \| ∥β[ ] j∈A[1]/S∥1 ⩽ 4 maxj /∈S \|ˆβ[1] j \| (∥β[ ] S −βS∥1 + ∥β[ ] A[1]/S∥1) + minj∈S \|ˆβ[1] j \| ∥β[ ] j −βj∥1 (32) (32) then take (31) into (32), we have then take (31) into (32), we have then take (31) into (32), we have ∥ˆβ[2] A[1]/S∥1 ⩽7∥ˆβ[2] S −βS∥1, ∥ˆβ[2] A[1]/S∥1 ⩽7∥ˆβ[2] S −βS∥1, ∥ˆβ[2] A[1]/S∥1 ⩽7∥ˆβ[2] S −βS∥1, which is ∥ˆu[2] A[1]/S∥1 ⩽7∥ˆu[2] S ∥1. ∥ˆu[2] A[1]/S∥1 ⩽7∥ˆu[2] S ∥1. Then we have RE condition holds (ˆu[2])TCˆu[2] ⩾K2∥ˆu[2]∥2 2. We write ˆβ[2] as ˆβ[2] = β + ˆu[2]/√n, where ˆu[2] = arg min V (uA[1]). Then we have We write ˆβ[2] as ˆβ[2] = β + ˆu[2]/√n, where ˆu[2] = arg min V (uA[1]). Then we have V (uA[1]) = 1 2uT A[1](C + λ22 n V TV )uA[1] −uT A[1]WA[1] + λ22 √nβT A[1]V TV uA[1] + λ21 X j∈A[1] (\|βj + ˆuj √n\| −\|βj\| \|ˆβ[1] j \| ) ⩾L1 + L2, 29 where L1 = K2 2 ∥uA[1]∥2 2 −uT S WS −λ21 √n ∥uS∥1 minj∈S \|ˆβ[1] j \| −λ21 4√n∥uS∥1, (33) (33) and L2 = λ21 √n ∥uA[1]/S∥ maxj /∈S \|β[1] j \| −uT A[1]/SWA[1]/S −λ21 4√n∥uA[1]/S∥1. (34) (34) Take (30), 2∥W∥∞⩽λ21 √n into (33), we have Take (30), 2∥W∥∞⩽λ21 √n into (33), we have Take (30), 2∥W∥∞⩽λ21 √n into (33), we have L1 ⩾K2 2 ∥uA[1]∥2 2 −2σ p q log n∥uS∥2 −4σ√log n K3n(c3−1)/2 √q∥uS∥2 −σ p log n√q∥uS∥2 ⩾∥uS∥2 K2 2 ∥uA[1]∥2 −3σ p q log n −4σ K3 p qn1−c3 log n . Take (28), 2∥W∥∞⩽λ21 √n into (34), we have Take (28), 2∥W∥∞⩽λ21 √n into (34), we have L2 ⩾ 4σ√log n 8σ K2K3 √qn−c3 log n∥uSc∥1 −3σ p log n∥uA[1]/S∥1 > 0. When ∥uA[1]∥2 > 8σ K2K3 p qn(1 −c3) log n, V (u) > 0, but we have V (u) ⩽0. Thus, with probability 1 −1/n, we have ∥ˆu[2]∥2 ⩽ 8σ K2K3 p qn1−c3 log n. Appendix (35) (35) Then we have Then we have Then we have P(S ⊆A[2]) ⩾1 −1/n. (36) (36) Similarly, (35) and (36) will hold at the k + 1th step when they hold at the kth step. Thus, by induction, they hold for k=3,4,5.... Conditional on the convergence A[1] ⊇A[2] ⊇· · · ⊇A[k] ⊇· · · ⊇A. Similarly, (35) and (36) will hold at the k + 1th step when they hold at the kth step. Thus, by induction, they hold for k=3,4,5.... Conditional on the convergence A[1] ⊇A[2] ⊇· · · ⊇A[k] ⊇· · · ⊇A. Similarly, (35) and (36) will hold at the k + 1th step when they hold at the kth step. Thus, by induction, they hold for k=3,4,5.... Conditional on the convergence A[1] ⊇A[2] ⊇· · · ⊇A[k] ⊇· · · ⊇A. Finally, we have Finally, we have Finally, we have Finally, we have Finally, we have ∥ˆβ −β∥2 ⩽ 8σ K2K3 p qn−c3 log n, ∥ˆβ −β∥2 ⩽ 8σ K2K3 p qn−c3 log n, and and P(sign(ˆβ) = sign(β)) ⩾1 −1/n. Thus, we have Theorem1 proved. 30 30
https://openalex.org/W2180238987	https://bmccancer.biomedcentral.com/track/pdf/10.1186/s12885-015-1906-5	English	null	Prognostic significance of ST3GAL-1 expression in patients with clear cell renal cell carcinoma	BMC cancer	2,015	cc-by	6,461	Abstract Background: Aberrant sialylated carbohydrate synthesis is frequently noted in various cancers. Sialyltransferase ST3GAL-1, which adds a sialic acid in an α-2,3 linkage to Gal β1,3 GalNAc, preforms an important role in modulating cellular behaviors. However, little is known about prognostic significance of ST3GAL-1 in clear cell renal cell carcinoma (ccRCC). In this study, we aimed to investigate the prognostic significance of sialyltransferase ST3GAL-1 and its correlation with clinical outcomes in patients with ccRCC. Methods: A total of 286 patients who underwent nephrectomy between 2005 and 2007 in a single academic center were recruited. Immunohistochemical staining was performed on tissue microarrays to assess the expression level. Kaplan-Meier method and Cox proportional hazard model were applied to assess the prognostic value of ST3GAL-1. Nomograms were generated as prediction model for overall survival and disease free survival at 5 and 8 years after nephrectomy. Results: The present results show high expression of ST3GAL-1 is associated with reduced overall survival (p = 0.013) and disease free survival (p = 0.004). In multivariate cox analyses, ST3GAL-1 was defined as an independent prognostic factor for overall survival (p = 0.006) and disease free survival (p = 0.001). After incorporation into the University of California Integrated Staging System (UISS) intermediate/high risk group for non-metastatic ccRCC, ST3GAL-1 could further distinguish patient with dismal prognosis (p = 0.015 and 0.002 for OS and DFS respectively). The nomograms revealed better predictive accuracy in predicting 5- and 8- year overall survival and disease free survival than the TNM stage alone. Conclusions: ST3GAL-1 is an independent adverse prognostic factor for recurrence and survival of patients with ccRCC. Conclusions: ST3GAL-1 is an independent adverse prognostic factor for recurrence and survival of patients with ccRCC. Keywords: Clear cell renal cell carcinoma, β-Galactoside α-2,3-Sialyltransferase 1, Prognosis, Nomogram, Overall survival, Disease free survival Keywords: Clear cell renal cell carcinoma, β-Galactoside α-2,3-Sialyltransferase 1, Prognosis, Nomogram, Overall survival, Disease free survival without any evidence of metastasis will develop relapse and/or metastatic RCC in the future [3]. Currently, TNM stage, Fuhrman grade, and Eastern Cooperative Oncology Group performance status (ECOG PS) have been widely used as predictors of clinical outcomes for patients with RCC. Several outcomes prognostic predic- tion algorithms like University of California Integrated Staging System (UISS), which incorporates TNM stage, Fuhrman grade, and ECOG PS, have been proposed pre- viously to stratify the clinical outcomes after nephrec- tomy [4, 5]. © 2015 Bai et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. RESEARCH ARTICLE Open Access * Correspondence: jjxufdu@fudan.edu.cn; guo.jianming@zs-hospital.sh.cn †Equal contributors 2Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China 1Department of Urology, Zhongshan Hospital, Fudan University, Shanghai 200032, China Bai et al. BMC Cancer (2015) 15:880 DOI 10.1186/s12885-015-1906-5 Bai et al. BMC Cancer (2015) 15:880 DOI 10.1186/s12885-015-1906-5 Abstract But the biological nature of ccRCC is kind of complex with an unpredictable course; even tumors Prognostic significance of ST3GAL-1 expression in patients with clear cell renal cell carcinoma Qi Bai1†, Li Liu1†, Yu Xia1†, Qilai Long1, Jiajun Wang1, Jiejie Xu2* and Jianming Guo1* Qi Bai1†, Li Liu1†, Yu Xia1†, Qilai Long1, Jiajun Wang1, Jiejie Xu2* and Jianming Guo1* Background Renal cell carcinoma (RCC) is the most common solid lesion within kidney and accounts for 2–3 % of all can- cers in adults [1]. The predominant histologic subtype is clear cell renal cell carcinoma (ccRCC) (70–85 %) [2]. Although most patients with localized tumors can be cured with surgical therapies, 20–30 % of the patients Bai et al. BMC Cancer (2015) 15:880 Page 2 of 9 Page 2 of 9 and baseline demographic characters include age, sex, tumor size, and necrosis were also collected retrospectively. Patients with localized RCC (n = 271) were treated with ei- ther nephron spare surgery or radical nephrectomy. Pa- tients with metastatic RCC (n = 15) at diagnosis received cytoreductive nephrectomy followed by interferon-α based immunotherapy. Tumor stage and postoperative histo- pathological type was determined according to the 2010 AJCC TNM classification [18]. The inclusion criteria were as follows: (1) the histopathological type proved to be ccRCC, (2) all the individuals had no history of anticancer therapy before the nephrectomy, and (3) had no history of other malignant before. If the histopathology was mostly necrosis (>80%) or the morphologic features represent a mixture type of clear cell RCC and other RCC were ex- cluded from the present study. The UISS predictive model was applied to all cases to stratify the risk groups. Most patients underwent regular follow-up every 6 months or earlier for the first 2 years right after the nephrectomy and every 12 months thereafter. Clinical Research Ethics Committee of Zhongshan Hospital, Fudan University had approved the study and granted permissions to access the patient records. Written, informed consent was obtained from each individual enrolled in the study. with the comparable stage or the same pathological type could show a wide variation in biological behavior and clinical outcomes. The improvement of the predictors of clinical outcomes for RCC is needed [6, 7]. Change in the structure of glycan chains is one of the most shared features of the malignant transformation [8]. The oligosaccharide chains of glycoprotein and glycolipid are often terminated by sialic acids, which play intensely important role in regulating physiologically and patho- logically interactions with ligands [9]. In types of cancers, abnormal high level of sialylated tumor associated carbo- hydrate antigens are frequently noted [10, 11]. The up- regulated sialylation may mediate key pathophysiological events during various steps of tumor progression [12–14]. Immunohistochemical staining and evaluation Hematoxylin and eosin-stained slides had been screened for optimal tumor content before we constructed tissue microarray (TMA) slides. Two cores were taken from the formalin-fixed, paraffin embedded surgical speci- mens with 2.0 mm diameter punch to make sure each array represented the dominant focus of ccRCC. Immu- nohistochemical staining was performed on TMA, and the primary antibody was rabbit polyclonal anti-SIAT4A antibody (ab96129, Abcam, Cambridge, MA, USA). The images of stained tissue were captured by the computer- ized image system composed of an Olympus CCD cam- era connected to a Nikon eclipse Ti-s microscope. The immunohistochemistry samples were scanned at high- Background β-galactoside α-2,3-sialyltransferase 1 (ST3GAL-1), which catalyzes the addition of sialic acid in an α-2,3 linkage to Gal β1,3 GalNAc, preforms important role in modulating cellular behavior. Increasing studies have revealed that the abnormal activity of ST3GAL-1 contributes to the tumor progression [10, 15–17]. However, few studies dedicated to evaluate the expression of ST3GAL-1 and its correl- ation with ccRCC pathological characters, making the issue elusive. In this study, we aimed to investigate the prognostic sig- nificance of sialyltransferase ST3GAL-1 expression and its correlation with clinical outcomes in patients with ccRCC. ST3GAL-1 expression was assessed by immunohistochem- istry, and nomograms integrating ST3GAL-1 with other prognostic parameters were generated to refine individual risk stratification in ccRCC patients. Methods Patients We retrospectively collected 286 patients who underwent nephrectomy between 2005 and 2007 at Zhongshan Hos- pital Fudan University (Shanghai, China). Patient records include TNM stage, Fuhrman grades, histology type, ECOG PS were extracted from the database of the institution, and all the data above were accessible. Other clinicopathological Fig. 1 Representative photographs of ST3GAL-1 immunostaining. ST3GAL-1 expression in normal kidney tissue (a); Low ST3GAL-1 expression in tumor tissue (b); High ST3GAL-1 expression in tumor tissue (c). Original magnification: ×200 Fig. 1 Representative photographs of ST3GAL-1 immunostaining. ST3GAL-1 expression in normal kidney tissue (a); Low ST3GAL-1 expression in tumor tissue (b); High ST3GAL-1 expression in tumor tissue (c). Original magnification: ×200 Bai et al. BMC Cancer (2015) 15:880 Page 3 of 9 Page 3 of 9 Table 1 Associations between ST3GAL-1 expression and clinicopathological characteristics in patients with ccRCC ST3gal-1 expression Characteristics All patients Low High p* Age (years) 0.656 Mean ± SD 55.0 ± 13.0 54.7 ± 13.6 55.4 ± 11.9 Gender 0.455 Female 90 57 33 Male 196 115 81 T stage 0.913 T1 180 106 74 T2 24 14 10 T3 79 50 29 T4 3 2 1 N stage 1.000 N0 284 171 113 N1 2 1 1 M stage 0.102 M0 273 167 106 M1 13 5 8 TNM stage 0.334 I 176 105 71 II 20 12 8 III 75 49 26 IV 15 6 9 Fuhrman nuclear grade grade 0.604 1 31 19 12 2 216 127 89 3 37 24 13 4 2 2 0 Necrosis 0.652 Absent 249 151 98 Present 37 21 16 ECOG PS 0.587 0 198 117 81 ≥1 88 55 33 Abbreviations: ECOG PS Eastern Cooperative Oncology Group performance status, ccRCC clear cell renal cell carcinoma χ2 test or t test was performed. p < 0.05 was regard as statistically significant Table 1 Associations between ST3GAL-1 expression and clinicopathological characteristics in patients with ccRCC power magnification (×200) and photographed by NIS- Elements F3.2 software. The staining intensity and extent were scored by two independent pathologists who were blind to the clinical outcomes using the integrated optical density (IOD) calculated by Image‐Pro Plus version 6.0 software (Media Cybernetics Inc., Bethesda, MD, USA). The intensity score was graded as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong); the extent score was calculated by the percentage of the positive cells (0–100 %). Methods Patients The staining intensity and extent were then multiplied to gen- erate the expression score ranging from 0 to 300. The score of 210 was selected as the cutoff point of high/low expression by the X-Tile software (Yale University School of Medicine, New Haven, CT, USA). Statistical analyses The correlation between ST3GAL-1 expression and clin- icopathological characteristics was analyzed with Fisher’s exact test, χ2 test or t test. Kaplan-Meier method was ap- plied to compare overall survival (OS) and disease free survival (DFS) for groups of low and high expression. Stat- istical significance was calculated using log-rank test. Multivariate Cox proportional hazard models were used to estimate and test the impact of demographic character- istics, clinical features and ST3GAL-1 expression on over- all survival and disease free survival. Hazard ratio (HR) and 95 % confidence interval (CI) were calculated. The nomograms were generated using R software with “rms” package (R Foundation for Statistical Computing, Vienna, Austria). Selection of variables included in no- mograms was based on statistical significance of multivari- ate analyses, and tumor size was also included in the model as continuous variable. For DFS, we divided T stage variable into T1a and T1b + T2, due to the clinical similar- ity for metastases after radical nephrectomy [19]. Calibra- tion plots for 5- and 8- year OS and DFS were generated to explore the performance characteristics of the predict- ive model. The Harrell’s concordance indices (c-indices) were used to measure the prognostic accuracy. All data analyses above were performed using SPSS version 19.0 (SPSS Inc., IL, Chicago, USA) and R software with “rms” package (R Foundation for Statistical Computing, Vienna, Austria). All statistical tests were two sided and consid- ered significant at two-sided p <0.05 levels. Abbreviations: ECOG PS Eastern Cooperative Oncology Group performance status, ccRCC clear cell renal cell carcinoma χ2 test or t test was performed. p < 0.05 was regard as statistically significant Results ST3GAL-1 expression and survival analyses in human ccRCC The representative images of immunostaining were present in Fig. 1. The ST3GAL-1 expression was very low or not detectable in normal kidney tissues (Fig. 1a). The positive staining of ccRCC predominantly appeared in the cell cyto- plasm (Fig. 1c). We analyzed a total of 286 patients in the present study. As Table 1 presented, the mean (SD) age was 55.0 (13.0) years old and the median follow up was 99 (range 2.63–120.47) months. Thirteen patients had distant metastasis, and two patients had regional lymph node me- tastasis at time of surgery. The Kaplan-Meier curves show that patients with high ST3GAL-1 expression level tend to have significantly adverse outcomes for OS (p = 0.013, Fig. 2a) than those with low ST3GAL-1 expression pa- tients. For patients with localized ccRCC, the differences Bai et al. BMC Cancer (2015) 15:880 Page 4 of 9 Fig. 2 Kaplan-Meier analyses for overall survival and disease free survival of patients with ccRCC according to ST3GAL-1 expression. Overall survival according to ST3GAL-1 expression in all ccRCC patients (a); overall survival according to ST3GAL-1 expression in localized ccRCC patients (b); disease free survival according to ST3GAL-1 expression in localized ccRCC patients (c); p-value was calculated by Log rank test, <0.05 was regarded as statistically significant Fig. 2 Kaplan-Meier analyses for overall survival and disease free survival of patients with ccRCC according to ST3GAL-1 expression. Overall survival according to ST3GAL-1 expression in all ccRCC patients (a); overall survival according to ST3GAL-1 expression in localized ccRCC patients (b); disease free survival according to ST3GAL-1 expression in localized ccRCC patients (c); p-value was calculated by Log rank test, <0.05 was regarded as statistically significant were still significant in both OS (p = 0.030, Fig. 2b) and DFS (p = 0.004, Fig. 2c). we sought to investigate whether incorporation of high/low ST3GAL-1 expression into UISS outcome algorithm would improve the predictive accuracy by calculating c-index. In the IR/HR group for localized ccRCC, significant differ- ence was found in OS (p = 0.015) and DFS (p = 0.002) ac- cording to the ST3GAL-1 expression (Fig. 3b, d), however ST3GAL-1 failed to further stratify patients with different prognosis in UISS LR patients (Fig. 3a, c). The c-indices of Multivariate cox regression analyses To investigate the prognostic impact of ST3GAL-1 on ccRCC, multivariate Cox regression models were applied (Table 2). In the multivariate cox models, patients with higher ST3GAL-1 expression showed a significantly re- duced OS (p = 0.006) and DFS (p = 0.001) compared with their counterparts. It was also confirmed that TNM stage (p < 0.001), Fuhrman grade (p = 0.044), Necrosis (p = 0.045), and ECOG PS (p = 0.047) were independent prognostic factors for OS, and T stage (p = 0.003), Fuhrman grade (p = 0.020) and necrosis (p = 0.015) were independent prognostic factors for DFS in ccRCC. It is now well-established that altered activity of sialyl- transferase is one of the key mechanisms to trigger aber- rant sialylation of glycans and expression of specific tumor associated carbohydrates in cancer [24]. However, re- searches focus on the sialylation glycans and sialyltransfer- ase in ccRCC is now devoid. So in this study, we put our focus on the sialyltransferase ST3GAL-1, which adds a si- alic acid in an α-2,3 linkage to Gal β1,3 GalNAc. In the study, we have revealed the dismal role of ST3GAL-1 and its impact on patients with ccRCC by survival analyses. The present results showed that ccRCC patients with higher level of ST3GAL-1 expression tend to have un- favorable clinical outcomes than the counterparts. We also demonstrated that incorporating ST3GAL-1 into UISS (non-metastatic) could refine the algorithm predic- tion. In multivariate cox regression model, it was proved Discussion Glycosylation is quite common in posttranslational mod- ifications. Almost all the cell surface proteins are glyco- sylated. The aberrant glycosylation, which is aroused by the altered expression and activity of glycosyltransferase, can dramatically affect the glycoprotein and then change the behavior of cell [20–22]. Sialic acids, a family of nine carbon sugars are one of the most important monosac- charide decorating the oligosaccharide chains of several classes of cell surface and secreted glycan molecules. The sialylation of the glycan plays very important role in regu- lating cellular interaction and signal transduction. The prominent position on cell membrane allows sialoglycan to effectively participate in cell-cell and cell-extracellular matrix interaction, including adhesion, migration, and im- mune recognition [23]. Hypersialylation of glycan in tumor cells is associated with metastatic behaviors including inva- sion and enhanced cell survival and correlates with a poor prognosis for patients with cancer [24]. Extension of the UISS (non-metastatic) prognostic model with ST3GAL-1 The UISS (non-metastatic) algorithm performed well in stratifying the risk group for overall survival and disease free survival in our ccRCC cohort (p < 0.001). Furthermore, Fig. 3 Kaplan-Meier analyses for overall survival and disease free survival of patients in UISS (non-metastatic) subgroups. Overall survival for patients in the UISS (non-metastatic) low risk group (a) and intermediate/high risk group (b) according to ST3GAL-1 expression; disease free survival for patients in the UISS (non-metastatic) low risk group (c) and intermediate/high risk group (d) according to ST3GAL-1 expression; UISS: University of California Los Angeles Integrated Staging System; p-value was calculated by Log rank test, <0.05 was regarded as statistically significant Fig. 3 Kaplan-Meier analyses for overall survival and disease free survival of patients in UISS (non-metastatic) subgroups. Overall survival for patients in the UISS (non-metastatic) low risk group (a) and intermediate/high risk group (b) according to ST3GAL-1 expression; disease free survival for patients in the UISS (non-metastatic) low risk group (c) and intermediate/high risk group (d) according to ST3GAL-1 expression; UISS: University of California Los Angeles Integrated Staging System; p-value was calculated by Log rank test, <0.05 was regarded as statistically significant Page 5 of 9 Page 5 of 9 Bai et al. BMC Cancer (2015) 15:880 the UISS were 0.65 and 0.63 for OS and DFS respectively, and improved to 0.69 and 0.68 when ST3GAL-1 was added. For patients with metastatic ccRCC, we didn’t use ST3GAL-1 to further refine the UISS (metastatic) algo- rithm due to the limited individuals. the UISS were 0.65 and 0.63 for OS and DFS respectively, and improved to 0.69 and 0.68 when ST3GAL-1 was added. For patients with metastatic ccRCC, we didn’t use ST3GAL-1 to further refine the UISS (metastatic) algo- rithm due to the limited individuals. free survival in ccRCC Nomograms were generated to predict OS (Fig. 4a) and DFS (Fig. 5a) at 5 and 8 years after nephrectomy. Total points were calculated to evaluate the clinical outcomes, with higher point indicating more adverse outcome probability. Calibration plots of the nomograms are shown for 5- and 8- year prediction of OS (Fig. 4b, c) and DFS (Fig. 5b,c). The Harrell’s c-indices, which indi- cated the accuracy of the prognostic model, were 0.753 (95 % CI, 0.701–0.805) and 0.745 (95 % CI 0.690–0.800) for OS and DFS respectively, higher than that of the TMN stage alone (0.703, 95 % CI: 0.647–0.759 and 0.660, 95 % CI: 0.599–0.721). Table 2 Multivariate cox regression analyses for overall survival and disease free survival in ccRCC patients Variables Overall survival Disease free survival HR 95 % CI p* HR 95 % CI p* TNM stage <0.001 0.003 III + IV vs I+II 2.76 1.75–4.34 2.13 1.30–3.51 Fuhrman 0.044 0.020 3 + 4 vs 1+2 1.85 1.02–3.37 2.19 1.13–4.23 Necrosis 0.045 0.015 Present vs Absent 1.81 1.01–3.25 2.16 1.16–4.00 ECOG PS 0.047 0.267 ≥1 vs 0 1.61 1.01–2.56 1.35 0.80–2.29 ST3GAL-1 0.006 0.001 High vs Low 1.88 1.20–2.94 2.30 1.40–3.77 Abbreviations: ECOG PS Eastern Cooperative Oncology Group performance status, CI confidence interval, HR hazard ratio *Data obtained from the Cox proportional hazards model; p <0.05 was regard as statistically significant Bai et al. BMC Cancer (2015) 15:880 Page 6 of 9 Fig. 4 Nomogram for predicting 5- and 8-year overall survival in patients with ccRCC. a Nomogram for predicting clinical outcomes integrated with Fuhrman grade (1/2/3 + 4), TNM stage (I + II/III + IV), tumor size (continuous variable), necrosis (absent/present), ECOG PS (0/≥1), and ST3GAL-1 expression (low/high); b Calibration plot for nomogram predicted and observed 5-year overall survival rate; c Calibration plot for nomogram predicted and observed 8-year overall survival rate. Line of dashes: ideal model, vertical bars: 95 % confident interval. ECOG PS = Eastern Cooperative Oncology Group performance status. Higher total point indicated a more adverse outcomes probability Fig. 4 Nomogram for predicting 5- and 8-year overall survival in patients with ccRCC. free survival in ccRCC a Nomogram for predicting clinical outcomes integrated with Fuhrman grade (1/2/3 + 4), TNM stage (I + II/III + IV), tumor size (continuous variable), necrosis (absent/present), ECOG PS (0/≥1), and ST3GAL-1 expression (low/high); b Calibration plot for nomogram predicted and observed 5-year overall survival rate; c Calibration plot for nomogram predicted and observed 8-year overall survival rate. Line of dashes: ideal model, vertical bars: 95 % confident interval. ECOG PS = Eastern Cooperative Oncology Group performance status. Higher total point indicated a more adverse outcomes probability and breast cancers [10, 17]. P. Videira found mRNA level of ST3GAL-1 was significantly higher in malignant bladder tumor [15]. It has also been confirmed that abnormal ST3GAL-1 activities could involve in malignant cell trans- formation and tumor progression. In breast carcinoma, constitutive ST3GAL-1 expression contributed to an unfavorable prognosis [16]. In colorectal carcinoma, ST3GAL-1 expression was associated with lymph node that ST3GAL-1 was an independent prognostic factor for OS and DFS. Unexpectedly, the N stage in multivariate analyses was not considered an independent prognostic factor, which was probably due to the limited number of N1 individuals (n = 2). Several studies have elucidated the association of ST3GAL-1 with tumor progression. Overexpression of ST3GAL-1 has been reported in types of tumors like colon Page 7 of 9 Page 7 of 9 Bai et al. BMC Cancer (2015) 15:880 Fig. 5 Nomogram for predicting 5- and 8-year disease free survival in patients with ccRCC. a Nomogram for predicting disease free survival integrated with T stage (T1a/T1b + T2/T3 + T4), Fuhrman grade (1/2/3 + 4), tumor size (continuous variable), necrosis (absent/present) and ST3GAL-1 expression (low/high); b Calibration plot for nomogram predicted and observed 5-year disease free survival rate; c Calibration plot for nomogram predicted and observed 8-year disease free survival rate. Line of dashes: ideal model, vertical bars: 95 % confident interval. Higher total point indicated a more adverse outcomes probability Fig. 5 Nomogram for predicting 5- and 8-year disease free survival in patients with ccRCC. a Nomogram for predicting disease free survival integrated with T stage (T1a/T1b + T2/T3 + T4), Fuhrman grade (1/2/3 + 4), tumor size (continuous variable), necrosis (absent/present) and ST3GAL-1 expression (low/high); b Calibration plot for nomogram predicted and observed 5-year disease free survival rate; c Calibration plot for nomogram predicted and observed 8-year disease free survival rate. Line of dashes: ideal model, vertical bars: 95 % confident interval. free survival in ccRCC Higher total point indicated a more adverse outcomes probability metastasis [10]. Previous studies have reported that EMT, which is essential for tumor progression, is asso- ciated with altered expression of sialoglycans [25]. Up- regulation of sialyltransferase and following expression of sialoglycan is an important step underlying the migratory phenotype during EMT. Moreover, the proto-oncogene, c- myc could up-regulate the ST3GAL-1, while the metastasis suppressor gene nm23-H1 plays an opposite role [25, 26], indicating the protumoral role of ST3Gal-1 in the develop- ment and progress of tumors. play an important role in mediating malignant cells dis- semination [12, 27]. Recent reports have been indicative of ST3GAL-1’s responsibility for the high expression of the sialoglycans sLex and sLea [25, 28]. Increased sLex was reported in liver metastasis of colorectal cancer, and was associated with poor patient survival and early re- currence in colorectal carcinoma [27, 29]. The interactions between selectins with sLex/a result in tumor cell ad- herence to blood vessels, thus facilitate extravasation into surrounding tissue, and trigger angiogenesis [30]. After tumors invade into vasculature, malignant cells with higher ST3GAL-1 were more likely to aggregate Selectins, receptors for sialyl Lewisx (sLex) and sialyl Lewisa (sLea) expressed on endothelial and blood cells Page 8 of 9 Page 8 of 9 Page 8 of 9 Bai et al. BMC Cancer (2015) 15:880 with platelets, and eventually lodge in the small vessels and distant organs [31]. Thus, it is conceivable that el- evated sLex/a antigen synthesis caused by ST3Gal-1 in the tumor favors tumor cells to bind to selectins and then extricate from the primary tumor enter into blood stream and promote metastasis formation. This could give a possible explanation for our finding that patients with higher ST3GAL-1 expression are more likely to suffer re- currence and metastasis in the future, and ST3GAL-1 ex- pression is also an independent risk factor for the disease free survival in ccRCC. Nowadays, increasing researches focusing on the sialylated glycan pathways have show a re- markable effectiveness for tumor therapy in preclinical and clinical studies [32–34], which might be suggestive that targeting sialyltransferase could be a novel approach to deal with RCC some day. Received: 26 August 2015 Accepted: 3 November 2015 References Adv Cancer Res. 1989;52:257–331. 9. Harduin-Lepers A, Vallejo-Ruiz V, Krzewinski-Recchi MA, Samyn-Petit B, Julien S, Delannoy P. The human sialyltransferase family. Biochimie. 2001;83(8):727–37. 10. Schneider F, Kemmner W, Haensch W, Franke G, Gretschel S, Karsten U, et al. Overexpression of sialyltransferase CMP-sialic Acid: Gal beta 1,3GalNAc- R alpha 6-sialyltransferase is related to poor patient survival in human colorectal carcinomas. Cancer Res. 2001;61(11):4605–11. 10. Schneider F, Kemmner W, Haensch W, Franke G, Gretschel S, Karsten U, et al. Overexpression of sialyltransferase CMP-sialic Acid: Gal beta 1,3GalNAc- R alpha 6-sialyltransferase is related to poor patient survival in human colorectal carcinomas. Cancer Res. 2001;61(11):4605–11. Abbreviations S G β G l 13. Ganzinger U, Deutsch E. Serum sialyltransferase levels as a parameter in the diagnosis and follow-up of gastrointestinal tumors. Cancer Res. 1980;40(4):1300–4. 13. Ganzinger U, Deutsch E. Serum sialyltransferase levels as a parameter in the diagnosis and follow-up of gastrointestinal tumors. Cancer Res. 1980;40(4):1300–4. ST3GAL-1: β-Galactoside α-2,3-Sialyltransferase 1; RCC: Renal cell carcinoma; ccRCC: Clear cell renal cell carcinoma; OS: Overall survival; DFS: Disease free survival; UISS: University of California Integrated Staging System; AJCC: American Joint Committee on Cancer; EMT: Epithelial-to-mesenchymal transition; TMA: Tissue microarray; C-index: Concordance index; CI: Confidence interval. 14. Schultz MJ, Swindall AF, Bellis SL. Regulation of the metastatic cell phenotype by sialylated glycans. Cancer Metast Rev. 2012;31(3-4):501–18. 14. Schultz MJ, Swindall AF, Bellis SL. Regulation of the metastatic cell phenotype by sialylated glycans. Cancer Metast Rev. 2012;31(3-4):501–18. 15. Videira PA, Correia M, Malagolini N, Crespo HJ, Ligeiro D, Calais FM, et al. ST3Gal.I sialyltransferase relevance in bladder cancer tissues and cell lines. BMC Cancer. 2009;9:357. 15. Videira PA, Correia M, Malagolini N, Crespo HJ, Ligeiro D, Calais FM, et al. ST3Gal.I sialyltransferase relevance in bladder cancer tissues and cell lines. BMC Cancer. 2009;9:357. Conclusion In conclusion, we have confirmed ST3GAL-1 is corre- lated with unfavorable outcomes and could be used as an independent prognosticator in patients with ccRCC. We also developed nomograms for OS and DFS, which could give a better prediction for patients with ccRCC after surgery. 11. Cohen AM, Allalouf D, Djaldetti M, Weigl K, Lehrer N, Levinsky H. Sialyltransferase activity in plasma-cells of multiple-myeloma. Eur J Haematol. 1989;43(3):191–4. 11. Cohen AM, Allalouf D, Djaldetti M, Weigl K, Lehrer N, Levinsky H. Sialyltransferase activity in plasma-cells of multiple-myeloma. Eur J Haematol. 1989;43(3):191–4. 12. Dimitroff CJ, Pera P, Dall’Olio F, Matta KL, Chandrasekaran EV, Lau JTY, et al. Cell surface N-acetylneuraminic acid alpha 2,3-galactoside-dependent intercellular adhesion of human colon cancer cells. Biochem Bioph Res Co. 1999;256(3):631–6. 12. Dimitroff CJ, Pera P, Dall’Olio F, Matta KL, Chandrasekaran EV, Lau JTY, et al. Cell surface N-acetylneuraminic acid alpha 2,3-galactoside-dependent intercellular adhesion of human colon cancer cells. Biochem Bioph Res Co. 1999;256(3):631–6. Authors’ contributions 17. Burchell J, Poulsom R, Hanby A, Whitehouse C, Cooper L, Clausen H, et al. An alpha 2,3 sialyltransferase (ST3Gal I) is elevated in primary breast carcinomas. Glycobiology. 1999;9(12):1307–11. QB carried out and conducted experiments, performed statistical analysis, and drafted the manuscript. LL participated in the collection of patient materials and drafting of the manuscript. YX carried out laboratory work and data analysis. QL participated in the study design and collection of related articles. JW performed laboratory work and participated in the correction of words in the manuscript. JX took charge of the study design and revising manuscript critically for important intellectual content. JG conceived of the study, and led the data analysis and oversaw the drafting of the manuscript. All authors read and approved the final manuscript. 18. Edge SB, Compton CC. The American Joint Committee on Cancer: the 7th Edition of the AJCC Cancer Staging Manual and the Future of TNM. Ann Surg Oncol. 2010;17(6):1471–4. 19. Leibovich BC, Blute ML, Cheville JC, Lohse CM, Frank I, Kwon ED, et al. Prediction of progression after radical nephrectomy for patients with clear cell renal cell carcinoma - A stratification tool for prospective clinical trials. Cancer-Am Cancer Soc. 2003;97(7):1663–71. 20. Hakomori S. Glycosylation defining cancer malignancy: New wine in an old bottle. Proc Natl Acad Sci U S A. 2002;99(16):10231–3. Received: 26 August 2015 Accepted: 3 November 2015 Videira PA, Correia M, Malagolini N, Crespo HJ, Ligeiro D, Calais FM, et al. ST3Gal.I sialyltransferase relevance in bladder cancer tissues and cell lines BMC Cancer. 2009;9:357. 16. Picco G, Julien S, Brockhausen I, Beatson R, Antonopoulos A, Haslam S, et Over-expression of ST3Gal-I promotes mammary tumorigenesis. Glycobiology. 2010;20(10):1241–50. 17. Burchell J, Poulsom R, Hanby A, Whitehouse C, Cooper L, Clausen H, et a An alpha 2,3 sialyltransferase (ST3Gal I) is elevated in primary breast carcinomas. Glycobiology. 1999;9(12):1307–11. 18. Edge SB, Compton CC. The American Joint Committee on Cancer: the 7t Edition of the AJCC Cancer Staging Manual and the Future of TNM. Ann Surg Oncol. 2010;17(6):1471–4. 19. Leibovich BC, Blute ML, Cheville JC, Lohse CM, Frank I, Kwon ED, et a Prediction of progression after radical nephrectomy for patients with clear cell renal cell carcinoma - A stratification tool for prospective clinical trials. Cancer-Am Cancer Soc. 2003;97(7):1663–71. 20. Hakomori S. Glycosylation defining cancer malignancy: New wine in an o bottle. Proc Natl Acad Sci U S A. 2002;99(16):10231–3. 21. Guo HB, Nairn A, Harris K, Randolph M, Alvarez-Manilla G, Moremen K, et Loss of expression of N-acetylglucosaminyltransferase Va results in altered gene expression of glycosyltransferases and galectins. Febs Lett. 2008;582(4):527–35. 22. Paulson JC, Blixt O, Collins BE. Sweet spots in functional glycomics. Nat Chem Biol. 2006;2(5):238–48. The authors declare no conflict of interest. The authors declare no conflict of interest. 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Patard JJ, Kim HL, Lam JS, Dorey FJ, Pantuck AJ, Zisman A, et al. Use of th University of California Los Angeles integrated staging system to predict survival in renal cell carcinoma: An International Multicenter Study. J Clin Oncol. 2004;22(16):3316–22. 6. Eichelberg C, Junker K, Ljungberg B, Moch H. Diagnostic and prognostic molecular markers for renal cell carcinoma: a critical appraisal of the curre state of research and clinical applicability. Eur Urol. 2009;55(4):851–63. 7. Sun M, Shariat SF, Cheng C, Ficarra V, Murai M, Oudard S, et al. Prognosti factors and predictive models in renal cell carcinoma: a contemporary review. Eur Urol. 2011;60(4):644–61. 8. Hakomori SI. Aberrant glycosylation in tumors and tumor-associated carbohydrate antigens. Adv Cancer Res. 1989;52:257–331. 9. Harduin-Lepers A, Vallejo-Ruiz V, Krzewinski-Recchi MA, Samyn-Petit B, Jul S, Delannoy P. The human sialyltransferase family. Biochimie. 2001;83(8):727– 10. 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Acknowledgments This work was supported by grants from National Natural Science Foundation of China (31100629, 31270863, 81471621, 81472227 and 81472376), Program for New Century Excellent Talents in University (NCET-13-0146), Shanghai Health and Family Planning Commission (14ZR1406300) and Shanghai Rising-Star Program (13QA1400300). All these study sponsors have no roles in the study design, in the collection, analysis, and interpretation of data. 21. Guo HB, Nairn A, Harris K, Randolph M, Alvarez-Manilla G, Moremen K, et al. Loss of expression of N-acetylglucosaminyltransferase Va results in altered gene expression of glycosyltransferases and galectins. Febs Lett. 2008;582(4):527–35. 22. Paulson JC, Blixt O, Collins BE. Sweet spots in functional glycomics. Nat Chem Biol. 2006;2(5):238–48. Page 9 of 9 Page 9 of 9 Bai et al. BMC Cancer (2015) 15:880 23. Varki A, Schauer R. Sialic acids. In: Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, Hart GW, Etzler ME, editors. 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Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit • Convenient online submission
https://openalex.org/W3006886430	https://europepmc.org/articles/pmc7332146?pdf=render	English	null	Value of CCNA1 promoter methylation in triaging ASC-US cytology	Asian Pacific journal of cancer prevention	2,020	cc-by	3,608	DOI:10.31557/APJCP.2020.21.2.473 CCNA1 for Triage ASC-US Cytology DOI:10.31557/APJCP.2020.21.2.473 CCNA1 for Triage ASC-US Cytology RESEARCH ARTICLE Editorial Process: Submission:09/05/2019 Acceptance:02/12/2020 Abstract Background: Using HPV testing to triage ASC-US still has some problems of unnecessary colposcopy in many cases. A previous study reported that methylation of CCNA1, a tumor suppressor gene, can differentiate between low and high grade lesions. This study was designed to evaluate the diagnostic values and application of CCNA1 methylation in the patients with ASC-US group. Materials and Methods: Cross sectional analytic study was conducted in the patients with ASC-US cytology. HPV DNA testing and CCNA1 promoter methylation testing were performed. The patients were sent for colposcopic examination and biopsy. Biopsy results were considered as gold standard. Diagnostic test of HPV test and CCNA1 methylation test were calculated for sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV), likelihood ratio for test positive and negative and 95% confidence interval. Results: One hundred and seventy patients were enrolled. Mean age was 39.7 years old. HR-HPV was positive in 70% of the patients. HPV type 16, type 18 and non-16,18 were 12.4%, 4.7% and 42.4%, respectively. CIN2+ were found in 12.4% (21 cases). CCNA1 promoter methylation was positive in 5 cases. CCNA1 had high specificity 99.3%, NPV 89.2% and PPV 80% in detection of CIN2+ but sensitivity was 19%. Likelihood ratio for positive test was 28.4 and likelihood ratio for negative test was 0.8. HPV test had sensitivity of 90.5% and NPV of 95.9% but low specificity and PPV as 31.5% and 15.7%, respectively. Conclusion: CCNA1 promoter methylation testing had very high specificity, likelihood ratio for the positive test and PPV (99.3%, 28.4 and 80.0, respectively). Therefore, CCNA1 promoter methylation test may be used in the HPV DNA positive cases to classify the urgency of colposcopy and the colposcopist should pay more attention to CCNA1 positive patients because of their higher chance to identify the significant lesions. Keywords: CCNA1- ASC-US- accuracy- diagnostic value- CIN2+ Asian Pac J Cancer Prev, 21 (2), 473-477 Asian Pac J Cancer Prev, 21 (2), 473-477 low positive predictive value (PPV) (25.1% and 17.5%, respectively) (Ko et al., 2006). Use of HPV testing still has some problems because positive rate of HPV testing in ASC-US patients are as high as 31.5% while the high grade lesion in ASC-US is only 5-10%. Therefore, HPV testing results in unnecessary colposcopy in many cases. Shina Oranratanaphan1, Kewalin Kobwitaya1,Wichai Termrungruanglert1, Surang Triratanachat1, Nakarin Kitkumthorn1,2, Apiwat Mutirangura1,3 Shina Oranratanaphan1, Kewalin Kobwitaya1,Wichai Termrungruanglert1, Surang Triratanachat1, Nakarin Kitkumthorn1,2, Apiwat Mutirangura1,3 1Department of Obstetrics and Gynecology, 3Center of Excellence in Molecular Genetics of Cancer and Human Diseases, Department of Anatomy, Faculty of Medicine, Chulalongkorn University, 2Department of Oral and Maxillofacial Pathology, Faculty of Dentistry, Mahidol University, Bangkok Thailand. For Correspondence: dr_shina@hotmail.com Asian Pacific Journal of Cancer Prevention, Vol 21 473 1Department of Obstetrics and Gynecology, 3Center of Excellence in Molecular Genetics of Cancer and Human Diseases, Department of Anatomy, Faculty of Medicine, Chulalongkorn University, 2Department of Oral and Maxillofacial Pathology, Faculty of Dentistry, Mahidol University, Bangkok Thailand. For Correspondence: dr_shina@hotmail.com Asian Pacific Journal of Cancer Prevention, Vol 21 Introduction Cervical cancer is the third most common gynecological cancer worldwide (Ferlay et al., 2015). The incidence of cervical cancer is 14 per 100,000-person year (Pongnikorn et al., 2015). It is the second most common cancer in Thai women. Atypical squamous cell of undetermined significant (ASC-US) cytology is the minor cytologic abnormality. Incidence of invasive cancer in ASC-US is low and varies from 0.1 to 1% (Sundstrom et al., 2017; Tokmak et al., 2014). According to American Society for Colposcopy and Cervical Pathology (ASCCP) guideline 2012 (Saslow et al., 2012), there are two options to manage ASC-US including follow up and reflect HPV testing. HPV testing is used to triage because the risk of cervical cancer in HPV negative patients is extremely low (Kjaer et al., 2010). Therefore, HPV negative patients should not be sent for colposcopy (de Sanjose et al., 2010). However, HPV testing has low specificity and Some studies reported that CyclinA1gene which is a tumor suppressor gene that was activated after E6 and E7 protein of HPV embedded in the host cells leaded to progress into cervical cancer. Some studies reported that the CCNA1 methylation was found in high grade lesions and cervical cancer but no CCNA1 promoter methylation in normal cervixes (Chujan et al., 2014; Yang et al., 2010; Yanatatsaneejit et al., 2011). The previous study reported that CCNA1 methylation in normal cervixes, LSIL and HSIL are different (0% vs. 2.88% vs. 83.33%, respectively) (Khunamornpong et al., 2014). Therefore, CCNA1 methylation may be used to triage atypical squamous cell of undetermined significance (ASC-US) 473 Asian Pacific Journal of Cancer Prevention, Vol 21 Shina Oranratanaphan et al template; the remaining volume was adjusted by adding milliQ DNase-free sterile water. Real-time PCRs were performed in duplicates using Applied Biosystem® 7500 Real-Time PCR System (Thermo Scientific™, Waltham, MA, USA). The PCR conditions were first denaturation at 95°C for 2 minutes then go after with 40 cycles as follows: denaturation at 95°C 15 seconds, followed by annealing at 60°C for 30 seconds. Negative control (dH20) and positive control (universal human methylated DNA (EpiTect®PCR control kit, Qaigen, USA)) were included in each PCR. A melting curve was generated to determine the specificity of the primers. Later, the threshold cycle (Ct) of the amplified methylation products was detected. The results of all samples must have Beta actin products as internal control. DNA bisulfite modification DNA concentration was measured by nanodrop and subsequently adjusted to 750 ng/µl. Bisulfite treatment to 20 µl of each sample was performed by using the EZ DNA Methylation Kit (Zymo Research). The converted DNA solutions were eluted in 20 µl of M-Elution Buffer and stored below -20°C for subsequent use. Materials and Methods This cross sectional analytic study was conducted in the patients with ASC-US cytology. The ASC-US specimens were reviewed by cytopathologists at King Chulalongkorn Memorial Hospital (KCMH). This study protocol has been approved by the Institutional Review Board (IRB), Faculty of Medicine, Chulalongkorn University (IRB No.603/59). All female patients with ASC-US cytology who came to colposcopy clinic at KCMH between February 2017 and January 2018 were recruited in this study. The patients who were performed Pap test from other hospitals were excluded. The participants signed their inform consent forms voluntarily. The process of cytologic specimen collection was done in the routine cervical cancer screening program by the residents, fellows and staff at KCMH. The collected cytologic specimen from BD SurePath™ liquid-based Pap test was sent for HPV DNA and CCNA1 promoter methylation testing. The cases with ASC-US cytology were sent to the colposcopy clinic for colposcopic examination and biopsy was performed at the most severe lesions. In case that no lesion was detected, random biopsy was performed. Introduction Data analysis reported positive if present of CCNA1 methylation product and reported negative if no CCNA1 methylation. (Stoler et al., 2011). This study was designed to evaluate the diagnostic values of CCNA1 methylation in the patients with ASC-US group and compare to HPV DNA test. Statistical analysis Statistical analysis was performed using SPSS software version 22.0 (IBM Corp, Armonk, N.Y., USA). Demographic data were analyzed by descriptive method. Diagnostic test was calculated both HPV test and CCNA1 methylation test for sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV) and 95% confidence interval. Sample size was calculated by infinite population proportion formula based on sensitivity of CCNA1 from the previous study which reported 83% (Chujan et al., 2014). Therefore, 170 participants were required in this study. HPV DNA test was performed with Cobas® HPV testing, fully automated test based on extraction of the HPV and cellular DNA then followed by real time PCR to detect 14 high risk HPVs which consisted of 16, 18 and other high risk types (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68). After that, the specimens were sent to CCNA1 promoter methylation testing at our lab. Gold standard The gold standard for diagnosis was the most severe pathologic results from colposcopic directed biopsy. Colposcopists in this study had to pass the colposcopic training course and had to pass the 50 validated cases before performed colposcopic examination in this study. Colposcopic examination reports were recorded. Colposcopic impression which meant the opinion of the colposcopist whether the lesion was high grade or low grade before biopsy. The biopsy results were reviewed by pathologists and recorded. Results All of the 211 patients of ASC-US cytology who underwent colposcopic directed biopsy at colposcopy clinic at King Chulalongkorn Memorial Hospital (KCMH), Bangkok, Thailand from February 2017 to January 2018. Forty-one cases were excluded because the cases had undergone Pap smear test from other hospitals. Accordingly, the remaining 170 cases were included to performed HPV DNA and CCNA1 methylation testing. Discussion The final outcomes of ASC-US in this study had high grade histology (CIN2+) 12.4%. This prevalence of high grade lesion in ASC-US is higher than the previous study in our institution in 2008-2012 which was 6.71% (Tantitamit et al., 2015). The different of prevalence may result from the widely use of reflect HPV based on ASCCP recommendation. Therefore, ASC-US with negative HPV was not sent to perform colposcopy. However, some of the patients insisted to perform colposcopy instead of HPV triage were also sent to colposcopic clinic. For those reason, the proportion of high grade histology in ASC-US in this study was higher than previous study. However, this prevalence concordance with the study in Northern part of Thailand that found high grade histology 10-20% in ASC-Us cytology (Khunamornpong et al., 2014). The colposcopic impression or the opinion of colposcopist in the ASC-US cytology was reported low-grade lesion 70.6% which concordance with the previous study which found low grade lesion in colposcopy in ASC-US cytology as 68.1% (Wentzensen et al., 2018). by colposcopic impression was only 9.4% (16 cases). Seventy percent of the cases had high risk HPV infection consisted of HPV type 16, type 18 and other high risk types were 12.4%, 4.7% and 42.4%, respectively. The final pathology after colposcopic biopsy was found benign lesion (HPV infection or chronic cervicitis) 67.1% (114 cases), CIN1 20.6% (35 cases) and CIN2+ 12.4% (21 cases). CIN 2+ cases consisted of 7cases of CIN2, 13 cases of CIN 3 and 1 case of AIS. There was no invasive lesion found in ASC-US cytology in this study. CCNA1 promoter methylation was test in 170 specimens and positive test was found in 5 cases. The positive cases consisted of CIN2 (2 cases) CIN3 (2 cases) and condyloma (1 case); details are shown in Table 2. Diagnostic value of the CCNA1 promoter methylation test in ASC-US cytology is shown in Table 2, 3. CCNA1 had high specificity 99.3%, negative predictive value (NPV) 89.2% and positive predictive value (PPV) 80% The diagnostic value of HPV testing in this study had high sensitivity 90.5% and high NPV 95.9% which was the high performance of screening test. Nevertheless, the specificity was low as 31.5%, which was concordance with the previous studies (Ko et al., 2006; Castle et al., 2015; Levi et al., 2003). CCNA1 real-time PCR CCNA1 real time PCR To detect CCNA1 promoter methylation, we designed two TaqMan™. probe real-time PCRs. The CCNA1 methylation set was composed of the forward primer (5’ GGTAGGAAGAGTAGGTGTGTG 3’), reverse primer (5’ ACAACCCCTAACAACCCCCTCTAA 3’) and probe (FAM-5’GGGTTAGAGTGGGTAG 3’-BHQ). The Beta actin set primers were designed in the area of no CpG island to serve as internal control. The Beta actin set consisted of forward primer, reverse primer and probe. Both PCR reactions were prepared in a volume of 20 µL containing 10 µL of 2X TaqMan GTXpress real-time PCR master mix, 0.4 µL of 10 µmoles of each primer as well as probes and 2 µL of bisulfite-treated DNA Demographic data are shown in Table1. Mean age of the subjects was 39.7 years old, standard deviation (SD) was 11.1. Most subjects were premenopausal status 80% (136 cases) and had more than 1 partner 52.9% (90 cases). Majority of the colposcopic impressions which meant the opinion of colposcopist before the final pathologic results report were low-grade lesion 70.6% (120 cases) and no lesion was found 10.6% (18 cases). High-grade lesion 474 Asian Pacific Journal of Cancer Prevention, Vol 21 74 DOI:10.31557/APJCP.2020.21.2.473 CCNA1 for Triage ASC-US Cytology DOI:10.31557/APJCP.2020.21.2.473 CCNA1 for Triage ASC-US Cytology Diagnostic value CCNA1 HPV Sensitivity % (95%CI) 19.0 (7.0-40.0) 90.5 (71.1-97.4) Specificity % (95%CI) 99.3 (96.3-99.8) 31.5 (24.6-39.4) Accuracy % (95%CI) 89.4 (83.9-93.2) 38.8 (31.8-46.3) PPV% (95%CI) 80.0 (37.5-96.4) 15.7 (10.3-23.2) NPV % (95%CI) 89.2 (83.5-93.0) 95.9 (86.2-98.9) LR for test positive 28.4 1.3 LR for test negative 0.8 0.3 Table 3. Diagnostic Value for CCNA1 and HPV Testing Character N (%) Age (years) (Mean + SD) 39.7 + 11.1 Menopause: N (%) Premenopause 136 (80%) Postmenopause 34 (20%) Partner (N (%)) Single 80 (47.1%) Multiple 90 (52.9%) Colposcopic findings (N (%)) No lesion 18 (10.6%) HPV or condyloma 16 (9.4%) Low grade lesion 120 (70.6%) High grade lesion 16 (9.4%) HPV DNA (N (%)) Negative 51 (30%) Type 16 21 (12.4%) Type 18 9 (4.7%) Non 16, 18, other high risk 72 (42.4%) More than 1 type 18 (10.5%) Histopathology (N (%)) Benign (HPV, chronic cervicitis) 114 (67.1%) CIN1 35 (20.6%) CIN2+ 21 (12.4%) Table 1. Demographic Data of the ASC-US Population (N=170) in detection of CIN2+. However, sensitivity of the test was only 19%. Likelihood ratio for positive test was 28.4 which was very high and likelihood ratio for negative test was 0.8. CCNA1 real-time PCR Results of HPV testing are shown in Table 2 and the diagnostic value of HPV test is shown in Table 3. We found HPV test had high sensitivity of 90.5% and NPV of 95.9% but low specificity and PPV as 31.5% and 15.7%, respectively (Table 3). Asian Pacific Journal of Cancer Prevention, Vol 21 475 References Castle PE, Eaton B, Reid J, Getman D, Dockter J (2015). Comparison of human papillomavirus detection by Aptima HPV and cobas HPV tests in a population of women referred for colposcopy following detection of atypical squamous cells of undetermined significance by Pap cytology. J Clin Microbiol, 53, 1277-81. Chujan S, Kitkumthorn N, Siriangkul S, Mutirangura A (2014). CCNA1 promoter methylation: a potential marker for grading Papanicolaou smear cervical squamous intraepithelial lesions. Asian Pac J Cancer Prev, 15, 7971-5. de Sanjose S, Quint WG, Alemany L, et al (2010). Human papillomavirus genotype attribution in invasive cervical cancer: a retrospective cross-sectional worldwide study. Lancet Oncol, 11, 1048-56. Ferlay J, Soerjomataram I, Dikshit R, et al (2015). Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer, 136, 359-86. Khunamornpong S, Settakorn J, Sukpan K, et al (2014). Performance of HPV DNA testing with hybrid capture 2 in triaging women with minor cervical cytologic abnormalities (ASC-US/LSIL) in Northern Thailand. Asian Pac J Cancer Prev, 15, 10961-6. Our strength of this study is the final pathological confirmation was performed in all cases which was the gold standard to evaluate the diagnostic values. There are some limitations in our study, however. First, this study had small amount of CIN2+ histopathology. Second, there was positive test of CCNA1 methylation only 3% which less than the other studies therefore the result still inconclusive to be used as triage model. For further study, we may increase the number of the patients with CIN2+ for evaluation. Hence, the result of diagnostic values and role of triage may be clearer. Kitkumthorn N, Yanatatsanajit P, Kiatpongsan S, et al (2006). Cyclin A1 promoter hypermethylation in human papillomavirus-associated cervical cancer. BMC Cancer, 8, 1-9. Kjaer SK, Frederiksen K, Munk C, Iftner T (2010). Long-term absolute risk of cervical intraepithelial neoplasia grade 3 or worse following human papillomavirus infection: role of persistence. J Nat Cancer Institut, 102, 1478-88. persistence. J Nat Cancer Institut, 102, 1478-88. Ko V, Tambouret RH, Kuebler DL, Black-Schaffer WS, Wilbur DC (2006). Human papillomavirus testing using hybrid capture II with SurePath collection: initial evaluation and longitudinal data provide clinical validation for this method. Cancer, 108, 468-74. Acknowledgements We acknowledge the staff members of the Center of Excellence in Molecular Genetics of Cancer and Human Diseases, Department of Anatomy, Faculty of Medicine, Chulalongkorn University, for their assistance in preparing DNA and performing the CCNA1 promoter methylation test. Sundström K, Lu D, Elfström KM, et al (2017). Follow-up of women with cervical cytological abnormalities showing atypical squamous cells of undetermined significance or low-grade squamous intraepithelial lesion: a nationwide cohort study. Am J Obstet Gynecol, 216, 1-48. Tantitamit T, Termrungruanglert W, Oranratanaphan S, et al (2015). Cost-effectiveness analysis of different management strategies for detection CIN2+ of women with Atypical squamous cells of undetermined significance (ASC-US) Pap smear in Thailand. Asian Pac J Cancer Prev, 16, 6857-62. Funding and support This research was supported by a 2019 Research Chair Grant from the National Science and Technology Development Agency (NSTDA), Thailand, and the Anantara Siam Bangkok Hotel, Four Seasons Hotel Care for Cancer Fun Run in coordination with the Thai Red Cross Society. Saslow D, Solomon D, Lawson HW, et al (2012). American society for colposcopy and cervical pathology, and American Society for Clinical Pathology screening guidelines for the prevention and early detection of cervical cancer. J Low Genit Tract Dis, 16, 175-204. Stoler MH, Wright TC Jr, Sharma A, et al (2011). High-risk human papillomavirus testing in women with ASC-US cytology: results from the ATHENA HPV study. Am J Clin Pathol, 135, 468-75. References In conclusion, CCNA1 promoter methylation test may not be a good screening test but CCNA1 may be useful when combined with HPV DNA test to allocate the urgency of colposcopy in the test positive patients and used as alert sign for carefully find the lesion when the test is positive. Levi AW, Kelly DP, Rosenthal DL, Ronnett BM (2003). Atypical squamous cells of undetermined significance in liquid-based cytologic specimens: results of reflex human papillomavirus testing and histologic follow-up in routine practice with comparison of interpretive and probabilistic reporting methods. Cancer, 99, 191-7. Pongnikorn D, Suwanrungruang K, Buasom R (2015), Cancer incidence in Thailand, in cancer in Thailand Vol. VIII, 2010- 2012. National Cancer Institute, Ministry of Public Health, Bangkok, pp 44-52. Discussion Previous studies showed HPV testing had high sensitivity, low specificity and PPV in ASC-US cytology range 89.4-93.4%, 25.1- 59.3% and 17.5-20.3%, respectively (Ko et al., 2006; Castle et al., 2015). Test CIN2+ CIN1- Total CCNA1 Positive 4 1 5 Negative 17 148 165 HPV Positive 19 102 121 Negative 2 47 49 Total 21 149 170 Table 2. CCNA1 Promoter Methylation and HPV Testing Results Table 2. CCNA1 Promoter Methylation and HPV Testing Results CCNA1 promoter methylation testing in this study had high specificity 99.3% which was the characteristic of the good diagnostic test. However, this test shown low sensitivity 19% which was too low to be a screening test. The data from the previous studies Chujan et al., (2014) and Kitkumthorn et al., (2006) showed that CCNA1 promotor methylation had high discrimination power to differential high grade lesion and invasive cervical Asian Pacific Journal of Cancer Prevention, Vol 21 47 Shina Oranratanaphan et al cancer from low grade lesion (Chujan et al., 2014; Yang et al., 2009). The Likelihood ratio for the positive test of CCNA1 in our study was very high (28.4) which was represented a good diagnostic test. Moreover, positive predictive value of CCNA1 was also as high as 80.0 (37.5-96.4). High likelihood for the positive test result and PPV told us that if CCNA1 positive there was high chance to identify the high grade lesion in the patient. These results may be used in clinical application. For example, CCNA1 promoter methylation test may be use in the HPV DNA positive cases to classify the urgent of colposcopy in the long waiting time situation. In CCNA1 positive patients, urgent colposcopy may be required. In case that HPV and CCNA1 results were negative, the patients may be omitting colposcopy because of high NPV 94.7% in this test. From our study, colposcopic results showed that 5 cases of high grade lesions were underestimated from colposcopic impression. CCNA1 may help in this situation. In the patients who had CCNA1 positive, the colposcopist should pay more attention to identify the lesions because of the high likelihood ratio for positive test. i References Asian Pacific Journal of Cancer Prevention, Vol 21 477 This work is licensed under a Creative Commons Attribution- Non Commercial 4.0 International License. DOI:10.31557/APJCP.2020.21.2.473 CCNA1 for Triage ASC-US Cytology Conflict of interest The authors, hereby, declare no conflicts of interest 476 Asian Pacific Journal of Cancer Prevention, Vol 21 76 DOI:10.31557/APJCP.2020.21.2.473 CCNA1 for Triage ASC-US Cytology DOI:10.31557/APJCP.2020.21.2.473 CCNA1 for Triage ASC-US Cytology Tokmak A, Guzel Al, Ozgu E, et al (2014). Clinical significance of atypical squamous cells of undetermined significance in detecting preinvasive cervical lesions in post- menopausal Turkish women. Asian Pac J Cancer Prev, 15, 6639-41. Wentzensen N, Walker J, Smith K, et al (2018). A prospective study of risk-based colposcopy demonstrates improved detection of cervical precancers. Am J Obstet Gynecol, 218, 604.e1-8. Yanatatsaneejit P, Mutirangura A, Kitkumthorn N (2011). Human papillomavirus’s physical state and cyclin A1 promoter methylation in cervical cancer. Int J Gynecol Cancer, 21, 902-6. Yang N, Eijsink JJ, Lendvai A, et al (2009). Methylation markers for CCNA1 and C13ORF18 are strongly associated with high-grade cervical intraepithelial neoplasia and cervical cancer in cervical scrapings. Cancer Epidemiol Biomarkers Prev, 18, 3000-7. Yang N, Nijhuis ER, Volders HH, et al (2010). Gene promoter methylation patterns throughout the process of cervical carcinogenesis. Cell Oncol, 32, 131-43. This work is licensed under a Creative Commons Attribution- Non Commercial 4.0 International License. This work is licensed under a Creative Commons Attribution- Non Commercial 4.0 International License.
https://openalex.org/W3135289365	https://www.researchsquare.com/article/rs-253081/v1.pdf?c=1631871418000	English	null	Biofilm Persister Cell Formation, and Relative Gene Expression Analysis of Type II Toxin-Antitoxin System in Pseudomonas Aeruginosa Strains in the Exponential and Stationary Phases	Research Square (Research Square)	2,021	cc-by	12,613	Bio¦lm Persister Cell Formation, and Relative Gene Expression Analysis of Type II Toxin-Antitoxin System in Pseudomonas Aeruginosa Strains in the Exponential and Stationary Phases Rezvan Golmoradi Zadeh Iran University of Medical Sciences Behrooz Sadeghi Kalani Ilam University of Medical Science, Ilam, Iran Marzie Mahdizade Ari Iran University of Medical Sciences Malihe Talebi Iran University of Medical Sciences Shabnam Razavi Iran University of Medical Sciences Faramarz Masjedian Jazi (  Masjedianf@gmail.com ) Iran University of Medical Sciences Research Article Keywords: Persister cell, Bio¦lm, Pseudomonas aeruginosa, TA systems, Real-time PCR, Exponential and Stationary phases Posted Date: March 2nd, 2021 DOI: https://doi.org/10.21203/rs.3.rs-253081/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Bio¦lm Persister Cell Formation, and Relative Gene Expression Analysis of Type II Toxin-Antitoxin System in Pseudomonas Aeruginosa Strains in the Exponential and Stationary Phases Rezvan Golmoradi Zadeh Iran University of Medical Sciences Behrooz Sadeghi Kalani Ilam University of Medical Science, Ilam, Iran Marzie Mahdizade Ari Iran University of Medical Sciences Malihe Talebi Iran University of Medical Sciences Shabnam Razavi Iran University of Medical Sciences Faramarz Masjedian Jazi (  Masjedianf@gmail.com ) Iran University of Medical Sciences Research Article Biofilm persister cell formation, and relative gene expression analysis of type II toxin- 1 antitoxin system in Pseudomonas aeruginosa strains in the exponential and stationary phases 2 3 4 5 Rezvan Golmoradi Zadeh1, Behrooz Sadeghi Kalani2, Marzie Mahdizade Ari3, Malihe 6 Talebi4, Shabnam Razavi4, Faramarz Masjedian Jazi,4 7 Author details 8 1M.Sc of Medical Microbiology, Department of Microbiology, School of Medicine, Iran 9 University of Medical Sciences, Tehran, Iran 10 2Ph.D. of Medical Microbiology, Department of Microbiology, school of medicine, Ilam 11 University of Medical Science, Ilam, Iran 12 3Ph.D. student of Medical Microbiology, Department of Microbiology, School of Medicine, 13 Iran University of Medical Sciences, Tehran, Iran 14 4Ph.D. of Medical Microbiology, Department of Microbiology, School of Medicine, Iran 15 University of Medical Sciences, Tehran, Iran 16 Corresponding Author: 17 Faramarz Masjedian Jazi, Ph.D. of Medical Microbiology, Department of Medical 18 Microbiology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran 19 Email: Masjedian.f@iums.ac.ir 20 Tel: + 982188058649 21 Fax: + 982188058649 22 23 24 25 26 27 28 29 30 31 Biofilm persister cell formation, and relative gene expression analysis of type II toxin- 1 antitoxin system in Pseudomonas aeruginosa strains in the exponential and stationary phases 2 3 4 Keywords: Persister cell; Biofilm; Pseudomonas aeruginosa; TA systems; Real-time PCR; 52 Exponential and Stationary phases 53 While antibiotics were thought to be able to eradicate infections, “the antibiotic resistance” 56 phenomena was emerged in recent years and led to bacterial resistance to a wide range of 57 antibiotics [1]. One of the virulence factors which involved in antibiotic resistance is the 58 production of self-produced matrix structures known as biofilm. Biofilm-related infections 59 currently are describing as serious problem worldwide [2]. Bacterial survival in biofilm led to 60 resistance to compounds such as biocides and antibiotics which result in uncontrolled behavior 61 of bacteria in foods industries and diseases [3]. Pseudomonas aeruginosa as a gram-negative 62 pathogen is one of the most important causes of nosocomial infections which able to form a 63 specific biofilm containing alginate. This pathogen has been described as a major cause of 64 death in patients who suffering from cystic fibrosis due to biofilm formation following 65 colonization in the lungs [4]. In these patients, P. aeruginosa is able to form persister cells as 66 a reservoir for chronic infections and led to decrease susceptibility of bacteria in biofilm [5-7]. 67 The development of bacterial biofilms in different organs often caused in chronic and recurrent 68 infections and makes the situation more difficult for patients. In patients with CF, the immune 69 system and antibiotics are almost inactive against the biofilm structure due to the presence of 70 persister cells which are antibiotic-tolerant factor within the biofilm [8, 9]. Persister cell 71 formation related to phenotypic changes and was induced following exposure of bacterial cells 72 to antibiotics and signal molecules which result in survival. However, the mechanisms of 73 formation of persister cell are not well defined but TA (toxin- antitoxin) systems were 74 suggested to have a major role in the formation of persister cells, as seen as in Escherichia coli 75 and Mycobacterium tuberculosis that involvement of TA systems in formation of persister cells 76 were reported [10, 11]. TA systems are classified into five groups based on the products that 77 the gene encodes [12, 13]. While toxins have an effect on RNA, ribosomes, and DNA gyrase 78 in the cell and cause bactericidal and bacteriostatic responses in the cell [14]. An antitoxin is a 79 RNA sequence or protein that binds to the toxin and blocks its activity [15]. Author details 8 1M.Sc of Medical Microbiology, Department of Microbiology, School of Medicine, Iran 9 University of Medical Sciences, Tehran, Iran 10 2Ph.D. of Medical Microbiology, Department of Microbiology, school of medicine, Ilam 11 University of Medical Science, Ilam, Iran 12 3Ph.D. student of Medical Microbiology, Department of Microbiology, School of Medicine, 13 Iran University of Medical Sciences, Tehran, Iran 14 4Ph.D. of Medical Microbiology, Department of Microbiology, School of Medicine, Iran 15 University of Medical Sciences, Tehran, Iran 16 Faramarz Masjedian Jazi, Ph.D. of Medical Microbiology, Department of Medical 18 Microbiology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran 19 Email: Masjedian f@iums ac ir 20 Fax: + 982188058649 22 Abstract 42 Chronic and persistent infections and therapy failure are concerning issues in patients with 43 Pseudomonas aeruginosa infections. Presence of persister cells in biofilm considers as one 44 from the causes for antibiotic resistance and treatment failure. Consequently, in this research, 45 the expression of TA type II systems into persister formation of biofilm was assessed in 46 presence of colistin ciprofloxacin antibiotics into exponential and stationary phases. The results 47 showed that TA systems at different stages of bacterial growth in biofilm have different 48 functions that subsequently, cause differences in the presence amount of persister cells at each 49 stage of bacterial growth. The expression of the systems were measured by Real-Time PCR 50 method. 51 Keywords: Persister cell; Biofilm; Pseudomonas aeruginosa; TA systems; Real-time PCR; 52 Exponential and Stationary phases 53 54 Introduction 55 While antibiotics were thought to be able to eradicate infections, “the antibiotic resistance” 56 phenomena was emerged in recent years and led to bacterial resistance to a wide range of 57 antibiotics [1]. One of the virulence factors which involved in antibiotic resistance is the 58 production of self-produced matrix structures known as biofilm. Biofilm-related infections 59 currently are describing as serious problem worldwide [2]. Bacterial survival in biofilm led to 60 resistance to compounds such as biocides and antibiotics which result in uncontrolled behavior 61 of bacteria in foods industries and diseases [3]. Pseudomonas aeruginosa as a gram-negative 62 pathogen is one of the most important causes of nosocomial infections which able to form a 63 specific biofilm containing alginate. This pathogen has been described as a major cause of 64 death in patients who suffering from cystic fibrosis due to biofilm formation following 65 colonization in the lungs [4]. Author details 8 In these patients, P. aeruginosa is able to form persister cells as 66 a reservoir for chronic infections and led to decrease susceptibility of bacteria in biofilm [5-7]. 67 The development of bacterial biofilms in different organs often caused in chronic and recurrent 68 infections and makes the situation more difficult for patients. In patients with CF, the immune 69 system and antibiotics are almost inactive against the biofilm structure due to the presence of 70 persister cells which are antibiotic-tolerant factor within the biofilm [8, 9]. Persister cell 71 formation related to phenotypic changes and was induced following exposure of bacterial cells 72 to antibiotics and signal molecules which result in survival. However, the mechanisms of 73 formation of persister cell are not well defined but TA (toxin- antitoxin) systems were 74 suggested to have a major role in the formation of persister cells, as seen as in Escherichia coli 75 and Mycobacterium tuberculosis that involvement of TA systems in formation of persister cells 76 were reported [10, 11]. TA systems are classified into five groups based on the products that 77 the gene encodes [12, 13]. While toxins have an effect on RNA, ribosomes, and DNA gyrase 78 in the cell and cause bactericidal and bacteriostatic responses in the cell [14]. An antitoxin is a 79 RNA sequence or protein that binds to the toxin and blocks its activity [15]. Since there is little 80 information about mechanism of persister cells formation and their presence in the biofilm, this 81 study aimed to evaluated persister cell formation following exposure to ciprofloxacin and 82 colistin antibiotics in the biofilm of P. aeruginosa PAO1 strain and a clinical isolate in 83 exponential and stationary phases. Moreover, due to the possible role of TA systems in 84 bacterial persistence the effects of 5 fold minimal inhibitory concentration (MIC) of 85 Abstract 42 Chronic and persistent infections and therapy failure are concerning issues in patients with 43 Pseudomonas aeruginosa infections. Presence of persister cells in biofilm considers as one 44 from the causes for antibiotic resistance and treatment failure. Consequently, in this research, 45 the expression of TA type II systems into persister formation of biofilm was assessed in 46 presence of colistin ciprofloxacin antibiotics into exponential and stationary phases. The results 47 showed that TA systems at different stages of bacterial growth in biofilm have different 48 functions that subsequently, cause differences in the presence amount of persister cells at each 49 stage of bacterial growth. The expression of the systems were measured by Real-Time PCR 50 method. 51 Keywords: Persister cell; Biofilm; Pseudomonas aeruginosa; TA systems; Real-time PCR; 52 Exponential and Stationary phases 53 Keywords: Persister cell; Biofilm; Pseudomonas aeruginosa; TA systems; Real-time PCR; 52 Exponential and Stationary phases 53 110 The optical density cut-off (ODc) was determined as three standard deviations higher than the 111 mean OD of the negative control. All the isolates were grouped into four groups based on the 112 adherence capabilities: Non-biofilm-former (OD ≤ ODc), weak biofilm former (ODc < OD ≤ 113 2 × ODc), moderate biofilm former (2ODc < OD ≤ 4 ODc), and strong biofilm former (OD > 114 4 ODc) [17]. 115 cells of P. aeruginosa PAO1 strain and clinical isolate biofilm in exponential and stationary 87 phases. 88 89 Material and methods: 90 91 Bacterial strain and growth Conditions 92 In this research, P. aeruginosa PAO1 strain and P. aeruginosa clinical isolate were taken from 93 the Microbiology Department of Iran University of Medical Sciences, Tehran, Iran. The 94 exponential phase of Planktonic bacteria were made by inoculating 105 CFU/ml of bacteria in 95 LB broth and were incubated for 3 hours at 37°C in an incubator shaker at 180 rpm. The 96 stationary phase of Planktonic bacteria were made by inoculating 105 CFU/ml of bacteria in 97 LB broth and were incubated for 18 hours at 37°C in incubator shaker at 180 rpm [7]. 98 99 Biofilm formation assay 100 To investigate biofilm formation, exponential and stationary phases culture of P. aeruginosa 101 isolates were diluted to an optical density (OD) 600 of 0.08 in 1% glucose trypticase soy broth 102 (TSB) medium and 200 µl of the bacterial suspension were inoculated in each well of 96-well 103 plate and the plates were incubated at 37 ˚C for 24 hours. Then the wells were washed with 104 PBS twice and were stained by 100µl of 1% crystal violet for 20 min and this process was 105 followed by washing twice with PBS. Then, 100 µl of 95% ethanol was added to each well and 106 incubated at room temperature for 15 min. The solubilized crystal violet and the cell density 107 were quantified by measuring OD at 570 nm by the Microplate reader Varioskan Flash 108 (Thermo Scientific, Billerica, MA, USA). Biofilm formation assay was done by three times for 109 both isolates and 1% glucose TSB medium without bacteria served as the negative control [16]. 110 The optical density cut-off (ODc) was determined as three standard deviations higher than the 111 mean OD of the negative control. Identification of type II Toxin–Antitoxin systems The nucleotide sequence of P. aeruginosa isolates was downloaded from NCBI and type II TA 118 systems were designated by The Toxin-antitoxin database (TADB). Moreover, specific primer 119 sequences were designed by primer3 software (Table1) [18, 19]. PCR assay was applied to 120 identify the presence of type II TA system genes (relBE, Xre-COG5654, vapBC, and Xre- 121 GNAT) in P. aeruginosa isolates. Shortly, a thermal cycler (PeqLab, Germany) and a final 122 volume of 25 μL was made and the following program were described as: initial denaturation 123 at 95 °C for 5 min which followed by 34 cycles of denaturation at 95 °C for 45 s, annealing at 124 58 °C for 35 s and extension at 72 °C for 30s. A final extension step was performed at 72 °C 125 for 5 min[20] . 126 Keywords: Persister cell; Biofilm; Pseudomonas aeruginosa; TA systems; Real-time PCR; 52 Exponential and Stationary phases 53 Since there is little 80 information about mechanism of persister cells formation and their presence in the biofilm, this 81 study aimed to evaluated persister cell formation following exposure to ciprofloxacin and 82 colistin antibiotics in the biofilm of P. aeruginosa PAO1 strain and a clinical isolate in 83 exponential and stationary phases. Moreover, due to the possible role of TA systems in 84 bacterial persistence, the effects of 5-fold minimal inhibitory concentration (MIC) of 85 ciprofloxacin and colistin were evaluated on the expression of type II TA system in persister 86 cells of P. aeruginosa PAO1 strain and clinical isolate biofilm in exponential and stationary 87 phases. 88 89 Material and methods: 90 91 Bacterial strain and growth Conditions 92 In this research, P. aeruginosa PAO1 strain and P. aeruginosa clinical isolate were taken from 93 the Microbiology Department of Iran University of Medical Sciences, Tehran, Iran. The 94 exponential phase of Planktonic bacteria were made by inoculating 105 CFU/ml of bacteria in 95 LB broth and were incubated for 3 hours at 37°C in an incubator shaker at 180 rpm. The 96 stationary phase of Planktonic bacteria were made by inoculating 105 CFU/ml of bacteria in 97 LB broth and were incubated for 18 hours at 37°C in incubator shaker at 180 rpm [7]. 98 99 Biofilm formation assay 100 To investigate biofilm formation, exponential and stationary phases culture of P. aeruginosa 101 isolates were diluted to an optical density (OD) 600 of 0.08 in 1% glucose trypticase soy broth 102 (TSB) medium and 200 µl of the bacterial suspension were inoculated in each well of 96-well 103 plate and the plates were incubated at 37 ˚C for 24 hours. Then the wells were washed with 104 PBS twice and were stained by 100µl of 1% crystal violet for 20 min and this process was 105 followed by washing twice with PBS. Then, 100 µl of 95% ethanol was added to each well and 106 incubated at room temperature for 15 min. The solubilized crystal violet and the cell density 107 were quantified by measuring OD at 570 nm by the Microplate reader Varioskan Flash 108 (Thermo Scientific, Billerica, MA, USA). Biofilm formation assay was done by three times for 109 both isolates and 1% glucose TSB medium without bacteria served as the negative control [16]. Keywords: Persister cell; Biofilm; Pseudomonas aeruginosa; TA systems; Real-time PCR; 52 Exponential and Stationary phases 53 All the isolates were grouped into four groups based on the 112 adherence capabilities: Non-biofilm-former (OD ≤ ODc), weak biofilm former (ODc < OD ≤ 113 2 × ODc), moderate biofilm former (2ODc < OD ≤ 4 ODc), and strong biofilm former (OD > 114 4 ODc) [17]. 115 Determination of MIC of colistin and ciprofloxacin 127 The broth Microdilution method was applied to determine the MIC of colistin and ciprofloxacin 128 sulfate antibiotics (Sigma Aldrich) according to the Clinical and Laboratory Standards Institute 129 (CLSI) [17]. For determining MIC, 0.5 McFarland concentration of overnight bacterial culture 130 was diluted by 1:100 in Mueller Hinton II Broth (cation-adjusted) containing colistin sulfate, 131 then incubated at 37 °C for 18 hours. The MIC was considered as the lowest concentration of 132 antibiotics that inhibited visible growth after incubation time. MIC assays were replicate three times [21]. Isolation persister cell of biofilm in the exponential and stationary phases with antibiotics ciprofloxacin and colistin p f Exponential and stationary phase culture of P. aeruginosa isolates were diluted to an OD 600 8 of 0.08 in 1% glucose TSB medium and 200 µl of the bacterial suspension were inoculated in 9 each wells of 96-well plate. The plates were incubated at 37ºC for 24 hours. Then, the content 0 of each well was removed and 200 µL of fresh medium added to each wells. To persister cells 1 formation in both exponential and stationary phases, the cultures of isolates were exposed to 2 5-fold MIC ciprofloxacin and colistin individually, then incubated for about 3.5 hours at 37°C 3 in 180 rpm incubator shaker and were washed twice with PBS. 200µl of fresh 1% glucose TSB 4 medium was added to each wells and biofilm cells were collected by sonication at times 1,3,5,7, 5 and 24 hours, then 10 µL of each sample were diluted in PBS solution and spread on LB agar 6 plates (1.5% agar). Also, bacterial culture without antibiotics was used as a control. The colony 7 counting was performed after overnight incubation at 37 ˚C [7]. 8 Gene expression analysis by Quantitative Real-time PCR (qRT-PCR) p y y Q (q ) The exponential and stationary cultures yielded by 3.5 hours of exposure to ciprofloxacin and 151 colistin, first were washed twice with PBS and total biofilm cells were collected by sonication. 152 RNA extraction from the biofilm and the control group (the untreated biofilm) were done using 153 the kit (Gene all, Korea) according to the manufacturer's protocol for evaluation of expression 154 of type II TA system genes in persister cells biofilm. The quality of obtained RNA was assessed 155 by NanoDrop spectrophotometer (Thermo Fisher Scientific, USA) and gel electrophoresis 156 analysis, then the RNA was treated with DNaseӀ (Roche, Germany) for eliminating residual 157 DNA according to the manufacturer’s protocol. The total RNA was reverse transcribed to 158 single-stranded cDNA by cDNA synthesis Kit besides to specific designed primers (Betabairn, 159 Germany). 160 Finally, qRT-PCR was performed in a Rotor-Gene thermal cycler (Corbett 6000; Australia) 161 according to SYBR Green method (AccuPower Green Star qPCR Master Mix; Bioneer; 162 Korea). Briefly, 2 μl of cDNA, 12.5 μl SYBR Green master mix, 4.5 μl nuclease-free water, 163 and 1 μl of each primer (5 pmol) were added to 20 μl of the final volume of the RT-qPCR 164 reaction and was run according to the following program: an initial activation step at 95 °C for 165 12 min, 40 cycles of denaturation at 95°C for 20 s, annealing at 58 for 35 s[20]. The 16s rRNA 166 was used to normalize mRNA quantities and fold changes of mRNA expression was calculated 167 by the 2−ΔΔCt method [22]. 168 181 Table 1. Primers used in this study 181 182 183 Result 184 Biofilm formation assay 185 Both strain of p. aeruginosa (PAO1, clinical) formed strong biofilm in the polystyrene plates 186 OD > 4 ODc. 187 PCR assay 188 Our results showed that p. aeruginosa strains (PAO1, clinical) were positive for all studied 189 type II TA systems genes (relBE, Xre-COG5654, vapBC, and Xre-GNAT). 190 191 Persister cell formation assay in exponential and stationary phases of biofilm and colony 192 count method 193 The antibiotic sensitivity of PAO1 and clinical isolates to colistin and ciprofloxacin sulfate was 194 observed according to CLSI (Table 2). Gene expression analysis by Quantitative Real-time PCR (qRT-PCR) To assess the effects of ciprofloxacin and colistin on 195 bacterial survival biofilm, exponential and stationary cultures of strains biofilm were exposed 196 to 5 fold MICs of ciprofloxacin and colistin for 3.5 hours. The result showed that a great portion 197 of cells were killed by ciprofloxacin and colistin however a small portion of cells (w1%) were 198 survived. The viable counts were measured for 1,3,5,7 and 24 hours intervals. The biofilm 199 formation of P. aeruginosa strains was strongly prevented in the presence of 5-fold MIC of 200 ciprofloxacin and colistin. After 1 hour, the number of viable cells reduced significantly. In 201 contrast, after 3 hours the number of live cells in the biofilm structure is almost constant and 202 the plateau-phase curves indicated the persister cells remained stable. PAO1 and clinical strains 203 biofilm in the exponential and stationary phases which treated with ciprofloxacin and colistin 204 antibiotics at times 1,3,5,7 and 24 hours showed significant different of the treated group 205 colony count in compare to control group (Figure-1 and 2). 206 Gene Names primers Sequence (5′→3′) Annealing temperature Product length T.relE F R GGACGCCATCTACGACCATT ACGATGTCCTGAACGGCTTT 58 °C 72 bp This study T. Xre- COG F R CAATTGGTCGACACCCTGGA GTAGAACAGGCTTGGCTCGT 58°C 177 bp This study vapBC /Xre-PIN F R CCCTGTGGGACGAATACGAG AGGGTTTCCAGCTTGTCCAG 58°C 76 bp This study -/Xre- GNAT F R CAAGCATGCCTTCGACAACC GTCGGTGATGCTGTAGACGA 58°C 178bp This study 16S. PSEU F R GCCCTCAAGTTCGATTTCGC ATGGAAGATCAGTGGCGTGG 58°C 99bp This study Table 1. Primers used in this study Gene Names primers Sequence (5′→3′) Annealing temperature Product length T.relE F R GGACGCCATCTACGACCATT ACGATGTCCTGAACGGCTTT 58 °C 72 bp This study T. Xre- COG F R CAATTGGTCGACACCCTGGA GTAGAACAGGCTTGGCTCGT 58°C 177 bp This study vapBC /Xre-PIN F R CCCTGTGGGACGAATACGAG AGGGTTTCCAGCTTGTCCAG 58°C 76 bp This study -/Xre- GNAT F R CAAGCATGCCTTCGACAACC GTCGGTGATGCTGTAGACGA 58°C 178bp This study 16S. PSEU F R GCCCTCAAGTTCGATTTCGC ATGGAAGATCAGTGGCGTGG 58°C 99bp This study Persister cell formation assay in exponential and stationary phases of biofilm and colony count method Table-2 Results obtained by MIC determination 208 strain Ciprofloxacin Colistin Standard strain 1(s) 1(s) Clinical isolate 1(s) 0.5(s) 209 210 211 13.2 13.4 13.6 13.8 14.0 14.2 14.4 T im e(H our) Control Treatment C iprofloxacin H1 H3 H5 H7 Log cfu/m l H24 14.0 14.5 15.0 15.5 C iprofloxacin T im e(H our) Control Treatment Log cfu/m l H1 H3 H5 H7 H24 212 213 13.0 13.5 14.0 14.5 15.0 C iprofloxacin Tim e(H our) Control Treatment Log cfu/m l H1 H3 H5 H7 H24 14.0 14.5 15.0 15.5 16.0 Ciprofloxacin Time(Hour) Log cfu/ml Control Treatment H1 H3 H5 H7 H24 214 Figure-1 Time-dependent killing of PAO1 and clinical strains biofilm exposed to ciprofloxacin. PAO1 strain biofilm. 215 Exponential phase (A) and stationary phase (B), clinical isolate biofilm of exponential phase (C) and stationary phase (D). The 216 bacterial culture without antibiotics treatment served as a control. The values are means of three biological replicates and error 217 bars indicate the standard deviation (SD). (Independent Samples T test, Repeated Measure, LSD) 218 219 A B C D 208 Table-2 Results obtained by MIC determination strain Ciprofloxacin Colistin Standard strain 1(s) 1(s) Clinical isolate 1(s) 0.5(s) t 14.0 14.5 15.0 15.5 C iprofloxacin T im e(H our) Control Treatment Log cfu/m l H1 H3 H5 H7 H24 B 13.2 13.4 13.6 13.8 14.0 14.2 14.4 T im e(H our) C iprofloxacin H1 H3 H5 H7 Log cfu/m l H24 A B 213 13.0 13.5 14.0 14.5 15.0 C iprofloxacin Tim e(H our) Control Treatment Log cfu/m l H1 H3 H5 H7 H24 14.0 14.5 15.0 15.5 16.0 Ciprofloxacin Time(Hour) Log cfu/ml Control Treatment H1 H3 H5 H7 H24 214 Figure-1 Time-dependent killing of PAO1 and clinical strains biofilm exposed to ciprofloxacin. PAO1 strain biofilm. 215 Exponential phase (A) and stationary phase (B), clinical isolate biofilm of exponential phase (C) and stationary phase (D). The 216 bacterial culture without antibiotics treatment served as a control. The values are means of three biological replicates and error 217 bars indicate the standard deviation (SD). Persister cell formation assay in exponential and stationary phases of biofilm and colony count method (Independent Samples T test, Repeated Measure, LSD) 218 219 C D 14.0 14.5 15.0 15.5 16.0 Ciprofloxacin Time(Hour) Log cfu/ml Control Treatment H1 H3 H5 H7 H24 D D 13.0 13.5 14.0 14.5 15.0 C iprofloxacin Tim e(H our) Control Treatmen Log cfu/m l H1 H3 H5 H7 H24 4 C Figure-1 Time-dependent killing of PAO1 and clinical strains biofilm exposed to ciprofloxacin. PAO1 strain biofilm. 215 Exponential phase (A) and stationary phase (B), clinical isolate biofilm of exponential phase (C) and stationary phase (D). The 216 bacterial culture without antibiotics treatment served as a control. The values are means of three biological replicates and error 217 bars indicate the standard deviation (SD). (Independent Samples T test, Repeated Measure, LSD) 218 219 220 13.0 13.5 14.0 14.5 Tim e(H our) Log cfu/m l Control Treatment C olistin H1 H3 H5 H7 H24 14.0 14.5 15.0 15.5 Colistin Tim e(H our) Log cfu/m l Control Treatment H1 H3 H5 H7 H24 221 A B t 14.0 14.5 15.0 15.5 Colistin Tim e(H our) Log cfu/m l Control Treatment H1 H3 H5 H7 H24 B 13.0 13.5 14.0 14.5 Tim e(H our) Log cfu/m l C olistin H1 H3 H5 H7 H24 A B 221 13.0 13.5 14.0 14.5 15.0 Colistin Tim e(H our) Log cfu/m l Control Treatment H1 H3 H5 H7 H24 14.0 14.5 15.0 15.5 Colistin Tim e(H our) Control Treatment Log cfu/m l H1 H3 H5 H7 H24 22 Figure-2 Time-dependent killing of PAO1 and clinical strains biofilm exposed to colistin. PAO1 strain biofilm exponential 23 phase (A) and stationary phase (B) and clinical isolate biofilm exponential phase (C) and stationary phase (D). The bacterial 24 culture without any antibiotic treatment served as a control. The values are means of three biological replicates and error bars 25 indicate the standard deviation (SD). (Independent Samples T test, Repeated Measure, LSD) 26 27 C D 13.0 13.5 14.0 14.5 15.0 Colistin Tim e(H our) Log cfu/m l Control Treatment H1 H3 H5 H7 H24 C 14.0 14.5 15.0 15.5 Colistin Tim e(H our) Control Treatment Log cfu/m l H1 H3 H5 H7 H24 D C D Figure-2 Time-dependent killing of PAO1 and clinical strains biofilm exposed to colistin. PAO1 strain biofilm exponential phase (A) and stationary phase (B) and clinical isolate biofilm exponential phase (C) and stationary phase (D). The bacterial culture without any antibiotic treatment served as a control. Persister cell formation assay in exponential and stationary phases of biofilm and colony count method The values are means of three biological replicates and error bars indicate the standard deviation (SD). (Independent Samples T test, Repeated Measure, LSD) 1 3 5 7 24 0 5 10 15 20 C iprofloxacin PA O 1 Tim e(H oure) Log cfu/m l Exponential Stationary * * * * * 1 3 5 7 24 0 5 10 15 20 C iprofloxacin C linical Tim e(H our) Log cfu/m l Exponential Stationary * * * * * 228 1 3 5 7 24 0 5 10 15 20 C olistin PA O 1 Tim e(H oure) Log cfu/m l Exponential Stationary * * * * * 1 3 5 7 24 0 5 10 15 20 C olistin C linical Tim e(H our) Log cfu/m l Exponential Stationary * * * 229 Figure-3 Time-dependent killing of PAO1 and clinical strains biofilm in the exponential and stationary phases exposed to 230 ciprofloxacin. PAO1 strain biofilm (A) and clinical isolate biofilm (B) and colistin PAO1 strain biofilm (C) and clinical isolate 231 biofilm (D). Graph data are indicated as the means±SD of three independent replicates. P < 0.05, P < 0.01, P < 0.001. 232 and *P < 0.0001 (statistical results obtained by independent samples T test). 233 . 234 A B C D al 1 3 5 7 24 0 5 10 15 20 C iprofloxacin C linical Tim e(H our) Log cfu/m l Exponential Stationary * * * * B 1 3 5 7 24 0 5 10 15 20 C iprofloxacin PA O 1 Tim e(H oure) Log cfu/m l Exponential Stationary * * * * * 1 3 5 7 24 0 5 10 15 20 C iprofloxacin C linical Tim e(H our) Log cfu/m l Exponential Stationary * * * * * 228 A B 228 ( ) ( ) 228 1 3 5 7 24 0 5 10 15 20 C olistin PA O 1 Tim e(H oure) Log cfu/m l Exponential Stationary * * * * * 1 3 5 7 24 0 5 10 15 20 C olistin C linical Tim e(H our) Log cfu/m l Exponential Stationary * * * 229 Figure-3 Time-dependent killing of PAO1 and clinical strains biofilm in the exponential and stationary phases exposed to 230 ciprofloxacin. PAO1 strain biofilm (A) and clinical isolate biofilm (B) and colistin PAO1 strain biofilm (C) and clinical isolate 231 biofilm (D). Persister cell formation assay in exponential and stationary phases of biofilm and colony count method 239 240 B C D A 1 3 5 7 24 0 5 10 15 Exponential C iprofloxacin Ti (H ) Log cfu/m l PAO1 Clinical 1 3 5 7 24 0 5 10 15 20 Stationary C iprofloxacin Ti (H ) Log cfu/m l PAO1 Clinical B A 1 3 5 7 24 0 Tim e(H our) 1 3 5 7 24 0 5 Tim e(H our) 235 1 3 5 7 24 0 5 10 15 Exponential C olistin Log cfu/m l PAO1 Clinical 1 3 5 7 24 0 5 10 15 20 Stationary C olistin Tim e(H our) Log cfu/m l PAO1 Clinical 236 Figure-4 Time-dependent killing of PAO1 and clinical strains biofilm exposed to ciprofloxacin exponential phase (A) and 237 stationary phase (B) and colistin exponential phase (C) and stationary phase (D). Graph data are indicated as the means±SD 238 C D Tim e(H our) Tim e(H our) 235 1 3 5 7 24 0 5 10 15 Exponential C olistin Log cfu/m l PAO1 Clinical 1 3 5 7 24 0 5 10 15 20 Stationary C olistin Tim e(H our) Log cfu/m l PAO1 Clinical 236 C D 235 236 237 238 239 240 241 242 Figure-4 Time-dependent killing of PAO1 and clinical strains biofilm exposed to ciprofloxacin exponential phase (A) and 237 stationary phase (B) and colistin exponential phase (C) and stationary phase (D). Graph data are indicated as the means±SD 238 of three independent replicates. P < 0.05, P < 0.01, P < 0.001 and *P < 0.0001 (by independent samples T test). Persister cell formation assay in exponential and stationary phases of biofilm and colony count method Graph data are indicated as the means±SD of three independent replicates. P < 0.05, P < 0.01, P < 0.001. 232 and **P < 0.0001 (statistical results obtained by independent samples T test). 233 . 234 C D 228 1 3 5 7 24 0 5 10 15 20 C olistin PA O 1 Tim e(H oure) Log cfu/m l Exponential Stationary * * * * 1 3 5 7 24 0 5 10 15 20 C olistin C linical Tim e(H our) Log cfu/m l Exponential Stationary * * * 229 C D Figure-3 Time-dependent killing of PAO1 and clinical strains biofilm in the exponential and stationary phases exposed to 230 ciprofloxacin. PAO1 strain biofilm (A) and clinical isolate biofilm (B) and colistin PAO1 strain biofilm (C) and clinical isolate 231 biofilm (D). Graph data are indicated as the means±SD of three independent replicates. P < 0.05, P < 0.01, P < 0.001. 232 and *P < 0.0001 (statistical results obtained by independent samples T test). 233 . 234 1 3 5 7 24 0 5 10 15 Exponential C iprofloxacin Tim e(H our) Log cfu/m l PAO1 Clinical 1 3 5 7 24 0 5 10 15 20 Stationary C iprofloxacin Tim e(H our) Log cfu/m l PAO1 Clinical 235 1 3 5 7 24 0 5 10 15 Exponential C olistin Log cfu/m l PAO1 Clinical 1 3 5 7 24 0 5 10 15 20 Stationary C olistin Tim e(H our) Log cfu/m l PAO1 Clinical 236 Figure-4 Time-dependent killing of PAO1 and clinical strains biofilm exposed to ciprofloxacin exponential phase (A) and 237 stationary phase (B) and colistin exponential phase (C) and stationary phase (D). Graph data are indicated as the means±SD 238 of three independent replicates. P < 0.05, P < 0.01, P < 0.001 and **P < 0.0001 (by independent samples T test). Persister cell formation assay in exponential and stationary phases of biofilm and colony count method 239 240 240 241 242 1 3 5 7 24 0 5 10 15 Exponential Phase PA O 1 Tim e(H our) Log cfu/m l C iprofloxacin Colistin 1 3 5 7 24 0 5 10 15 20 Stationary Phase PA O 1 Tim e(H our) Log cfu/m l Ciprofloxacin Colistin 243 244 A B 241 242 1 3 5 7 24 0 5 10 15 Exponential Phase PA O 1 Tim e(H our) Log cfu/m l C iprofloxacin Colistin 1 3 5 7 24 0 5 10 15 20 Stationary Phase PA O 1 Tim e(H our) Log cfu/m l Ciprofloxacin Colistin 243 244 A B 242 1 3 5 7 24 0 5 10 15 Exponential Phase PA O 1 Tim e(H our) Log cfu/m l C iprofloxacin Colistin 1 3 5 7 24 0 5 10 15 20 Stationary Phase PA O 1 Tim e(H our) Log cfu/m l Ciprofloxacin Colistin 243 244 A B 242 1 3 5 7 24 0 5 10 15 Exponential Phase PA O 1 Tim e(H our) Log cfu/m l C iprofloxacin Colistin 1 3 5 7 24 0 5 10 15 20 Stationary Phase PA O 1 Tim e(H our) Log cfu/m l Ciprofloxacin Colistin 243 A B B A 1 3 5 7 24 0 5 10 15 Exponential Phase Clinical Tim e(H our) Log cfu/m l Ciprofloxacin Colistin 1 3 5 7 24 0 5 10 15 20 Stationary Phase Clinical Tim e(H our) Log cfu/m l Ciprofloxacin Colistin Figure-5 Time-dependent killing of ciprofloxacin and colistin in P. aeruginosa strain PAO1 exponential phase (A) and stationary phase (B) and the P. aeruginosa clinical isolate biofilm exponential phase (C) and stationary phase (D). Graph data are indicated as the means±SD of three independent replicates. P < 0.05, P < 0.01, P < 0.001 and **P < 0.0001 (by independent samples T test). C D 1 3 5 7 24 0 5 10 15 Exponential Phase Clinical Tim e(H our) Log cfu/m l Ciprofloxacin Colistin 1 3 5 7 24 0 5 10 15 20 Stationary Phase Clinical Tim e(H our) Log cfu/m l Ciprofloxacin Colistin C D Figure-5 Time-dependent killing of ciprofloxacin and colistin in P. aeruginosa strain PAO1 exponential phase (A) and stationary phase (B) and the P. aeruginosa clinical isolate biofilm exponential phase (C) and stationary phase (D). Persister cell formation assay in exponential and stationary phases of biofilm and colony count method Graph data are indicated as the means±SD of three independent replicates. P < 0.05, P < 0.01, P < 0.001 and **P < 0.0001 (by independent samples T test). Effects of ciprofloxacin and colistin on the expression of type II TA system genes in P. aeruginosa strains persister cells in exponential and stationary phases of biofilm 50 Effects of ciprofloxacin and colistin on the expression of type II TA system genes in P. 51 aeruginosa strains persister cells in exponential and stationary phases of biofilm 52 aeruginosa strains persister cells in exponential and stationary phases of biofilm 252 TA systems (relBE, Xre-COG5654, vapBC and Xre-GNAT) in persister cell formation of P. 253 aeruginosa strains biofilm showed different expression in the exponential and stationary 254 cultures. There was a significant difference between the expression of TA system genes (relBE, 255 Xre-COG5654, vapBC, and Xre-GNAT) in the PAO1 strain biofilm and clinical isolate biofilm 256 in P. aeruginosa. Statistical analysis of the expression of type II TA system genes in the 257 persister cells of biofilm following exposure to ciprofloxacin showed an increase levels in the 258 expression of relBE/RHH-RelE, /Xre-COG5654, /Xre-GNAT, and vapBC /Xre-PIN TA 259 systems in the biofilm of PAO1 strain in exponential phase. Biofilm of clinical isolate same as 260 biofilm of PAO1 strain showed an increase in expression of relBE/RHH-RelE, /Xre-GNAT, 261 vapBC /Xre-PIN systems but there’s not any increase in /Xre-COG5654 system. In the 262 stationary phase, the expression of the /Xre-GNAT and vapBC /Xre-PIN decreased in the 263 biofilm of clinical isolate while for the PAO1 strain biofilm the expression of the /Xre-GNAT 264 was decreased (Figure-6). 265 Analyzing the expression of type II TA systems genes in exponential phase in the persister 266 cells of biofilm following exposure to colistin showed that the expression of the vapBC /Xre- 267 PIN was increased in biofilm of PAO1 strain. Also, the expression of relBE/RHH-RelE, /Xre- 268 COG5654, /Xre-GNAT and vapBC /Xre-PIN showed an increase in biofilm of clinical isolate. 269 In the stationary phase, the expression of relBE/RHH-RelE system was decreased in biofilm of 270 PAO1 strain and expression of the relBE/RHH-RelE, /Xre-COG5654, /Xre-GNAT and vapBC 271 /Xre-PIN were increased in biofilm of clinical isolate (Figure-6). 50 Effects of ciprofloxacin and colistin on the expression of type II TA system genes in P. 51 aeruginosa strains persister cells in exponential and stationary phases of biofilm 52 272 273 relBE/RHH-RelE vapBC /Xre-PIN /Xre-COG5654 /Xre-GNAT -500 0 500 1000 1500 C iprofloxacin Log2 Fold C hange Exponential Stationary ** relBE/RHH-RelE vapBC /Xre-PIN /Xre-COG5654 /Xre-GNAT -1500 -1000 -500 0 500 C iprofloxacin Log2 Fold C hange Exponential Stationary * ** * 274 275 relBE/RHH-RelE vapBC /Xre-PIN /Xre-COG5654 /Xre-GNAT -15 -10 -5 0 5 10 Colistin Log2 Fold C hange Exponential Stationary ** relBE/RHH-RelE vapBC /Xre-PIN /Xre-COG5654 /Xre-GNAT relBE/RHH-RelE vapBC /Xre-PIN /Xre-COG5654 /Xre-GNAT 0 50 100 150 Colistin Log2 Fold C hange Exponential Stationary 276 Figure-6 Expression levels of type II TA system genes in the exponential and stationary phases in the presence of 5-fold MIC 277 of ciprofloxacin PAO1 strain biofilm (A) clinical isolate biofilm (B) and colistin PAO1 strain biofilm (C) clinical isolate 278 biofilm (D). Graph data are indicated as the means±SD of three independent replicates. P < 0.05, P < 0.01, P < 0.001 279 and *P < 0.0001 (by independent samples T test). 50 Effects of ciprofloxacin and colistin on the expression of type II TA system genes in P. 51 aeruginosa strains persister cells in exponential and stationary phases of biofilm 52 280 281 A B C D relBE/RHH-RelE vapBC /Xre-PIN /Xre-COG5654 /Xre-GNAT -500 0 500 1000 1500 C iprofloxacin Log2 Fold C hange Exponential Stationary ** relBE/RHH-RelE vapBC /Xre-PIN /Xre-COG5654 /Xre-GNAT -1500 -1000 -500 0 500 C iprofloxacin Log2 Fold C hange Exponential Stationary * ** * 274 275 A B relBE/RHH-RelE vapBC /Xre-PIN /Xre-COG5654 /Xre-GNAT -500 0 500 1000 1500 C iprofloxacin Log2 Fold C hange Expon Statio * 274 275 A A ential nary relBE/RHH-RelE vapBC /Xre-PIN /Xre-COG5654 /Xre-GNAT -1500 -1000 -500 0 500 C iprofloxacin Log2 Fold C hange Exponential Stationary * ** * B B al relBE/RHH-RelE vapBC /Xre-PIN /Xre-COG5654 /Xre-GNAT relBE/RHH-RelE vapBC /Xre-PIN /Xre-COG5654 /Xre-GNAT 0 50 100 150 Colistin Log2 Fold C hange Exponential Stationary D 275 relBE/RHH-RelE vapBC /Xre-PIN /Xre-COG5654 /Xre-GNAT -15 -10 -5 0 5 10 Colistin Log2 Fold C hange Exponential Stationary ** relBE/RHH-RelE vapBC /Xre-PIN /Xre-COG5654 /Xre-GNAT relBE/RHH-RelE vapBC /Xre-PIN /Xre-COG5654 /Xre-GNAT 0 50 100 150 Colistin Log2 Fold C hange Exponential Stationary 276 Figure-6 Expression levels of type II TA system genes in the exponential and stationary phases in the presence of 5-fold MIC 277 of ciprofloxacin PAO1 strain biofilm (A) clinical isolate biofilm (B) and colistin PAO1 strain biofilm (C) clinical isolate 278 biofilm (D) Graph data are indicated as the means±SD of three independent replicates P < 0 05 P < 0 01 *P < 0 001 279 C D 275 relBE/RHH-RelE vapBC /Xre-PIN /Xre-COG5654 /Xre-GNAT -15 -10 -5 0 5 10 Colistin Log2 Fold C hange Expone Station * ** 276 C D C Exponential Stationary 276 277 278 279 280 281 Figure-6 Expression levels of type II TA system genes in the exponential and stationary phases in the presence of 5-fold MIC of ciprofloxacin PAO1 strain biofilm (A) clinical isolate biofilm (B) and colistin PAO1 strain biofilm (C) clinical isolate biofilm (D). Graph data are indicated as the means±SD of three independent replicates. P < 0.05, P < 0.01, P < 0.001 and *P < 0.0001 (by independent samples T test). 50 Effects of ciprofloxacin and colistin on the expression of type II TA system genes in P. 51 aeruginosa strains persister cells in exponential and stationary phases of biofilm 52 281 relBE/RHH-RelE vapBC /Xre-PIN /Xre-COG5654 /Xre-GNAT -2000 -1000 0 1000 2000 C iprofloxacin Log2 Fold C hange PAO1 Clinical * relBE/RHH-RelE vapBC /Xre-PIN /Xre-COG5654 /Xre-GNAT -5 0 5 10 15 C iprofloxacin Log2 Fold C hange PAO1 Clinical * 282 283 A B A B relBE/RHH-RelE vapBC /Xre-PIN /Xre-COG5654 /Xre-GNAT -50 0 50 100 150 Colistin Log2 Fold C hange PAO1 Clinical * * relBE/RHH-RelE vapBC /Xre-PIN /Xre-COG5654 /Xre-GNAT -2 0 2 4 6 8 C olistin Log2 Fold C hange PAO1 Clinical * 284 Figure-7 Expression levels of type II TA system genes in PAO1 and clinical strains biofilm in the presence of 5-fold MIC of 285 ciprofloxacin exponential phase (A), stationary phase (B) and colistin exponential phase (C) stationary phase (D). Graph data 286 are indicated as the means±SD of three independent replicates. P < 0.05, P < 0.01, P < 0.001 and *P < 0.0001 (by 287 independent samples T test). 288 289 D C relBE/RHH-RelE vapBC /Xre-PIN /Xre-COG5654 /Xre-GNAT -50 0 50 100 150 Colistin Log2 Fold C hange PAO1 Clinical ** * C relBE/RHH-RelE vapBC /Xre-PIN /Xre-COG5654 /Xre-GNAT -2 0 2 4 6 8 C olistin Log2 Fold C hange PAO1 Clinical * D Figure-7 Expression levels of type II TA system genes in PAO1 and clinical strains biofilm in the presence of 5-fold MIC of ciprofloxacin exponential phase (A), stationary phase (B) and colistin exponential phase (C) stationary phase (D). Graph data are indicated as the means±SD of three independent replicates. P < 0.05, P < 0.01, P < 0.001 and *P < 0.0001 (by independent samples T test). 289 PAO1 Clinical -1000 -500 0 500 1000 Exponential Phase Log2 Fold C hange Ciprofloxacin Colistin PAO1 Clinical 0 2 4 6 8 Stationary Phase Log2 Fold C hange Ciprofloxacin Colistin 290 Figure-8 Expression levels of type II TA system genes in the presence of 5-fold MIC of ciprofloxacin and colistin exponential 291 phase (A) stationary phase (B) in PAO1 and clinical strains biofilm. Graph data are indicated as the means±SD of three 292 independent replicates. P < 0.05, P < 0.01, P < 0.001 and **P < 0.0001 (by independent samples T test). 50 Effects of ciprofloxacin and colistin on the expression of type II TA system genes in P. 51 aeruginosa strains persister cells in exponential and stationary phases of biofilm 52 293 294 295 A B acin PAO1 Clinical 0 2 4 6 8 Stationary Phase Log2 Fold C hange Ciprofloxacin Colistin B B B Figure-8 Expression levels of type II TA system genes in the presence of 5-fold MIC of ciprofloxacin and colistin exponential phase (A) stationary phase (B) in PAO1 and clinical strains biofilm. Graph data are indicated as the means±SD of three independent replicates. P < 0.05, P < 0.01, P < 0.001 and **P < 0.0001 (by independent samples T test). Discussion The type of antibiotic used in 349 study did not cause any significant difference in formation persister cells of biofilm strains. 350 Keren et al examined the persister cells formation in biofilm of P. aeruginosa, Staphylococcus 351 aureus and E. coli in exponential and stationary phases indicate that the rate of persister cell 352 formation are depend on bacterial growth stage [33]. D Shah et al observed that the physiology 353 of the produced persister cells in exponential and stationary phases in E. coli was more different 354 to bacterial population that have potential to re-growth and induce recurrence of the infection 312 after treatment [27]. Toxin-antitoxin systems is one of the factors involved in the formation of 313 persister cells. The toxin have potential to inhibit protein synthesis, cell wall and DNA 314 replication which lead to delay in the growth of bacteria and induction of persister cell [28]. 315 Five categories of TA systems are known based on the mode of action in bacterial cell that type 316 II is more common [29]. The first TA system that identified to have main role in biofilm 317 formation is a type II TA system and known as the MqsR system [30]. This study aimed to 318 evaluate the possible role of type II TA systems in persister cell formation of clinical and 319 standard (PAO1) P. aeruginosa strains biofilm by analyzing the expression of type II TA 320 systems genes (relBE/RHH-RelE, vapBC /Xre-PIN, /Xre-COG5654 and /Xre-GNAT) in the 321 exponential and stationary phases following exposure to ciprofloxacin and colistin antibiotics. 322 As shown in fig-6, the results indicated that the expression of type II TA systems which 323 involved in the formation of persister cells in P. aeruginosa strains biofilm in the exponential 324 phase was different in compare to the stationary phase that shows in each phases of bacterial 325 growth, the function of TA systems in the formation of persister cells were different. According 326 to the fig-7, the expression of TA systems in the persister cells of biofilm of clinical isolate 327 showed more differences than the PAO1 strain which implies in depending on variation of the 328 performance of TA systems in different type of strain. Discussion Biofilm formation occurs in chronic infections and sometimes can lead to death in patients. In 299 patients with cystic fibrosis, P. aeruginosa is resistant to a variety of antibiotics due to its ability 300 to form biofilms, which can lead to treatment failure and death [23]. Resistance factors involved 301 in biofilm structure are not fully understood, but persister cells can play an important role in 302 resistant profile [9]. Biofilm related chronic infections on burning wounds often formed by 303 extracellular polymeric substances (EPS) as effective shielder and presence of a subset of the 304 bacterial population known as “persister cells” which give tolerance profile against 305 bacteriostatic antibiotics. These events reduce the penetration of antibiotics into the biofilm 306 which along with a decrease in metabolic activity and bacterial growth lead to persister cells 307 development and antibiotic ineffectiveness. Since Gram negative bacterial cell membrane 308 composed of lipopolysaccharides, increased persister cell formation was more common among 309 this bacterial population biofilms which led to further limitation antibiotic penetration into the 310 bacterial cells [24-26] Persister cells are phenotypically distinct but genetically were identical 311 to bacterial population that have potential to re-growth and induce recurrence of the infection 312 after treatment [27]. Toxin-antitoxin systems is one of the factors involved in the formation of 313 persister cells. The toxin have potential to inhibit protein synthesis, cell wall and DNA 314 replication which lead to delay in the growth of bacteria and induction of persister cell [28]. 315 Five categories of TA systems are known based on the mode of action in bacterial cell that type 316 II is more common [29]. The first TA system that identified to have main role in biofilm 317 formation is a type II TA system and known as the MqsR system [30]. This study aimed to 318 evaluate the possible role of type II TA systems in persister cell formation of clinical and 319 standard (PAO1) P. aeruginosa strains biofilm by analyzing the expression of type II TA 320 systems genes (relBE/RHH-RelE, vapBC /Xre-PIN, /Xre-COG5654 and /Xre-GNAT) in the 321 exponential and stationary phases following exposure to ciprofloxacin and colistin antibiotics. 322 As shown in fig-6, the results indicated that the expression of type II TA systems which 323 involved in the formation of persister cells in P. Discussion aeruginosa strains biofilm in the exponential 324 phase was different in compare to the stationary phase that shows in each phases of bacterial 325 growth, the function of TA systems in the formation of persister cells were different. According 326 to the fig-7, the expression of TA systems in the persister cells of biofilm of clinical isolate 327 showed more differences than the PAO1 strain which implies in depending on variation of the 328 performance of TA systems in different type of strain. According to the fig-8, in the presence 329 of ciprofloxacin and colistin, a non-significant difference in the type of TA systems expression 330 in the exponential phase persister cells of biofilm were observed in both strains. Moreover, in 331 the stationary phase, there was a non-significant difference in the type and amount of 332 expression of TA systems. 333 The results of this study showed that the TA system function’s involved in the formation of 334 persister cells of biofilm depends on the growth stage of the strains, the type of antibiotic, and 335 strain. Narimisa et al evaluated the expression of type TA systems ( relE1/relB1, relE2/relB2, 336 hipA/hipB, vapB/vapC, phd/doc and mazF/mazE) in the persister cell formation of biofilm 337 which except for phd/doc system, all level of TA systems expression were increased [31]. 338 Chenglong Sun et al observed that the MqsR/MqsA system is involved in the formation of 339 persister cells of biofilm Pseudomonas putida KT2440 [32]. According to the fig-3, the colony 340 counts results in the exponential and stationary phases for evaluating the formation of persister 341 cell in exposure to ciprofloxacin and colistin in biofilm of both strains showed that more 342 persister cells formation in stationary phase in compare to exponential phase in biofilm of both 343 strains. Also, as seen in fig-4, the number of persister cells that formed in both clinical and 344 PAO1 biofilm of strains of P. aeruginosa were similar and have same number with persister 345 cells which found in exponential phase of both strains biofilms. No differences were observed 346 in the number of persister cells that formed in biofilm of stationary phase of both strains. 347 Furthermore, fig-5 demonstrates that the number of persister cells in biofilm of both strains 348 were similar following exposure to ciprofloxacin and colistin. Discussion According to the fig-8, in the presence 329 of ciprofloxacin and colistin, a non-significant difference in the type of TA systems expression 330 in the exponential phase persister cells of biofilm were observed in both strains. Moreover, in 331 the stationary phase, there was a non-significant difference in the type and amount of 332 expression of TA systems. 333 The results of this study showed that the TA system function’s involved in the formation of 334 persister cells of biofilm depends on the growth stage of the strains, the type of antibiotic, and 335 strain. Narimisa et al evaluated the expression of type TA systems ( relE1/relB1, relE2/relB2, 336 hipA/hipB, vapB/vapC, phd/doc and mazF/mazE) in the persister cell formation of biofilm 337 which except for phd/doc system, all level of TA systems expression were increased [31]. 338 Chenglong Sun et al observed that the MqsR/MqsA system is involved in the formation of 339 persister cells of biofilm Pseudomonas putida KT2440 [32]. According to the fig-3, the colony 340 counts results in the exponential and stationary phases for evaluating the formation of persister 341 cell in exposure to ciprofloxacin and colistin in biofilm of both strains showed that more 342 persister cells formation in stationary phase in compare to exponential phase in biofilm of both 343 strains. Also, as seen in fig-4, the number of persister cells that formed in both clinical and 344 PAO1 biofilm of strains of P. aeruginosa were similar and have same number with persister 345 cells which found in exponential phase of both strains biofilms. No differences were observed 346 in the number of persister cells that formed in biofilm of stationary phase of both strains. 347 Furthermore, fig-5 demonstrates that the number of persister cells in biofilm of both strains 348 were similar following exposure to ciprofloxacin and colistin. The type of antibiotic used in 349 study did not cause any significant difference in formation persister cells of biofilm strains. 350 Keren et al examined the persister cells formation in biofilm of P. aeruginosa, Staphylococcus 351 aureus and E. coli in exponential and stationary phases indicate that the rate of persister cell 352 formation are depend on bacterial growth stage [33]. D Shah et al observed that the physiology 353 of the produced persister cells in exponential and stationary phases in E. coli was more different 354 than non-persister cells E. coli [34]. Discussion Conlon et al observed more persister cell formation in 355 stationary phase of Staphylococcus aureus due to release of intracellular adenosine 356 triphosphate (ATP) in this phase that contributes to bacterial resistance and persister cell 357 formation [35]. According to our study, the level of persister cells formation in biofilm depends 358 on the bacterial growth stage. In addition to this, in the stationary phase more persister cell was 359 formed than exponential phase which may due to different functions of TA systems in the 360 stationary phase which needs further investigation. 361 than non-persister cells E. coli [34]. Conlon et al observed more persister cell formation in 355 stationary phase of Staphylococcus aureus due to release of intracellular adenosine 356 triphosphate (ATP) in this phase that contributes to bacterial resistance and persister cell 357 formation [35]. According to our study, the level of persister cells formation in biofilm depends 358 on the bacterial growth stage. In addition to this, in the stationary phase more persister cell was 359 formed than exponential phase which may due to different functions of TA systems in the 360 stationary phase which needs further investigation. 361 Conclusion: 362 The results of this study showed that the expression of type II TA system genes in persister 363 cells of biofilm P. aeruginosa significantly increased in compared to the control group in 364 exponential and stationary phases that maybe a reason for different persister cell formation 365 levels and their presence in exponential and stationary phases. 366 367 Declaration 368 369 Consent for publication 370 Not applicable 371 372 Availability of data and material 373 All the datasets supporting the conclusions of this article are available. Additional data of this 374 paper can be obtained upon request. The corresponding author (Faramarz Masjedian Jazi: 375 Masjedian.f@iums.ac.ir) should be contacted if someone wants to request the data from this 376 study. 377 378 Competing interests 379 The authors declare that they have no competing interests. 380 381 Funding 382 This study was financially supported by a research grant (No.13546) for an M.Sc thesis at Iran 383 University of Medical Sciences (Tehran, Iran), for which we are very grateful 384 385 Authors 'contributions 386 This article was part of a study that contributed to an MSC thesis by Rezvan Golmoradi zadeh. 387 Behrooz Sadeghi kalani and Faramarz Masjedian jazi designed the study. Discussion Rezvan Golmoradi 388 zadeh and Marzie Mahdizade Ari performed experiments of the research and drafted the 389 Conclusion: 362 Hill, Ectopic overexpression of wild-type and mutant hipA 429 genes in Escherichia coli: effects on macromolecular synthesis and persister 430 manuscript and Malihe Talebi, Shabnam Razavi performed the statistical analysis. All authors 390 read and approved the final manuscript. 391 392 Conflicts of interest 393 We have no conflicts of interest to disclose. 394 395 Acknowledgements 396 We would like to thank the members of the Microbiology Department for their valuable 397 contributions for this research project. 398 399 400 References 401 1. Maeda, T., et al., Quorum quenching quandary: resistance to antivirulence 402 compounds. The ISME journal, 2012. 6(3): p. 493-501. 403 2. Ciofu, O. and T. Tolker-Nielsen, Tolerance and resistance of Pseudomonas 404 aeruginosa biofilms to antimicrobial agents—How P. aeruginosa can escape 405 antibiotics. Frontiers in microbiology, 2019. 10: p. 913. 406 3. Roberts, M.E. and P.S. Stewart, Modelling protection from antimicrobial agents in 407 biofilms through the formation of persister cells. Microbiology, 2005. 151(1): p. 75- 408 80. 409 4. Maurice, N.M., B. Bedi, and R.T. Sadikot, Pseudomonas aeruginosa biofilms: host 410 response and clinical implications in lung infections. American journal of respiratory 411 cell and molecular biology, 2018. 58(4): p. 428-439. 412 5. Goodyear, A., et al., Persistent gastric colonization with Burkholderia pseudomallei 413 and dissemination from the gastrointestinal tract following mucosal inoculation of 414 mice. PLoS One, 2012. 7(5): p. e37324. 415 6. Fauvart, M., V.N. De Groote, and J. Michiels, Role of persister cells in chronic 416 infections: clinical relevance and perspectives on anti-persister therapies. Journal of 417 medical microbiology 2011 60(6): p 699-709 418 We have no conflicts of interest to disclose. 394 395 Acknowledgements 396 We would like to thank the members of the Microbiology Department for their valu 397 contributions for this research project. 398 399 400 References 401 1. Maeda, T., et al., Quorum quenching quandary: resistance to antivirulence 402 compounds. The ISME journal, 2012. 6(3): p. 493-501. 403 2. Ciofu, O. and T. Tolker-Nielsen, Tolerance and resistance of Pseudomonas 404 aeruginosa biofilms to antimicrobial agents—How P. aeruginosa can escape 405 antibiotics. Frontiers in microbiology, 2019. 10: p. 913. 406 3. Roberts, M.E. and P.S. Stewart, Modelling protection from antimicrobial agents in 407 1. Maeda, T., et al., Quorum quenching quandary: resistance to antivirulence 402 compounds. The ISME journal, 2012. 6(3): p. 493-501. 403 2. Ciofu, O. and T. 11. Korch, S.B. and T.M. 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Keren, I., et al., Persister cells and tolerance to antimicrobials. FEMS microbiology 482 letters, 2004. 230(1): p. 13-18. 483 34. Shah, D., et al., Persisters: a distinct physiological state of E. coli. BMC 484 microbiology, 2006. 6(1): p. 1-9. 485 35. Conlon, B.P., et al., Persister formation in Staphylococcus aureus is associated with 486 ATP depletion. Nature microbiology, 2016. 1(5): p. 1-7. 487 488 33. Keren, I., et al., Persister cells and tolerance to antimicrobials. FEMS microbiology 482 letters, 2004. 230(1): p. 13-18. 483 33. Keren, I., et al., Persister cells and tolerance to antimicrobials. FEMS microbiology 482 letters, 2004. 230(1): p. 13-18. 483 34. Shah, D., et al., Persisters: a distinct physiological state of E. coli. BMC 484 microbiology, 2006. 6(1): p. 1-9. 485 35. Conlon, B.P., et al., Persister formation in Staphylococcus aureus is associated with 486 ATP depletion. Nature microbiology, 2016. 1(5): p. 1-7. 487 488 489 490 491 492 34. Shah, D., et al., Persisters: a distinct physiological state of E. coli. BMC 484 microbiology, 2006. 6(1): p. 1-9. 485 35. Conlon, B.P., et al., Persister formation in Staphylococcus aureus is associated with 486 ATP depletion. Nature microbiology, 2016. 1(5): p. 1-7. 487 35. Conlon, B.P., et al., Persister formation in Staphylococcus aureus is associated with 486 ATP depletion. Nature microbiology, 2016. 1(5): p. 1-7. 487 35. Conlon, B.P., et al., Persister formation in Staphylococcus aureus is associated with 486 ATP depletion. Nature microbiology, 2016. 1(5): p. 1-7. 487 Figures Figure 1 Time-dependent killing of PAO1 and clinical strains bio¦lm exposed to cipro§oxacin. PAO1 strain bio¦lm. Exponential phase (A) and stationary phase (B), clinical isolate bio¦lm of exponential phase (C) and stationary phase (D). The bacterial culture without antibiotics treatment served as a control. The values are means of three biological replicates and error bars indicate the standard deviation (SD). (Independent Samples T test, Repeated Measure, LSD) Figure 1 Figure 1 Time-dependent killing of PAO1 and clinical strains bio¦lm exposed to cipro§oxacin. PAO1 strain bio¦lm. Exponential phase (A) and stationary phase (B), clinical isolate bio¦lm of exponential phase (C) and stationary phase (D). The bacterial culture without antibiotics treatment served as a control. The values are means of three biological replicates and error bars indicate the standard deviation (SD). (Independent Samples T test, Repeated Measure, LSD) Figure 2 Time-dependent killing of PAO1 and clinical strains bio¦lm exposed to colistin. PAO1 strain bio¦lm exponential phase (A) and stationary phase (B) and clinical isolate bio¦lm exponential phase (C) and stationary phase (D). The bacterial culture without any antibiotic treatment served as a control. The values are means of three biological replicates and error bars indicate the standard deviation (SD). (Independent Samples T test, Repeated Measure, LSD) Time-dependent killing of PAO1 and clinical strains bio¦lm exposed to colistin. PAO1 strain bio¦lm exponential phase (A) and stationary phase (B) and clinical isolate bio¦lm exponential phase (C) and stationary phase (D). The bacterial culture without any antibiotic treatment served as a control. The values are means of three biological replicates and error bars indicate the standard deviation (SD). (Independent Samples T test, Repeated Measure, LSD) Figure 3 Time-dependent killing of PAO1 and clinical strains bio¦lm in the exponential and stationary phases exposed to cipro§oxacin. PAO1 strain bio¦lm (A) and clinical isolate bio¦lm (B) and colistin PAO1 strain bio¦lm (C) and clinical isolate bio¦lm (D). Graph data are indicated as the means±SD of three independent replicates. P < 0.05, P < 0.01, P < 0.001. and **P < 0.0001 (statistical results obtain by independent samples T test). Figure 3 Time-dependent killing of PAO1 and clinical strains bio¦lm in the exponential and stationary phases exposed to cipro§oxacin. PAO1 strain bio¦lm (A) and clinical isolate bio¦lm (B) and colistin PAO1 strain bio¦lm (C) and clinical isolate bio¦lm (D). Graph data are indicated as the means±SD of three independent replicates. P < 0.05, P < 0.01, P < 0.001. and **P < 0.0001 (statistical results obtained by independent samples T test). Figure 4 Time-dependent killing of PAO1 and clinical strains bio¦lm exposed to cipro§oxacin exponential phase (A) and stationary phase (B) and colistin exponential phase (C) and stationary phase (D). Graph data a indicated as the means±SD of three independent replicates. P < 0.05, P < 0.01, P < 0.001 and *** < 0.0001 (by independent samples T test). Figure 4 Figure 4 Time-dependent killing of PAO1 and clinical strains bio¦lm exposed to cipro§oxacin exponential phase (A) and stationary phase (B) and colistin exponential phase (C) and stationary phase (D). Graph data are indicated as the means±SD of three independent replicates. P < 0.05, P < 0.01, P < 0.001 and *P < 0.0001 (by independent samples T test). igure 5 Time-dependent killing of cipro§oxacin and colistin in P. aeruginosa strain PAO1 exponential phase (A) nd stationary phase (B) and the P. aeruginosa clinical isolate bio¦lm exponential phase (C) and tationary phase (D). Graph data are indicated as the means±SD of three independent replicates. P < .05, P < 0.01, P < 0.001 and **P < 0.0001 (by independent samples T test). Figure 5 Time-dependent killing of cipro§oxacin and colistin in P. aeruginosa strain PAO1 exponential phase (A) and stationary phase (B) and the P. aeruginosa clinical isolate bio¦lm exponential phase (C) and stationary phase (D). Graph data are indicated as the means±SD of three independent replicates. P < 0.05, P < 0.01, P < 0.001 and **P < 0.0001 (by independent samples T test). Time-dependent killing of cipro§oxacin and colistin in P. aeruginosa strain PAO1 exponential phase (A) and stationary phase (B) and the P. aeruginosa clinical isolate bio¦lm exponential phase (C) and stationary phase (D). Graph data are indicated as the means±SD of three independent replicates. P < 0.05, P < 0.01, P < 0.001 and **P < 0.0001 (by independent samples T test). Figure 6 Expression levels of type II TA system genes in the exponential and stationary phases in the presence o 5-fold MIC of cipro§oxacin PAO1 strain bio¦lm (A) clinical isolate bio¦lm (B) and colistin PAO1 strain bio¦lm (C) clinical isolate bio¦lm (D). Graph data are indicated as the means±SD of three independent replicates. P < 0.05, P < 0.01, P < 0.001 and **P < 0.0001 (by independent samples T test). Figure 6 Expression levels of type II TA system genes in the exponential and stationary phases in the presence o 5-fold MIC of cipro§oxacin PAO1 strain bio¦lm (A) clinical isolate bio¦lm (B) and colistin PAO1 strain bio¦lm (C) clinical isolate bio¦lm (D). Graph data are indicated as the means±SD of three independent replicates. P < 0.05, P < 0.01, P < 0.001 and **P < 0.0001 (by independent samples T test). Figure 6 Expression levels of type II TA system genes in the exponential and stationary phases in the presence of 5-fold MIC of cipro§oxacin PAO1 strain bio¦lm (A) clinical isolate bio¦lm (B) and colistin PAO1 strain bio¦lm (C) clinical isolate bio¦lm (D). Graph data are indicated as the means±SD of three independent replicates. P < 0.05, P < 0.01, P < 0.001 and **P < 0.0001 (by independent samples T test). gure 7 xpression levels of type II TA system genes in PAO1 and clinical strains bio¦lm in the presence of 5-f IC of cipro§oxacin exponential phase (A), stationary phase (B) and colistin exponential phase (C) ationary phase (D). Graph data are indicated as the means±SD of three independent replicates. Figure 5 P < 05, P < 0.01, P < 0.001 and **P < 0.0001 (by independent samples T test). Figure 7 Expression levels of type II TA system genes in PAO1 and clinical strains bio¦lm in the presence of 5-fold MIC of cipro§oxacin exponential phase (A), stationary phase (B) and colistin exponential phase (C) stationary phase (D). Graph data are indicated as the means±SD of three independent replicates. P < 0.05, P < 0.01, P < 0.001 and **P < 0.0001 (by independent samples T test). Expression levels of type II TA system genes in PAO1 and clinical strains bio¦lm in the presence of 5-fold MIC of cipro§oxacin exponential phase (A), stationary phase (B) and colistin exponential phase (C) stationary phase (D). Graph data are indicated as the means±SD of three independent replicates. P < 0.05, P < 0.01, P < 0.001 and **P < 0.0001 (by independent samples T test). Figure 8 Expression levels of type II TA system genes in the presence of 5-fold MIC of cipro§oxacin and colistin exponential phase (A) stationary phase (B) in PAO1 and clinical strains bio¦lm. Graph data are indicated as the means±SD of three independent replicates. P < 0.05, P < 0.01, P < 0.001 and **P < 0.0001 (by independent samples T test). Figure 8 Expression levels of type II TA system genes in the presence of 5-fold MIC of cipro§oxacin and colistin exponential phase (A) stationary phase (B) in PAO1 and clinical strains bio¦lm. Graph data are indicated as the means±SD of three independent replicates. P < 0.05, P < 0.01, P < 0.001 and ***P < 0.0001 (by independent samples T test).
https://openalex.org/W2797222877	https://zenodo.org/records/2349682/files/article.pdf	English	null	St. Patrick's (R. C.) Cathedral, Armagh	The musical times/Musical times	1,921	public-domain	2,966	St. Patrick's (R. C.) Cathedral, Armagh Author(s): W. Wooding Starmer Source: The Musical Times, Vol. 62, No. 946 (Dec. 1, 1921), pp. 827-830 Published by: Musical Times Publications Ltd. Stable URL: http://www.jstor.org/stable/908545 Accessed: 11-02-2016 21:45 UTC St. Patrick's (R. C.) Cathedral, Armagh Author(s): W. Wooding Starmer Source: The Musical Times, Vol. 62, No. 946 (Dec. 1, 1921), pp. 827-830 Published by: Musical Times Publications Ltd. Stable URL: http://www.jstor.org/stable/908545 Accessed: 11-02-2016 21:45 UTC St. Patrick's (R. C.) Cathedral, Armagh Author(s): W. Wooding Starmer Source: The Musical Times, Vol. 62, No. 946 (Dec. 1, 1921), pp. 827-830 Published by: Musical Times Publications Ltd. Stable URL: http://www.jstor.org/stable/908545 Accessed: 11-02-2016 21:45 UTC Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at http://www.jstor.org/page/ info/about/policies/terms.jsp JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact support@jstor.org. Musical Times Publications Ltd. is collaborating with JSTOR to digitize, preserve and extend access to The Musical Times. http://www.jstor.org AND SINGING-CLASS CIRCULAR This noble and imposing pile is built on an eminence known as Sandy Hill--a position to the north of the city commanding the entire neighbourhood. DECEMBER I 1921 In our article in the November Musical Times on 'rThe Musical Press'we pointed out the difficul position of a monthly journal in regard to news matter. We showed that, owing to the increased prominence given to music in the daily and weekly press, provincial as well as London, a monthly organ could contain nothing fresh, and was bound to publish a good deal that was stale. 'It seems likely [we said] that such journals will eventually reduce their news department to a bare record of important events at home and abroad for purposes of reference.' t g The foundation-stone was laid in I840, the completed structure being consecrated in 1904. The foundations, in some instances 6o-ft. deep, cost a large sum on account of the friable nature of the surface strata. y y , - - s As in the case of so many other ecclesiastical buildings, the original plans were not carried out. The first architect, Mr. Duff, produced a perpendicular Gothic design strongly reminiscen of York Minster. When the walls were well advanced (the architect having then been dead for some years) a rising young architect named McCarthy was consulted, with the result that many changes were made. He produced an entirely new design in the 14th century style of decorated Gothic, and this was adopted. Five Primates spent their lives in advancing the construction of the Cathedral, but the finishing of the whole conception, particularly with regard to the interior decoration and adorn- ment, has fallen to the lot of the present Primate, Cardinal Logue, who has accomplished his task with the greatest success. Most of the recent additions have been made to the designs of Messrs. Ashlin & Coleman. The building of the Cathedral has necessitated the expenditure of over G17 5,000. It is cruciform in plan, and the dimensions are : But even a bare record is a formidable affair so great is the amount of musical activity through out the country. Our provincial news section is already double the size of that of ten years ago. This content downloaded from 142.51.1.212 on Thu, 11 Feb 2016 21:45:05 UTC All use subject to JSTOR Terms and Conditions This content downloaded from 142.51.1.212 on Thu, 11 Feb 2016 21:45:05 UTC All use subject to JSTOR Terms and Conditions THE MUSICAL TIMES-DECEMBER I 1921 827 THE EXTERIOR g We have been considering this news question for some months, and it seems that the beginning of our 1922 volume offers a good opportunity for an attempt to solve the problem. We are therefor making arrangements by means of which the Musical Times will continue to furnish the record that has made its volumes so useful in the past; but it will do so, we trust, at the cost of far less space than at present. Similar compression will be applied to our foreign correspondence and, in a lesser degree, to our London news. The columns thus saved will enable us to widen the scope of the journal in several directions impossible hitherto. g e The exterior is a successful example of 14th century Gothic. The two lofty and elegant spires add much dignity to the general design, which as a whole is imposing. AND SINGING-CLASS CIRCULAR Roughly it now fills nearly a quarter of the letter press space, and if we acceded to all the requests from readers who wish to act as our correspondent at home and abroad the Musical Times would contain little but news--mostly a month old. Obviously the time has come for a drastic step. The time-honoured policy of this paper has been to present a bird's-eye view of musical doings throughout the country, and that policy must be maintained so far as possible. It can be done, we think, by steadily holding in view the object of such columns, viz., the recording of the chief provincial concerts, partly as a matter of interest but chiefly for future reference. Total length, 212-ft. Width of transepts, 12o-ft. Length of nave, 114-ft. 9 chancel, 6o-ft. Width of nave and chancel, 75-ft. Exterior roof-height, Ioo-ft. Interior ,, 8I-ft. Two western towers, with spires 2Io-ft. high. Total length, 212-ft. Width of transepts, 12o-ft. Length of nave, 114-ft. 9 chancel, 6o-ft. Width of nave and chancel, 75-ft. Exterior roof-height, Ioo-ft. Interior ,, 8I-ft. Two western towers, with spires 2Io-ft. high. The value of such a record lies in facts rather than in views-that is to say, it should be concerned with the matter performed rather than with the manner of its performance. The former is indisputable; the latter is a mere opinion. There may be some interest in reading and discussing the varying views of critics on the day after a concert, but each succeeding day lessens the interest, and by the time the criticism appears in a monthly journal the performanc itself has often been forgotten. m e The principal entrance is the deeply-recessed west door, which is approached by a magnificen seven-terraced flight of steps, 225-ft. long, leading from the entrance gates up to the spacious piazza fronting the building. This content downloaded from 142.51.1.212 on Thu, 11 Feb 2016 21:45:05 UTC All use subject to JSTOR Terms and Conditions E 0e lusical Eimes By W. WOODING STARMER TIHE INTERIOR , The interior is one of the most beautiful of any modern ecclesiastical building in the kingdom, both in regard to general proportions and detailed decoration. The whole of Europe has been laid under contribution for precious marbles of varied colours, which have been employed on a very extensive scale and with beautiful effect. This content downloaded from 142.51.1.212 on Thu, 11 Feb 2016 21:45:05 UTC All use subject to JSTOR Terms and Conditions 828 THE MUSICAL TIMES-DECEMBER I 192I r n SWELL ORGAN Ft. Ft. Bourdon ... .... ... 6 Mixture .... ...(ranks) 3 Open diapason ...... 8 Cornopean ... .. 8 Dulciana... .. ... 8 Oboe ... ... . ... 8 Rohr flute ... ... 8 Clarion ... ... ... 4 Octave .... .... ... 4 Tremulant Fifteenth... ... ... 2 CIOIR ORGAN Ft. Ft. Salicional . ... 8 Dulcet .... ....... 4 Viol di gamba ...... 8 Piccolo .... .... ... 2 Lieblich gedackt ... 8 Clarinet .... ....... 8 Flauto traverso ... ... 4 Dulciana ... ... ... 8 PEIDATL ORGAN Fl. Ft. Open diapason ... ... 16 Quint ........ ... Io* Bourdon .... .... ... 16 Posaune... ... .... 16 Open diapason ....... 8 Trumpet ... ... 8 Octave .... .... ... 8 COUPLERS Swell to Pedal. Swell to Great. Great to Pedal. Swell to Choir. Choir to Pedal. Choir to Great. Swell Super-Octave. I I Combination Pistons. Pedal Helps to Swell Great and Choir Organs. The mosaics are remarkable, particularly at the crossing where the high altar stands. The six spandrils over the great arches (8o-ft. high and 4o-ft. wide) are inlaid with figured subjects in gold and colour with gorgeous effect. The groining of the aisles, side chapels, baptistery, and tower porches is of Bath stone. The mosaics are remarkable, particularly at the crossing where the high altar stands. The six spandrils over the great arches (8o-ft. high and 4o-ft. wide) are inlaid with figured subjects in gold and colour with gorgeous effect. The groining of the aisles, side chapels, baptistery, and tower porches is of Bath stone. p THE ORGAN The organ, by Messrs. Telford, of Dublin, is erected on the west gallery, and contains 2,453 pipes. The oak case, of excellent design, rises to a height of 30o-ft. at the sides, and in the centre is recessed to a depth of Io-ft. in order to prevent any obstruction of the large west window The console faces west. The action is tubular-pneumatic. An excellent wind supply is provided by an electric installation placed in the south-west tower. THE ORGAN The organ, by Messrs. Telford, of Dublin, is erected on the west gallery, and contains 2,453 pipes. THE ORGAN The organ, by Messrs. Telford, of Dublin, is erected on the west gallery, and contains 2,453 pipes. This content downloaded from 142.51.1.212 on Thu, 11 Feb 2016 21:45:05 UTC All use subject to JSTOR Terms and Conditions p p The oak case, of excellent design, rises to a height of 30o-ft. at the sides, and in the centre is recessed to a depth of Io-ft. in order to prevent any obstruction of the large west window The console faces west. The action is tubular-pneumatic. An excellent wind supply is provided by an electric installation placed in the south-west tower. The action is tubular-pneumatic. An excellent wind supply is provided by an electric installation placed in the south-west tower. SPECIF-ICATION COMPASS Manuals CC to G ... ... 56 notes. Pedals CCC to F 30 notes. GREAT ORGAN Ft. Ft Double open diapason ... 16 Octave ... ...... 4 Open diapason ... ... 8 Twelfth ... ... 2. Harmonic diapason ... 8 Fifteenth ... 2 Gamba ... .... 8 Mixture... "(ranks) 5 Hohl flute ... .. 8 Trumpet .. ... 8 Harmonic flute ... ... 4 Orchestral oboe ... 8 GREAT ORGAN Ft. Ft. Double open diapason ... 16 Octave ... ...... 4 Open diapason ... ... 8 Twelfth ... ... 2. Harmonic diapason ... 8 Fifteenth ... 2 Gamba ... .... 8 Mixture... "(ranks) 5 Hohl flute ... .. 8 Trumpet .. ... 8 Harmonic flute ... ... 4 Orchestral oboe ... 8 The organist and choirmaster is Mr. J. W. Holden, who has held this position for the past eighteen years. x gki ,~~iRr d ~ ~ s~ aeg~Bi ~ I~iii r' z:, x? ax, MOWi jA4 Ip 1 Photo bj1, INTERIOR: LOOKING EAST [H. Allison Son, Armnagh Photo bj1, to bj1, INTERIOR: LOOKING EAST [H. Allison Son, Armnagh INTERIOR: LOOKING EAST [H. Allison Son, Armnagh INTERIOR: LOOKING EAST This content downloaded from 142.51.1.212 on Thu, 11 Feb 2016 21:45:05 UTC All use subject to JSTOR Terms and Conditions This content downloaded from 142.51.1.212 on Thu, 11 Feb 2016 21:45:05 UTC All use subject to JSTOR Terms and Conditions THE MUSICAL TIMES-DECEMBER 1 1921 829 Photo by] THE WESTERN TOWERS [H. Allison and Son, Aormagh Photo by] THE WESTERN TOWERS [H. Allison and Son, Aormagh THE WESTERN TOWERS time. There are no chime tunes. The carillon consists of thirty-nine bells with clavier (three octaves chromatic), with the two largest bells diatonic. k time. There are no chime tunes. The carillon consists of thirty-nine bells with clavier (three octaves chromatic), with the two largest bells diatonic. A new powerful clock with quarter chimes (Westminster), containing the most modern improvements in construction, has been put in by Messrs. Potts, of Leeds. The hour is struck on the great bell, the first stroke being the exact g g SCALE All soundhig one octave higier. e" The founders are Messrs. Taylor, of Loughborough. The founders are Messrs. Taylor, of Loughborough. III. ~~!i-liii:i:i iiiiiiiiliiiii:iilii:i:liii??:li:?'iiii iiiiil:?aiiiiiii:ili:ii-iii i:iiiiiiiii?iliiii?iii:iiiiiiiiiiiiii?i :::;.i' Eli I~ i:lii~ :-::: : ::::::i:l:::::I.:,x:: : : :i .. p::::-:::::::j:i:fllli:ii'i ~:::::::'::?*P::: :::::::::::::::::::: I"?i~~?h~::??;?:i~:l??: 1:1:i:::::::1: :::::::::::.:.:::.:-:-:::: -1:: ::?:::::::::?:i?: ::'::::: i:::;::::::: ::r:: ::: :::::::::::::::::::::::: ::::::::::::: ~~iiB-:i:i:i:::'::::: .::..j ::::i~:j::l:::ljlj ::::~~:??~~i-i:::si:~'ii i:i-i:i:- :::::::::::::::::: ::::;`:iiiiii::ii:iiii :`::::: :~:?::::?::::: :::::::::::::::::::::::: ';?'?::?:::';I'???.:?il ~6~88~DB~~'~"'BD'~?~: ::-:::::: ::::::::::::::::::i:,,-,-,~r~?~s::;?.~.? ::::::::::::::::::::: ::w:::~?:::::: iriiirX:i~~3iiaFiriEiliii'i~~iiiii~~i :::: :?:::::::::xi?:::::: THE LARGEST AND SMALLEST BELLS ?:?:?::: I:-:: iii-i-i It will easily be realised that when Kcechlin resorts to recondite or apparently complicated modes of expression, he does so not in consequence of a taste for the far-fetched or the exceptional, but with a steadfast purpose and for well-weighed reasons. For instance, the most searching exami- nation will fail to discover in 'Paysages et Marines' a single passage of which it might be said that it could have been written more simply without anything vital being sacrificed. y g g If the same can hardly apply to the pianoforte accompaniments to certain of his songs, it is for the reason that most of these were originally conceived for various combinations of instrument A survey of the three books of Songs, and three of Rondels, will show that the composer's favourite medium for accompaniments is the orchestra Of seventeen songs in the first book, only three are not published with orchestral accompani- ment. In the second book we see that out of fourteen songs, four exist with orchestra accompaniment, another four with accompaniment by string quartet and pianoforte. The whole contents of the third book are orchestrated. And in all likelihood it is the orchestral form that represents the composer's original conception. THE LARGEST AND SMALLEST BELLS The carillon is placed in the north-west tower in an excellent position acoustically. p y The inauguration of this most recent addition to the Cathedral took place on Sunday, November 6, when His Eminence Cardinal Logue was present at the Pontifical M1ass. A sermon on bells was preached by the Rev. Dr. Beecher, of Maynooth. Immediately after the service M. Antoine Nauwelaerts, the city carillonneur of Bruges, who came over specially for the occasion, gave the opening recital with the following pro- gramme : p p g p At times an extra solo part, or an optional choir, are provided for. It may happen that these added voices do little but duplicate the principal part (e.g., the female choir in 'Promenade Galante' in the first book). SCALE All soundhig one octave higier. y g g The founders are Messrs. Taylor, of Loughborough This content downloaded from 142.51.1.212 on Thu, 11 Feb 2016 21:45:05 UTC All use subject to JSTOR Terms and Conditions 830 TIIE MUSICAL TIMES-DECEMBER I 1921 All these items were listened to with rapt attention by a very large gathering. All these items were listened to with rapt attention by a very large gathering. This splendid set of bells-perfect as to accuracy of tune and of rich resonant tone-constitutes one of the finest modern carillons in Europe, and in one respect the best extant. Messrs. Taylor have introduced many improvements in the action-work connecting the clavier with the bells, making the touch so easy and light that a child could play every note without undue exertion, and also giving the player absolute control of the tone from pianissimo to for/issznmo. This is a great advance on anything existing in Holland or Belgium, where every carillon demands very considerable physical strength for its effective manipulation. k y y g g g M. Nauwelaerts is one of the best represen tative carillonneurs of the modern Belgian school. His playing is most brilliant, with well-defined rhythm, and to him technical difficulties are non- existent. His expressive powers were amply displayed in the music of Schubert and Beethoven, which revealed to the listener the extraordinary possibilities of the instrument in the hands of such a virtuoso. Recitals were given in the afternoon and evening, also on Monday. strength manipulation. The total weight of the bells is eleven tons. g y Ireland now possesses two magnificent carillons, at Queenstown and Armagh, both made by the same founders. g Particulars of the three largest bells are as follows : NOTE. WEIGHT INSCRIPTION. CWTS. C 432 Gloria in excelsis Deo-192o. D 3o0 B. Maria Virgine Mater Dei. E 211 B. Michaeli Archangelo. By M.-D. CALVOCORESSI. (Cozfinuedfrom November number, fage 761) III. The smallest bell weighs twenty pounds. III. Likewise in 'Le Ninuphar' (second book) a solo flute is required more for the sake of colour than in order to provide special patterns. Some of the things contained in these books-for instance, 'La Jeune Tarentine,' 'N6ere,'' Juin,' 'Midi,' 'Le Sommeil de Canope' -are not songs in the usual sense of the word, but big tone-poems for voice and orchestra, in which the pianoforte part can hardly do justice to the composer's intentions. g Prelude: 'The Cuckoo' ... ... Vanden G/eyn ' Ave Maria'... ... ... ... ... Sc/izber Sonata .. ... Van Hoey Adagio ('Pathetic' Sonata) ... Beethove ' Le Retour de la Fauvette' ....... de Boeck Irish Folk-Tunes: (a) 'Londonderry Air.' (b) ' Snowy-breasted Pearl.' (c) ' Foggy Dew.' ' Rubens March' ...... . ... Benoit g Prelude: 'The Cuckoo' ... ... Vanden G/eyn ' Ave Maria'... ... ... ... ... Sc/izber Sonata .. ... Van Hoey Adagio ('Pathetic' Sonata) ... Beethove ' Le Retour de la Fauvette' ....... de Boeck I i hF lk T (a) 'Londonderry Air.' ( ) ' ( ) Londonderry (b) ' Snowy-breasted Pearl.' ( ) ' ' y (c) ' Foggy Dew.' ' Rubens March' ...... . ... Benoit This content downloaded from 142.51.1.212 on Thu, 11 Feb 2016 21:45:05 UTC All use subject to JSTOR Terms and Conditions
https://openalex.org/W3203963369	https://repozitorij.uni-lj.si/Dokument.php?id=155856&dn=	English	null	Integrative Map of HIF1A Regulatory Elements and Variations	Genes	2,021	cc-by	10,661	Keywords: cancer; erythrocytosis; hypoxia-inducible factor (HIF); HIF1A; hypoxia genes G C A T T A C G G C A T genes Article Integrative Map of HIF1A Regulatory Elements and Variations Tanja Kunej Department of Animal Science, Biotechnical Faculty, University of Ljubljana, 1000 Ljubljana, Slovenia; tanja.kunej@bf.uni-lj.si; Tel.: +386-1-2303-890 Department of Animal Science, Biotechnical Faculty, University of Ljubljana, 1000 Ljubljana, Slovenia; tanja.kunej@bf.uni-lj.si; Tel.: +386-1-2303-890 Abstract: Hypoxia-inducible factor (HIF) family of transcription factors (HIF1A, EPAS1, and HIF3A) are regulators of the cellular response to hypoxia. They have been shown to be involved in de- velopment of various diseases such as cancer, diabetes, and erythrocytosis. A complete map of connections between HIF family of genes with various omics types has not yet been developed. The main aim of the present analysis was to construct the integrative map of genomic elements associated with HIF1A gene and prioritize potentially deleterious variants. Various genomic databases and bioinformatics tools were used, including Ensembl, MirTarBase, STRING, Cytoscape, MethPrimer, CADD, SIFT, and UALCAN. Integrative HIF1A gene map was visualized and includes transcrip- tional and post-transcriptional regulators, downstream targets, and genetic variants. One CpG island overlaps transcription start site of the HIF1A gene. Out of over 450 missense variants, four have predicted deleterious effect on protein function by at least ﬁve bioinformatics tools. Currently there are 85 miRNAs reported to target HIF1A. HIF1A downstream targets include protein-coding genes, long noncoding RNAs, and microRNAs (hypoxamiRs). The study presents the ﬁrst integration of heterogeneous molecular interactions associated with HIF1A gene enabling a holistic view of the gene and lays the groundwork for supplementing the data in the future. Citation: Kunej, T. Integrative Map of HIF1A Regulatory Elements and Variations. Genes 2021, 12, 1526. https://doi.org/10.3390/ genes12101526 Academic Editor: Mary Frances McMullin Received: 20 August 2021 Accepted: 24 September 2021 Published: 28 September 2021 Citation: Kunej, T. Integrative Map of HIF1A Regulatory Elements and Variations. Genes 2021, 12, 1526. https://doi.org/10.3390/ genes12101526 Academic Editor: Mary Frances McMullin Received: 20 August 2021 Accepted: 24 September 2021 Published: 28 September 2021 genes G C A T T A C G G C A T genes G C A T T A C G G C A T genes G C A T T A C G G C A T 1. Introduction Hypoxia-inducible factor (HIF) family of transcription factors are master regulators of the cellular response to low oxygen availability (hypoxia). They coordinate hypoxic response in cells, ensuring metabolic and vascular adaptation to shortage of oxygen. Heterodimeric HIF transcription factors consist of the α and β subunits. The α subunit of the HIF heterodimer is O2-sensitive, and in hypoxia, it functions as a master regulator of various genes involved in hypoxia pathway. The β subunit of HIF heterodimer (HIF1B, HIF-1β) with the ofﬁcial gene name aryl hydrocarbon nuclear translocator (ARNT) is constitutively expressed [1,2]. y p HIF α paralogs dimerize with the same β subunit (HIF-1β). Three paralogs of α subunit are known: hypoxia-inducible factor 1 subunit α (HIF1A, HIF-1α), endothelial PAS domain-containing protein 1 (EPAS1, HIF2A, HIF-2α), and hypoxia-inducible factor 3 subunit α (HIF3A; Figure 1). Among HIF family of genes HIF1A and EPAS1 genes have been a topic of several studies, however, HIF3A is less studied [3,4]. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Under normoxia, the HIF signaling pathway is inhibited due to degradation of HIFα subunits. Two proline residues of HIF1A and EPAS1 are hydroxylated by prolyl hydroxy- lase domain protein 2 (PHD2), also named egl-9 family hypoxia-inducible factor 1 (EGLN1). Modiﬁcation by prolyl hydroxylation is a regulatory event that targets HIF subunits for ubiquitin-mediated proteolysis via binding to the Hippel–Lindau tumor suppressor (VHL). In hypoxic conditions, the HIF α subunit is activated and stabilized, resulting in activation of HIF target genes and various activities (Figure 2) [5]. Copyright: © 2021 by the author. 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/). The bHLH-PAS motifs at N-terminus are essential for heterodimer formation between HIF α and β subunits and for binding to the sequence on the target genes. Conserved HLH domain is involved in DNA binding to hypoxia response element (HRE) in various target https://www.mdpi.com/journal/genes Genes 2021, 12, 1526. https://doi.org/10.3390/genes12101526 Genes 2021, 12, 1526 2 of 14 ion be- on be- Co genes. HIF α paralogs have both unique and common downstream targets in hypoxic gene regulation. The HIF1A and EPAS subunits have two transactivation domains (TAD), N-TAD and C-TAD, which are responsible for transcriptional activity. 1. Introduction C-TAD interacts with co-activators such as CBP/p300 to modulate gene transcription under hypoxia. N- TAD is responsible for stabilizing HIFα against degradation. HIFα subunits have an oxygen-dependent degradation (ODD) domain (ODDD) overlapping N-TAD. The ODDD is important in mediating O2 regulation stability [6,7]. various target genes. HIF α paralogs have both unique and common downstream targets in hypoxic gene regulation. The HIF1A and EPAS subunits have two transactivation do- mains (TAD), N-TAD and C-TAD, which are responsible for transcriptional activity. C- TAD interacts with co-activators such as CBP/p300 to modulate gene transcription under hypoxia. N-TAD is responsible for stabilizing HIFα against degradation. HIFα subunits have an oxygen-dependent degradation (ODD) domain (ODDD) overlapping N-TAD. The ODDD is important in mediating O2 regulation stability [6,7]. various target genes. HIF α paralogs have both unique and common downstream targets in hypoxic gene regulation. The HIF1A and EPAS subunits have two transactivation do- mains (TAD), N-TAD and C-TAD, which are responsible for transcriptional activity. C- TAD interacts with co-activators such as CBP/p300 to modulate gene transcription under hypoxia. N-TAD is responsible for stabilizing HIFα against degradation. HIFα subunits have an oxygen-dependent degradation (ODD) domain (ODDD) overlapping N-TAD. The ODDD is important in mediating O2 regulation stability [6,7]. Figure 1. Terminology of the HIF gene family. Figure 1. Terminology of the HIF gene family. Figure 1. Terminology of the HIF gene family. Figure 1. Terminology of the HIF gene family. Figure 2. Structure of the HIF1α protein. bHLH: basic helix–loop–helix; PAS: Per-ARNT-Sim do main; ODDD: oxygen-dependent degradation domain; TAD: transactivation domain. Figure 1. Terminology of the HIF gene family. Figure 1. Terminology of the HIF gene family. Figure 2. Structure of the HIF1α protein. bHLH: basic helix–loop–helix; PAS: Per-ARNT-Sim do- main; ODDD: oxygen-dependent degradation domain; TAD: transactivation domain. Figure 2. Structure of the HIF1α protein. bHLH: basic helix–loop–helix; PAS: Per-ARNT-Sim domain; ODDD: oxygen-dependent degradation domain; TAD: transactivation domain. Figure 1. Terminology of the HIF gene family. Figure 1. Terminology of the HIF gene family. Figure 1. Terminology of the HIF gene family. Figure 2. Structure of the HIF1α protein. bHLH: basic helix–loop–helix; PAS: Per-ARNT-Sim do- main; ODDD: oxygen-dependent degradation domain; TAD: transactivation domain. Figure 2. Structure of the HIF1α protein. bHLH: basic helix–loop–helix; PAS: Per-ARNT-Sim do- main; ODDD: oxygen-dependent degradation domain; TAD: transactivation domain. Figure 2. Structure of the HIF1α protein. bHLH: basic helix–loop–helix; PAS: Per-ARNT-Sim domain; ODDD: oxygen-dependent degradation domain; TAD: transactivation domain. 1. Introduction Figure 2. Structure of the HIF1α protein. bHLH: basic helix–loop–helix; PAS: Per-ARNT-Sim do- main; ODDD: oxygen-dependent degradation domain; TAD: transactivation domain. Figure 2. Structure of the HIF1α protein. bHLH: basic helix–loop–helix; PAS: Per-ARNT-Sim do- main; ODDD: oxygen-dependent degradation domain; TAD: transactivation domain. Figure 2. Structure of the HIF1α protein. bHLH: basic helix–loop–helix; PAS: Per-ARNT-Sim domain; ODDD: oxygen-dependent degradation domain; TAD: transactivation domain. Asparagine 803 located in the transactivation domain is hydroxylated in oxygenated cells by factor inhibiting HIF-1 (FIH-1; HIF1AN; hypoxia-inducible factor 1 subunit α in- hibitor), which blocks the binding of the co-activators p300 and CBP (EP300; E1A binding protein/CREBBP; CREB binding protein) [7]. Under hypoxic conditions, the prolyl and asparaginyl hydroxylation reactions are inhibited by oxygen deprivation [8 9] Asparagine 803 located in the transactivation domain is hydroxylated in oxygenated cells by factor inhibiting HIF-1 (FIH-1; HIF1AN; hypoxia-inducible factor 1 subunit α in- hibitor), which blocks the binding of the co-activators p300 and CBP (EP300; E1A binding protein/CREBBP; CREB binding protein) [7]. Under hypoxic conditions, the prolyl and asparaginyl hydroxylation reactions are inhibited by oxygen deprivation [8,9]. Asparagine 803 located in the transactivation domain is hydroxylated in oxygenated cells by factor inhibiting HIF-1 (FIH-1; HIF1AN; hypoxia-inducible factor 1 subunit α inhibitor), which blocks the binding of the co-activators p300 and CBP (EP300; E1A binding protein/CREBBP; CREB binding protein) [7]. Under hypoxic conditions, the prolyl and asparaginyl hydroxylation reactions are inhibited by oxygen deprivation [8,9]. asparaginyl hydroxylation reactions are inhibited by oxygen deprivation [8,9]. HIF genes have been shown to be involved in the development of various diseases such as cancer, diabetes, and erythrocytosis [7,10]. Inhibiting the interaction between tran- scription factor HIF-1α and coactivator p300/CBP represents one of the possible ap- oa he fo blo ki hy o ia ath ay i tu o [11] p g y y y y yg p [ , ] HIF genes have been shown to be involved in the development of various diseases such as cancer, diabetes, and erythrocytosis [7,10]. Inhibiting the interaction between tran- scription factor HIF-1α and coactivator p300/CBP represents one of the possible ap- proaches for blocking hypoxia pathway in tumors [11]. HIF genes have been shown to be involved in the development of various diseases such as cancer, diabetes, and erythrocytosis [7,10]. Inhibiting the interaction between transcription factor HIF-1α and coactivator p300/CBP represents one of the possible approaches for blocking hypoxia pathway in tumors [11]. 1. Introduction proaches for blocking hypoxia pathway in tumors [11]. proaches for blocking hypoxia pathway in tumors [11]. pp g yp p y HIF genes are involved in the complex interplay of molecular interactions at various omics levels, including genomics, transcriptomics, proteomics, epigenomics, and miR- Nomics. Gene regulatory networks (GRN) are sets of proteins and RNAs that interact to control the level of expression of various genes. The main players in regulatory networks Genes 2021, 12, 1526 3 of 14 3 of 14 are DNA-binding protein transcription factors as they modulate the ﬁrst step in gene expression. Upstream regulators include transcription factors (TFs) and any gene or small molecule that has been observed experimentally to affect gene expression (Li et al., 2015). Various epigenetic mechanisms regulate gene expression, including DNA methyla- tion/hydroxymethylation, post-translational modiﬁcations of histones, chromatin remod- eling, and regulation by noncoding RNAs. Noncoding RNAs are divided into two major groups, short ncRNA (for example miRNAs) and long noncoding RNAs (lncRNAs) [12]. MicroRNAs that are under the control of HIF genes are termed hypoxia-regulated miRNAs (HRMs) or hypoxia-induced miRNAs (hypoxamiR)—for example, miR-210. Hypoxia is therefore a powerful stimulus regulating the expression of a speciﬁc subset of miRNAs, which are regulators of the cell responses to decreased oxygen tension. miR-210, for example, is elevated in patients with ischemic diseases and most solid tumors [13]. The number of publications on HIF transcription factor family is increasing, and therefore efforts toward integration and organization of published knowledge are an urgent need to facilitate research developments of this study ﬁeld. A map of HIF regulatory elements would enable more planned and coordinated development of this topic. The aim of the present study was therefore to review the main elements involved in the molecular interplay associated with HIF1A gene and visualize the information as the map of regulatory elements including upstream regulatory elements regulating HIF1A expression, post-transcriptional regulators, and its downstream targets. 2. Materials and Methods Relevant genomics data regarding the HIF1A gene in humans was extracted from genomics databases and publications. The terminology for the gene names and sym- bols was edited according to the HUGO gene nomenclature committee (HGNC) (https: //www.genenames.org/ (accessed on 2 August 2021). The HIF1A gene structure and genetic variations were obtained from the Ensembl genome browser [14], release 104 (May 2021). Prediction of the effect of genetic variants on protein function was performed using six bioinformatics tools integrated in the Ensembl database: SIFT (sorting intolerant from tolerant), PolyPhen (polymorphism phenotyping), CADD (combined annotation- dependent depletion), REVEL (rare exome variant ensemble learner), MetaLR, and Muta- tion Assessor. Experimentally validated microRNA-target (MTI) interactions were obtained from the MirTarBase [15] and visualized using Cytoscape software [16]. Protein–protein interaction (PPI) networks were obtained from the STRING tool [17] using the setting network type/physical network, in which the edges indicate that the proteins are part of a physical complex. The MethPrimer tool was used for CpG island prediction using the fol- lowing criteria: CpG island size >200, GC percent >50.0, and observed/expected CpG ratio >0.6 [18]. Phenotype associations were extracted from the Ensembl database, which imports data from the Cancer Gene Census (CGC; https://cancer.sanger.ac.uk/census) (accessed on 2 August 2021)) [19], Zebraﬁsh Information Network (ZFIN; https://zﬁn.atlassian. net/wiki/spaces/general/overview?mode=global (accessed on 2 August 2021)) [20], Rat Genome Database (RGD; https://rgd.mcw.edu/ (accessed on 2 August 2021)) [21], and Mouse Genome Informatics (MGI; http://www.informatics.jax.org/ (accessed on 2 Au- gust 2021) [22]. Cancer OMICS data were extracted from the UALCAN interactive web resource, which includes the Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC) data [23,24]. The UALCAN database also includes the TargetScan tool (release 7.2), which was used for the prediction of the miRNA target sites within the 3′ UTR region of the HIF1A gene [25]. 3.1. Characteristics of the HIF1A Gene 3.1. Characteristics of the HIF1A Gene Human HIF1A gene is associated with the following gene identiﬁers (Gene IDs): ENSG00000100644 (Ensembl ID), 3091 (NCBI ID), and 4910 (HGNC ID). According to the latest release of the Ensembl database, the HIF1A gene has 12 annotated transcripts (splice Genes 2021, 12, 1526 4 of 14 variants), with the following biotypes: four protein-coding, two with retained intron, and six processed transcripts—transcripts that do not contain an open reading frame (ORF). Two protein-coding transcripts are marked as the golden path (merged Ensembl/Havana): ENST00000323441.10 (HIF1A-201) and ENST00000337138.9 (HIF1A-202). The longest transcript, ENST00000337138.9, consists of 15 coding exons, the transcript length is 3946 bps, the translation length is 826 residues, and it is associated with 14,136 variant alleles (Figure 3). The shorter transcript ENST00000323441.10 has 14 exons, the transcript length is 3555 bp, the translation length is 826 aa, and it is associated with 14,018 variant alleles. In humans, the HIF1A gene has seven paralogs: NPAS4, EPAS1, NPAS3, NPAS1, SIM1, SIM2, and HIF3A. The HIF1A gene currently has 226 annotated orthologues—for example, Hif1a (ENSMUSG00000021109) in mouse, hif1aa (ENSDARG00000006181) in zebraﬁsh, and HIF1A (ENSBTAG00000020935) in cow. Human HIF1A gene is associated with the following gene identifiers (Gen ENSG00000100644 (Ensembl ID), 3091 (NCBI ID), and 4910 (HGNC ID). According latest release of the Ensembl database, the HIF1A gene has 12 annotated transcripts variants), with the following biotypes: four protein-coding, two with retained intro six processed transcripts—transcripts that do not contain an open reading frame Two protein-coding transcripts are marked as the golden path (merged Ensembl/Ha ENST00000323441.10 (HIF1A-201) and ENST00000337138.9 (HIF1A-202). The l transcript, ENST00000337138.9, consists of 15 coding exons, the transcript length i bps, the translation length is 826 residues, and it is associated with 14,136 variant (Figure 3). The shorter transcript ENST00000323441.10 has 14 exons, the transcript is 3555 bp, the translation length is 826 aa, and it is associated with 14,018 variant In humans, the HIF1A gene has seven paralogs: NPAS4, EPAS1, NPAS3, NPAS1, SIM2 and HIF3A (a) Figure 3. Cont. (a) Figure 3. Cont. 5 of 14 Genes 2021, 12, 1526 Genes 2021 (b) (c) man HIF1A gene structure and genetic variants. (a) Gene sequence from 5′ UTR/exon 1 to e from intron 9 to exon 15/3′UTR. Partial sequences of introns are shown; start and end. (c) C btained from the Ensembl genome browser. Figure 3. Human HIF1A gene structure and genetic variants. 3.2. Genetic Variability of the HIF1A Gene 3.2. Genetic Variability of the HIF1A Gene 3.2. Genetic Variability of the HIF1A Gene 3.2. Genetic Variability of the HIF1A Gene The HIF1A gene includes genetic variants with various biotypes, including missense and synonymous variants present in all exons, and frameshift variants in exons 2, 5, 8, 10, 11, and 14 (Figure 3). Several splice variants, marked in orange in Figure 3, are present in the HIF1A gene. Splice variants overlap splice sites at the exon/intron borders and are deﬁned as the changes located either within 1–3 nucleotides of the exon or 3–8 nucleotides of the intron. Besides several intronic splice variants, there are also splice variants located within exons; within the ﬁrst three nucleotides of exons 5, 11, 13, and 14 and within the last three nucleotides of exon 2. Additionally, the HIF1A gene also has three splice donor variants, located within the ﬁrst two nucleotides of the intron (introns 6, 7, and 11) and one splice acceptor variant located in the last two nucleotides of the introns (introns 7 and 12). The HIF1A gene currently does not have variants within the start codon. However, two variants are located in the stop-codon: stop lost (rs1389320297; /R) and stop retained variant (rs770333662; /). The gene also has nine stop gained variants in exons 8, 9, 10, and 15. The HIF1A gene includes genetic variants with various biotypes, including missense and synonymous variants present in all exons, and frameshift variants in exons 2, 5, 8, 10, 11, and 14 (Figure 3). Several splice variants, marked in orange in Figure 3, are present in the HIF1A gene. Splice variants overlap splice sites at the exon/intron borders and are defined as the changes located either within 1–3 nucleotides of the exon or 3–8 nucleotides of the intron. Besides several intronic splice variants, there are also splice variants located within exons; within the first three nucleotides of exons 5, 11, 13, and 14 and within the last three nucleotides of exon 2. Additionally, the HIF1A gene also has three splice donor variants, located within the first two nucleotides of the intron (introns 6, 7, and 11) and one splice acceptor variant located in the last two nucleotides of the introns (introns 7 and 12). The HIF1A gene currently does not have variants within the start codon. However, two variants are located in the stop-codon: stop lost (rs1389320297; /R) and stop retained variant (rs770333662; /). 3.2. Genetic Variability of the HIF1A Gene 3.2. Genetic Variability of the HIF1A Gene The gene also has nine stop gained variants in exons 8, 9, 10, and 15 Figure 4 presents the HIF1A nucleotide sequence of exon 12 with marked genetic variants. The location of two frequently studied missense variants at the protein positions 582 and 588 are marked: rs11549465 (p.Pro582Ser) and rs11549467 (p.Ala588Thr). These two variants are located within the ODD domain. and 15. Figure 4 presents the HIF1A nucleotide sequence of exon 12 with marked genetic variants. The location of two frequently studied missense variants at the protein positions 582 and 588 are marked: rs11549465 (p.Pro582Ser) and rs11549467 (p.Ala588Thr). These two variants are located within the ODD domain. Figure 4. HIF1A nucleotide sequence of exon 12 with marked genetic variants. The green arrow denotes a synonymous variant C > T at the proline residue at position 564 (P564); the site of hydroxylation by HIF prolyl hydroxylase EGLN1. The black arrows denote missense variants at the protein positions 582 and 588. Legend: P/S: Pro/Ser; A/T: Ala/Thr. Figure 4. HIF1A nucleotide sequence of exon 12 with marked genetic variants. The green arrow denotes a synonymous variant C > T at the proline residue at position 564 (P564); the site of hydroxylation by HIF prolyl hydroxylase EGLN1. The black arrows denote missense variants at the protein positions 582 and 588. Legend: P/S: Pro/Ser; A/T: Ala/Thr. Figure 4. HIF1A nucleotide sequence of exon 12 with marked genetic variants. The green arrow denotes a synonymous variant C > T at the proline residue at position 564 (P564); the site of hydroxylation by HIF prolyl hydroxylase EGLN1. The black arrows denote missense variants at the protein positions 582 and 588. Legend: P/S: Pro/Ser; A/T: Ala/Thr. Figure 4. HIF1A nucleotide sequence of exon 12 with marked genetic variants. The green arrow denotes a synonymous variant C > T at the proline residue at position 564 (P564); the site of hydroxylation by HIF prolyl hydroxylase EGLN1. The black arrows denote missense variants at the protein positions 582 and 588. Legend: P/S: Pro/Ser; A/T: Ala/Thr. The analysis for the presence of variants located at the sites of protein hydroxylation revealed that there are variations located at all three sites: P402, P564, and N803. 3.1. Characteristics of the HIF1A Gene (a) Gene sequence from 5′ to exon 9. (b) HIF1A gene sequence from intron 9 to exon 15/3′ UTR. Partial sequenc are shown; start and end. (c) Color legend. The ﬁgures were obtained from the Ensem (b) (c) an HIF1A gene structure and genetic variants. (a) Gene sequence from 5′ UTR/exon 1 to exon 9. (b) HIF1 from intron 9 to exon 15/3′UTR. Partial sequences of introns are shown; start and end. (c) Color legend. T tained from the Ensembl genome browser. Figure 3. Human HIF1A gene structure and genetic variants. (a) Gene sequence from 5′ UTR/exon 1 to exon 9. (b) HIF1A gene sequence from intron 9 to exon 15/3′ UTR. Partial sequences of introns are shown; start and end. (c) Color legend. The ﬁgures were obtained from the Ensembl genome browser. (b) (c) n HIF1A gene structure and genetic variants. (a) Gene sequence from 5′ UTR/exon 1 to exon 9. (b) HIF rom intron 9 to exon 15/3′UTR. Partial sequences of introns are shown; start and end. (c) Color legend. T tained from the Ensembl genome browser. Figure 3. Human HIF1A gene structure and genetic variants. (a) Gene sequence from 5′ UTR/exon 1 to exon 9. (b) HIF1A gene sequence from intron 9 to exon 15/3′ UTR. Partial sequences of introns are shown; start and end. (c) Color legend. The ﬁgures were obtained from the Ensembl genome browser. (b) (c) (c) n HIF1A gene structure and genetic variants. (a) Gene sequence from 5′ UTR/exon 1 to exon 9. (b) HIF om intron 9 to exon 15/3′UTR. Partial sequences of introns are shown; start and end. (c) Color legend. T ained from the Ensembl genome browser. Figure 3. Human HIF1A gene structure and genetic variants. (a) Gene sequence from 5′ UTR/exon 1 to exon 9. (b) HIF1A gene sequence from intron 9 to exon 15/3′ UTR. Partial sequences of introns are shown; start and end. (c) Color legend. The ﬁgures were obtained from the Ensembl genome browser. Genes 2021, 12, 1526 Genes 2021, 12, x FOR 6 of 14 6 of 15 3.2. Genetic Variability of the HIF1A Gene 3.2. Genetic Variability of the HIF1A Gene Two synonymous variations encoding for same amino acid are present at the site of proline hydroxylation by HIF prolyl hydroxylase EGLN1 (P402 and P564) and a missense variant The analysis for the presence of variants located at the sites of protein hydroxylation revealed that there are variations located at all three sites: P402, P564, and N803. Two synonymous variations encoding for same amino acid are present at the site of proline hydroxylation by HIF prolyl hydroxylase EGLN1 (P402 and P564) and a missense vari- Genes 2021, 12, 1526 Genes 2021, 12, x FOR 7 of 14 7 of 15 ant is located at the site of asparagine hydroxylation (N803). The ﬁrst site of proline hydroxylation (P402) is located in exon 9, and there is a synonymous variant within this codon (rs750187829 A > G; codons CCA > CCG; Figure 3). The second site of proline hydroxylation is located in exon 12; at the proline residue at position 564 (P564). A genetic variant, rs41492849 C > T, is located within this codon (Figure 4). Additionally, the missense variation rs1220369809 T > G is located at the asparagine 803 (N803) in exon 15, the site of hydroxylation by HIF1AN. This variation is predicted to have a deleterious effect on protein function or, it is likely disease-causing by four bioinformatics tools: SIFT, PolyPhen, REVEL, and MetaLR. ylation (P402) is located in exon 9, and there is a synonymous variant within this codon (rs750187829 A > G; codons CCA > CCG; Figure 3). The second site of proline hydroxyla- tion is located in exon 12; at the proline residue at position 564 (P564). A genetic variant, rs41492849 C > T, is located within this codon (Figure 4). Additionally, the missense vari- ation rs1220369809 T > G is located at the asparagine 803 (N803) in exon 15, the site of hydroxylation by HIF1AN. This variation is predicted to have a deleterious effect on pro- tein function or, it is likely disease-causing by four bioinformatics tools: SIFT, PolyPhen, REVEL, and MetaLR. The current release of the Ensembl database lists over 450 missense variants of the The current release of the Ensembl database lists over 450 missense variants of the HIF1A gene with calculated predictions on protein function. 3.2. Genetic Variability of the HIF1A Gene 3.2. Genetic Variability of the HIF1A Gene The missense variant rs1566567018 G > A at protein position 29 has been predicted to have a deleterious effect by all six tools: deleterious (SIFT), probably damaging (PolyPhen), likely deleterious (CADD), likely disease-causing (REVEL), damaging (MetaLR), and high functional impact (Mutation Assessor). Additionally, three polymorphisms that have predicted a deleterious effect using ﬁve bioinformatics tools are located on protein position 27 (rs754062510), 29 (rs746540920), and 129 (rs1594871023). Examples of ﬁve HIF1A variants with a high predicted deleterious effect using bioinformatics tools are shown in Figure 5. These ﬁve polymorphisms are located at protein position 17 (rs904387412 and rs1163104034), position 29 (rs1566567018), position 129 (rs1594871023), and position 545 (rs1339195532). HIF1A gene with calculated predictions on protein function. The missense variant rs1566567018 G > A at protein position 29 has been predicted to have a deleterious effect by all six tools: deleterious (SIFT), probably damaging (PolyPhen), likely deleterious (CADD), likely disease-causing (REVEL), damaging (MetaLR), and high functional im- pact (Mutation Assessor). Additionally, three polymorphisms that have predicted a dele- terious effect using five bioinformatics tools are located on protein position 27 (rs754062510), 29 (rs746540920), and 129 (rs1594871023). Examples of five HIF1A variants with a high predicted deleterious effect using bioinformatics tools are shown in Figure 5. These five polymorphisms are located at protein position 17 (rs904387412 and rs1163104034), position 29 (rs1566567018), position 129 (rs1594871023), and position 545 (rs1339195532). Figure 5. Five HIF1A polymorphisms with predicted effect on protein function. The analysis was performed using six bioinformatics tools included in the Ensembl genome browser. Figure 5. Five HIF1A polymorphisms with predicted effect on protein function. The analysis was performed using six bioinformatics tools included in the Ensembl genome browser. Figure 5. Five HIF1A polymorphisms with predicted effect on protein function. The analysis was performed using six bioinformatics tools included in the Ensembl genome browser. Figure 5. Five HIF1A polymorphisms with predicted effect on protein function. The analysis was performed using six bioinformatics tools included in the Ensembl genome browser. Figure 5. Five HIF1A polymorphisms with predicted effect on protein function. The analysis was performed using six bioinformatics tools included in the Ensembl genome browser. Figure 5. Five HIF1A polymorphisms with predicted effect on protein function. The analysis was performed using six bioinformatics tools included in the Ensembl genome browser. 3.3.1. HIF Target Genes The HIF family of g 3.3.1. HIF Target Genes The HIF family of genes has been shown to target protein-coding genes as well as several classes of noncoding RNAs. The number of reported HIF target genes exceeds 1000 and is increasing [26,27]; however, a complete database of target genes does not yet exist. The preliminary catalog of HIF1A targets consists of 98 genes that were described in 51 published papers. Those target genes were shown to be associated with 20 pathways, in- cluding metabolism of carbohydrates and pathways in cancer. Additionally, reanalysis of genomic coordinates of hypoxia response elements (HREs) revealed six polymorphisms within HRE sites (termed as HRE-SNPs) of four genes: ABCG2, ACE, CA9, and CP [28]. The list of target genes is not yet complete, and further studies are needed to reveal the complete HIF1A targetome. Large heterogeneity of result presentation in scientific litera- ture reporting HIF1A targets was observed, and therefore a minimal checklist for report- ing HIF1A targets was suggested; it consisted of 10 relevant data types: official symbols of the target genes, species, methodology, cell lines, expression of the target gene, ge- nomics location of HRE sites, presence of HRE polymorphisms, associated phenotypes or diseases, and available identification numbers of genes, diseases, and species [28]. Stand- ardized reporting of HIF targets would facilitate data curation and enable faster develop- ment of a complete catalog of HIF targetome. Th h i l k i l d i f i di d The HIF family of genes has been shown to target protein-coding genes as well as several classes of noncoding RNAs. The number of reported HIF target genes exceeds 1000 and is increasing [26,27]; however, a complete database of target genes does not yet exist. The preliminary catalog of HIF1A targets consists of 98 genes that were described in 51 published papers. Those target genes were shown to be associated with 20 pathways, including metabolism of carbohydrates and pathways in cancer. Additionally, reanalysis of genomic coordinates of hypoxia response elements (HREs) revealed six polymorphisms within HRE sites (termed as HRE-SNPs) of four genes: ABCG2, ACE, CA9, and CP [28]. The list of target genes is not yet complete, and further studies are needed to reveal the complete HIF1A targetome. 3.3. A Map of HIF Regulatory Elements 3.3. A Map of HIF Regulatory Elements The HIF1A gene has been studied at various omics levels, including genomics (DNA level), transcriptomics (RNA level), proteomics, epigenomics, interactomics, and miRNomics levels. The HIF1A gene is involved in a complex interplay of interactions, and a subset of regulatory elements associated with the HIF1A gene is shown in Figure 6. HIF1A acts as a transcription factor of several downstream targets; however, HIF1A is under the control of various transcription factors (TFs) binding to transcription factor binding sites (TFBS). Additionally, miRNA regulate the expression of the HIF1A gene through interaction of their seed region with the target region, primarily at the 3′UTR re- gion. The HIF1A transcription factor drives the expression of a large set of target genes, The HIF1A gene has been studied at various omics levels, including genomics (DNA level), transcriptomics (RNA level), proteomics, epigenomics, interactomics, and miR- Nomics levels. The HIF1A gene is involved in a complex interplay of interactions, and a subset of regulatory elements associated with the HIF1A gene is shown in Figure 6. HIF1A acts as a transcription factor of several downstream targets; however, HIF1A is under the control of various transcription factors (TFs) binding to transcription factor binding sites (TFBS). Additionally, miRNA regulate the expression of the HIF1A gene through interaction of their seed region with the target region, primarily at the 3′ UTR region. The HIF1A transcription factor drives the expression of a large set of target genes, including protein-coding genes, long noncoding RNAs (lncRNAs), and short ncRNAs, including miRNAs (HRMs, hypoxamiRs). Genes 2021, 12, 1526 8 of 14 As, in- Figure 6. The map of regulatory elements associated with the HIF transcription factor family. The HIF1A gene structure was obtained from the Ensembl genome browser. Figure 6. The map of regulatory elements associated with the HIF transcription factor family. The HIF1A gene structure was obtained from the Ensembl genome browser. Figure 6. The map of regulatory elements associated with the HIF transcription factor family. The HIF1A gene structure was obtained from the Ensembl genome browser. Figure 6. The map of regulatory elements associated with the HIF transcription factor family. The HIF1A gene structure was obtained from the Ensembl genome browser. 3.3.1. HIF Target Genes The HIF family of g 3.3.1. HIF Target Genes Large heterogeneity of result presentation in scientiﬁc literature reporting HIF1A targets was observed, and therefore a minimal checklist for reporting HIF1A targets was suggested; it consisted of 10 relevant data types: ofﬁcial symbols of the target genes, species, methodology, cell lines, expression of the target gene, genomics location of HRE sites, presence of HRE polymorphisms, associated phenotypes or diseases, and available identiﬁcation numbers of genes, diseases, and species [28]. Standardized reporting of HIF targets would facilitate data curation and enable faster development of a complete catalog of HIF targetome. The hypoxic regulatory network includes transcripts of protein-coding genes and a class of long noncoding RNAs termed T-UCRs (transcribed ultraconserved regions). The hypoxia-induced noncoding ultraconserved transcript (HINCUT1) is located in the re- tained intron of the OGT gene and has been shown to be upregulated in colon cancer and involved in protein glycosylation in hypoxia [29]. HIF1A also promotes the expression of several hypoxamiRs, including miR-210, miR-146a, miR145, miR-382, miR-191, miR-363, miR-421 in tumor cells, miR-204 in neuronal cells, and miR-30a and miR-21 in cardiomy- ocytes (reviewed in [30]). The hypoxic regulatory network includes transcripts of protein-coding genes and a class of long noncoding RNAs termed T-UCRs (transcribed ultraconserved regions). The hypoxia-induced noncoding ultraconserved transcript (HINCUT1) is located in the retained intron of the OGT gene and has been shown to be upregulated in colon cancer and involved in protein glycosylation in hypoxia [29]. HIF1A also promotes the expression of several hypoxamiRs, including miR-210, miR-146a, miR145, miR-382, miR-191, miR-363, miR-421 in tumor cells, miR-204 in neuronal cells, and miR-30a and miR-21 in cardiomyocytes (reviewed in [30]). 3.3.3. Epigenomics 3.3.3. Epigenomics The HIF1A gene is under the control of various epigenetic mechanisms, including DNA methylation, histone modiﬁcations, and miRNAs. A large CpG island comprising 1431 bp is present around the transcription start site of the HIF1A gene (Figure 7). Hypomethylation non-CpG/CpG sites in the promoter of the HIF1A gene in combination with increased H3K9Ac modiﬁcation was shown to contribute to increased transcription and expression in breast cancer [31]. The HIF1A gene is under the control of various epigenetic mechanisms, including DNA methylation, histone modifications, and miRNAs. A large CpG island comprising 1431 bp is present around the transcription start site of the HIF1A gene (Figure 7). Hypo- methylation non-CpG/CpG sites in the promoter of the HIF1A gene in combination with increased H3K9Ac modification was shown to contribute to increased transcription and expression in breast cancer [31]. Figure 7. CpG island comprising 1431 bp associated with the HIF1A gene. Figure 7. CpG island comprising 1431 bp associated with the HIF1A gene. Figure 7. CpG island comprising 1431 bp associated with the HIF1A gene. Figure 7. CpG island comprising 1431 bp associated with the HIF1A gene. Ten-eleven translocation (TET) methylcytosine dioxygenases catalyze the conversion of 5-mC to 5′-hmC. TET1-mediated 5-hmC changes have been shown to be an epigenetic component of the hypoxic response. Hypoxia increases global 5-hmC levels, and accumu- lation of 5-hmC sites has been identified at hypoxia response genes. 5-hmC gains are also located at HRE sites, facilitating DNA demethylation and HIF binding. Hypoxia results in transcriptional activation of TET1, and induction of hypoxia-responsive genes and global 5-hmC increases require TET1. 5-hmC increases and TET1 upregulation in hypoxia are HIF1 dependent [32] Ten-eleven translocation (TET) methylcytosine dioxygenases catalyze the conversion of 5-mC to 5′-hmC. TET1-mediated 5-hmC changes have been shown to be an epigenetic component of the hypoxic response. Hypoxia increases global 5-hmC levels, and accumu- lation of 5-hmC sites has been identiﬁed at hypoxia response genes. 5-hmC gains are also located at HRE sites, facilitating DNA demethylation and HIF binding. Hypoxia results in transcriptional activation of TET1, and induction of hypoxia-responsive genes and global 5-hmC increases require TET1. 5-hmC increases and TET1 upregulation in hypoxia are HIF1 dependent [32]. HIF1 dependent [32]. Lysine methyltransferases G9a and G9a-like protein (GLP) methylate HIF1A protein and inhibit HIF1A activity within solid tumors, making it unable to bind to the HRE of its target genes, resulting in inhibition of the downstream HIF pathway. 3.3.3. Epigenomics 3.3.3. Epigenomics G9a and GLP cata- lyze mono- and di-methylation of HIF-1α at lysine (K) 674 in vitro and in vivo [3,33]. In- terestingly, the present analysis revealed that the missense polymorphism is located at this position (rs779328210 Lys674Asn) and might affect regulation of HIF1A activity p Lysine methyltransferases G9a and G9a-like protein (GLP) methylate HIF1A protein and inhibit HIF1A activity within solid tumors, making it unable to bind to the HRE of its target genes, resulting in inhibition of the downstream HIF pathway. G9a and GLP catalyze mono- and di-methylation of HIF-1α at lysine (K) 674 in vitro and in vivo [3,33]. Interestingly, the present analysis revealed that the missense polymorphism is located at this position (rs779328210, Lys674Asn) and might affect regulation of HIF1A activity. this position (rs779328210, Lys674Asn) and might affect regulation of HIF1A activity. It has been reported that HIF1A employs diverse cofactors to regulate different sub- sets of target genes. For example, HIF1A acts on KAT5 (lysine acetyltransferase 5; TIP60), which leads to chromatin histone acetylation and then to the activation of polymerase II, which acti ates the transcription of HIF 1α target genes [34] It has been reported that HIF1A employs diverse cofactors to regulate different subsets of target genes. For example, HIF1A acts on KAT5 (lysine acetyltransferase 5; TIP60), which leads to chromatin histone acetylation and then to the activation of polymerase II, which activates the transcription of HIF-1α target genes [34]. 3.3.2. Upstream Regulation of HIF1A Signaling Various pathways have been shown to regulate HIF activity by regulating HIF syn- thesis [6]. The HIF family of genes is affected by various factors, including hormones, growth factors, and cytokines on transcriptional and post-transcriptional levels [3]. For example, the PI3K/Akt/mTOR pathway plays a signiﬁcant role in regulating HIF activity. Genes 2021, 12, 1526 9 of 14 y mones, 3] F 9 of 14 y mones, 3] F Phosphoinositide 3-kinase (PI3K), pyruvate dehydrogenase kinase (PDK), and protein kinase B (PKB; Akt) activation induced by growth factors (GFs) activate the mammalian target of the rapamycin (mechanistic target of rapamycin kinase; mTOR) pathway, which results in elevated HIF-1α transcriptional activity [6]. Other pathways/factors control- ling HIF regulation include the TNF signaling/NF-κB (nuclear factor kappa-B) pathway, RAS/RAF/MEK/ERK kinase cascade, mouse double minute 2 homolog (MDM2), and heat shock protein 90 (HSP90) [3,6]. Phosphoinositide 3-kinase (PI3K), pyruvate dehydrogenase kinase (PDK), and protein ki- nase B (PKB; Akt) activation induced by growth factors (GFs) activate the mammalian target of the rapamycin (mechanistic target of rapamycin kinase; mTOR) pathway, which results in elevated HIF-1α transcriptional activity [6]. Other pathways/factors controlling HIF regulation include the TNF signaling/NF-κB (nuclear factor kappa-B) pathway, RAS/RAF/MEK/ERK kinase cascade, mouse double minute 2 homolog (MDM2), and heat shock protein 90 (HSP90) [3,6]. Phosphoinositide 3-kinase (PI3K), pyruvate dehydrogenase kinase (PDK), and protein kinase B (PKB; Akt) activation induced by growth factors (GFs) activate the mammalian target of the rapamycin (mechanistic target of rapamycin kinase; mTOR) pathway, which results in elevated HIF-1α transcriptional activity [6]. Other pathways/factors control- ling HIF regulation include the TNF signaling/NF-κB (nuclear factor kappa-B) pathway, RAS/RAF/MEK/ERK kinase cascade, mouse double minute 2 homolog (MDM2), and heat shock protein 90 (HSP90) [3,6]. Phosphoinositide 3-kinase (PI3K), pyruvate dehydrogenase kinase (PDK), and protein ki- nase B (PKB; Akt) activation induced by growth factors (GFs) activate the mammalian target of the rapamycin (mechanistic target of rapamycin kinase; mTOR) pathway, which results in elevated HIF-1α transcriptional activity [6]. Other pathways/factors controlling HIF regulation include the TNF signaling/NF-κB (nuclear factor kappa-B) pathway, RAS/RAF/MEK/ERK kinase cascade, mouse double minute 2 homolog (MDM2), and heat shock protein 90 (HSP90) [3,6]. 3 3 4 Mi RNA R l ti 3.3.4. MicroRNA Regulation 3.3.4. MicroRNA Regulation HIF1A has been shown to be under the control of several miRNAs. Based on the miRTarBase, a genomic resource of experimentally validated MTIs, there are currently 85 HIF1A has been shown to be under the control of several miRNAs. Based on the miRTarBase, a genomic resource of experimentally validated MTIs, there are currently 85 miRNAs reported to be in interaction with the HIF1A gene (Figure 8). Some interactions between miRNAs and HIF1A mRNA have been reported in more than one publication and 116 miRNA-HIF1A interactions have been described in 45 scientiﬁc papers. Most of these interactions were identiﬁed using methodology considered as weak validation status, such as photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP), crosslinking, ligation, and sequencing of hybrids (CLASH), high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP), or qRT- Genes 2021, 12, 1526 10 of 14 precipi- ), high- 10 of 14 precipi- ), high- PCR. However, 36 of the reported interactions have a strong validation status and were conﬁrmed using approaches such as luciferase reporter assay or western blot. CLIP), or qRT PCR. However, 36 of the reported interactions have a strong validation sta tus and were confirmed using approaches such as luciferase reporter assay or western blot. PCR. However, 36 of the reported interactions have a strong validation status and were conﬁrmed using approaches such as luciferase reporter assay or western blot. ), q , p g tus and were confirmed using approaches such as luciferase reporter assay or western blot. PCR. However, 36 of the reported interactions have a strong validation status and were conﬁrmed using approaches such as luciferase reporter assay or western blot. ), q , p g tus and were confirmed using approaches such as luciferase reporter assay or western blot. Figure 8. MicroRNAs reported to regulate the HIF1A gene. The data obtained from the miRTar- Base. Figure 8. MicroRNAs reported to regulate the HIF1A gene. The data obtained from the miRTarBase. 3 3 5 P t i I t ti Figure 8. MicroRNAs reported to regulate the HIF1A gene. The data obtained from the miRTar- B Figure 8. MicroRNAs reported to regulate the HIF1A gene. The data obtained from the miRTarBase. Base. 3.3.5. Protein Interactions 3.3.5. Protein Interactions Figure 9a presents the PPI network associated with HIF1A protein using the STRING tool. The network includes 11 nodes, and it has significantly more interactions than ex- pected (PPI enrichment p-value: 5.39 × 10−6). The HIF1A PPI network consists of the fol- lowing interacting partners: EGLN3, ARNT, ARNT2, EP300, VHL, CREBBP, TCEB1, TCEB2, CUL2, and EGLN1. Adding more nodes to the network resulted in the extended network consisting of 21 proteins (Figure 9b). An extensive list of additional HIF1A pro- Figure 9a presents the PPI network associated with HIF1A protein using the STRING tool. The network includes 11 nodes, and it has signiﬁcantly more interactions than expected (PPI enrichment p-value: 5.39 × 10−6). The HIF1A PPI network consists of the following interacting partners: EGLN3, ARNT, ARNT2, EP300, VHL, CREBBP, TCEB1, TCEB2, CUL2, and EGLN1. Adding more nodes to the network resulted in the extended network consisting of 21 proteins (Figure 9b). An extensive list of additional HIF1A protein interacting partners was reviewed by Semenza, and the list continues to grow rapidly [2]. anslational Modifications (PTMs) 3.4. Association of HIF1A with Diseases ylation has been described as the major modification of the HIF-α subunits. several other post-translational modifications play a role in HIF localization, activity: acetylation, methylation, S-nitrosylation, phosphorylation, sumola- quitination [2,35]. n of HIF1A with Diseases variations and dysregulation of the HIF1A gene have been shown to be asso- he development of several diseases. Our previous review of the literature NPs with significant associations with 40 different phenotypes, including 6 Genetic variations and dysregulation of the HIF1A gene have been shown to be associated with the development of several diseases. Our previous review of the literature revealed 16 SNPs with signiﬁcant associations with 40 different phenotypes, including 6 SNPs associated with 14 cancer types [10]. Genetic variants of the HIF1A gene have been shown to be associated with cardiovascular system diseases such as ischemic heart disease, coronary artery disease (CAD), premature coronary artery disease, pre-eclampsia, and acute myocardial infarction. Moreover, HIF1A SNPs have been shown to be involved in metabolic disorders such as diabetic nephropathy. Missense SNPs rs11549465 (p.Pro582Ser) and rs11549467 (p.Ala588Thr) within the ODD domain are most frequently studied. NPs with significant associations with 40 different phenotypes, including 6 ted with 14 cancer types [10]. Genetic variants of the HIF1A gene have been associated with cardiovascular system diseases such as ischemic heart dis- y artery disease (CAD), premature coronary artery disease, pre-eclampsia, ocardial infarction. Moreover, HIF1A SNPs have been shown to be involved disorders such as diabetic nephropathy Missense SNPs rs11549465 According to the Ensembl/Cancer Gene Census database, the HIF1A gene is associated with 62 phenotypes in humans (Table S1). Additionally, orthologues of this gene in other species have also been associated with several phenotype, disease, and trait annotations. HIF1A–phenotype associations from the Ensembl browser, which integrates the data from ZFIN, MGI, and RGD, are shown in Table S2. disorders such as diabetic nephropathy. Missense SNPs rs11549465 and rs11549467 (p.Ala588Thr) within the ODD domain are most frequently ng to the Ensembl/Cancer Gene Census database, the HIF1A gene is associ- phenotypes in humans (Table S1). Additionally, orthologues of this gene in have also been associated with several phenotype, disease, and trait annota- –phenotype associations from the Ensembl browser, which integrates the IN, MGI, and RGD, are shown in Table S2. anslational Modifications (PTMs) 3.4. Association of HIF1A with Diseases 1 presents HIF1A-associated data, extracted from the UALCAN database, an eb resource for analyzing cancer omics data: (1) expression of HIF1A across Figure S1 presents HIF1A-associated data, extracted from the UALCAN database, an interactive web resource for analyzing cancer omics data: (1) expression of HIF1A across TCGA cancers with tumor and normal samples (Figure S1A), (2) effect of HIF1A expression level on bladder urothelial carcinoma (BLCA) patient survival (Figure S1B), (3) HIF1A proteomic expression proﬁle based on sample types (Figure S1C), and (4) conserved miRNA mir-199-5p target site within the 3′ UTR region of the HIF1A gene (Figure S1D). The present study presents only part of the HIF1A data available in the UALCAN database, and additional in-depth study could contribute to revealing the unknown mechanisms of HIF1A involvement in the development of various cancer types. eb resource for analyzing cancer omics data: (1) expression of HIF1A across rs with tumor and normal samples (Figure S1A), (2) effect of HIF1A expres- n bladder urothelial carcinoma (BLCA) patient survival (Figure S1B), (3) omic expression profile based on sample types (Figure S1C), and (4) con- A mir-199-5p target site within the 3′ UTR region of the HIF1A gene (Figure esent study presents only part of the HIF1A data available in the UALCAN d additional in-depth study could contribute to revealing the unknown mech- F1A involvement in the development of various cancer types. ce in molecular mechanisms of the hypoxia-inducible factor-erythropoietin athway can result in hematological disorders [36]. Regulation of the EPO raises the possibility that variants in all three HIFA paralogs play a role in Imbalance in molecular mechanisms of the hypoxia-inducible factor-erythropoietin (HIF-EPO) pathway can result in hematological disorders [36]. Regulation of the EPO gene by HIF raises the possibility that variants in all three HIFA paralogs play a role in disorders of erythropoiesis, such as erythrocytosis. EPAS1 was shown to be a key regulator of EPO production, and variants of the EPAS1 gene have been reported as a cause of familial erythrocytosis type 4 (ECYT4) [7]. To date, 20 EPAS1 variants identiﬁed in patients with erythrocytosis or associated with its symptoms are known [7]. By contrast, only two variants of the HIF3A gene are associated with FE [37]. The missense variant p.Pro582Ser located in the HIF1A gene was found to be associated with higher Hb and ferritin levels in male blood donors. tein interacting partners was reviewed by Seme [2] 3.3.6. Post-Translational Modiﬁcations (PTMs) [2]. Hydroxylation has been described as the major modiﬁcation of the HIF-α subunits. Additionally, several other post-translational modiﬁcations play a role in HIF localiza- tion, stability, and activity: acetylation, methylation, S-nitrosylation, phosphorylation, sumolation, and ubiquitination [2,35]. 11 of 14 Genes 2021, 12, 1526 FOR PEER REVIEW Figure 9. The PPI network associated with HIF1A protein visualized using the STRING tool. (a) HIF1A direct interacting partners; the network includes 11 nodes. (b) HIF1A extended interaction network with first neighbors consisting of 21 proteins. Figure 9. The PPI network associated with HIF1A protein visualized using the STRING tool. (a) HIF1A direct interacting partners; the network includes 11 nodes. (b) HIF1A extended interaction network with ﬁrst neighbors consisting of 21 proteins. Figure 9. The PPI network associated with HIF1A protein visualized using the STRING tool. (a) HIF1A direct interacting partners; the network includes 11 nodes. (b) HIF1A extended interaction network with first neighbors consisting of 21 proteins. Figure 9. The PPI network associated with HIF1A protein visualized using the STRING tool. (a) HIF1A direct interacting partners; the network includes 11 nodes. (b) HIF1A extended interaction network with ﬁrst neighbors consisting of 21 proteins. anslational Modifications (PTMs) 3.4. Association of HIF1A with Diseases This polymorphism affects red blood cell and iron homeostasis after blood loss and is associated with a resistance to anemia in males [38]. It was also shown Genes 2021, 12, 1526 12 of 14 12 of 14 that the Pro582Ser variant does not impair HIF1A prolyl hydroxylation in the ODD domain and it does not diminish the association of HIF1A with VHL [39]. Current literature shows that EPAS1 has an important role in erythropoiesis; however, the role of HIF1A and HIF3A in the development of erythrocytosis should be further investigated [7]. 3.5. Future Developments The present data synthesis on the HIF1A gene presents the ﬁrst step toward the construction of a map of regulatory elements for the HIF1A gene and aims to establish a holistic view of the HIF1A gene integrating data from various genomics resources and publications. However, the integrative map is not yet complete, and additional data should be integrated from published and upcoming studies. Some of research directions may include: Some of research directions may include: • Update of the review on associations between HIF genes and diseases. • Development of a catalog of regulatory elements regulating HIF1A expression (up- stream regulators). • Update of the catalog of reported HIF1A target genes (downstream signaling cas- cades). • Update of the catalog of reported HIF1A target genes (downstream signaling cas- cades). • Identiﬁcation of novel HIF1A target genes. • Multi-omics view in understanding HIF1A regulation, including genomics–DNA level, transcriptomics–RNA level, proteomics, glycomics, epigenomics, and miRNomics. p p g y p g • Identiﬁcation of therapeutic strategies targeting the HIF signaling pathway for therapy in various diseases, including cancer. • Analysis of potential functional effect of polymorphisms located within sites of HIF1A protein hydroxylation and methylation. • Analysis of potential functional effect of polymorphisms located within sites of HIF1A protein hydroxylation and methylation. 4. Conclusions Data obtained from the Ensembl genome browser. Funding: This research was funded by Slovenian Research Agency grant number L3-9279 and research program P4-0220. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: For preparation of the manuscript, the following publicly available databases were used: HGNC (https://www.genenames.org/) (accessed on 2 August 2021). Ensembl genome browser (https://www.ensembl.org/index.html) (accessed on 2 August 2021). MirTarBase (https://mirtarbase.cuhk.edu.cn/) (accessed on 2 August 2021) STRING (https://string-db.org/) accessed on 2 August 2021. MethPrimer (https://www.urogene.org/methprimer/) accessed on 2 August 2021. UALCAN (http://ualcan.path.uab.edu/index.html) accessed on 20 September 2021. All the data are presented within the article and in the supplementary material. Conﬂicts of Interest: The author declares no conﬂict of interest. Conﬂicts of Interest: The author declares no conﬂict of interest. References Genet. Dev. 2010, 20, 51–56. [CrossRef] [ b d] 9. Kaelin, W.G.; Ratcliffe, P.J. Oxygen sensing by metazoans: The central role of the HIF hydroxylase pathway. Mol. Cell 2008, 30, 393–402. [CrossRef] 10. Gladek, I.; Ferdin, J.; Horvat, S.; Calin, G.A.; Kunej, T. HIF1A gene polymorphisms and human diseases: Graphical review of 97 association studies. Genes Chromosomes Cancer 2017, 56, 439–452. [CrossRef] 11. Wei, J.; Yang, Y.; Lu, M.; Lei, Y.; Xu, L.; Jiang, Z.; Xu, X.; Guo, X.; Zhang, X.; Sun, H.; et al. Recent Advances in the Discovery of HIF-1α-p300/CBP Inhibitors as Anti-Cancer Agents. Mini Rev. Med. Chem. 2018, 18, 296–309. [CrossRef] [PubMed] 12. Pirih, N.; Kunej, T. 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Conclusions The main aim of this study was to summarize the current state of the HIF1A gene knowledge and discuss future directions. The current data synthesis is the ﬁrst step toward creating a map of regulatory factors for the HIF1A gene, with the intention of building a holistic perspective of the HIF1A gene by combining data from publications and diverse genomics resources such as Ensembl, STRING, and UALCAN. The summary of genomic databases and bioinformatics tools used in the present analysis is presented in Figure S2. Out of over 450 missense variants, four have predicted a deleterious effect on protein function by at least ﬁve bioinformatics tools. Additionally, 85 miRNAs have been reported to target HIF1A. HIF1A downstream targets include protein-coding genes, long noncoding RNAs, and miRNAs. Because of the increasing research interest in the HIF family of genes, systematic reviews are needed in the future. Development of databases including all known molecular interactions and genetic variations associated with HIF1A would substantially contribute to the development of the ﬁeld. One of the major obstacles that hinder the development of HIF1A research is the heterogeneity of published results. The knowledge related to the HIF1A gene is presently scattered across various publications and databases and does not enable a systems view of this research topic. For a better understanding of the research ﬁeld, an integration of these diverse data is needed and a complete map regulatory elements associated to all HIF paralogs should be developed. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/genes12101526/s1: Figure S1: HIF1A data, extracted from the UALCAN database, an interactive web resource for analyzing cancer omics data. (A) Expression of HIF1A across TCGA cancers with tumor and normal samples. 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